WO2024133584A2 - Antigen binding proteins - Google Patents
Antigen binding proteins Download PDFInfo
- Publication number
- WO2024133584A2 WO2024133584A2 PCT/EP2023/087131 EP2023087131W WO2024133584A2 WO 2024133584 A2 WO2024133584 A2 WO 2024133584A2 EP 2023087131 W EP2023087131 W EP 2023087131W WO 2024133584 A2 WO2024133584 A2 WO 2024133584A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- antigen binding
- amino acid
- sequence
- substantially homologous
- Prior art date
Links
- 102000025171 antigen binding proteins Human genes 0.000 title claims abstract description 599
- 108091000831 antigen binding proteins Proteins 0.000 title claims abstract description 599
- 239000000427 antigen Substances 0.000 claims abstract description 361
- 108091007433 antigens Proteins 0.000 claims abstract description 361
- 102000036639 antigens Human genes 0.000 claims abstract description 361
- 230000027455 binding Effects 0.000 claims abstract description 355
- 102210042925 HLA-A*02:01 Human genes 0.000 claims abstract description 242
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims abstract description 148
- 229940127121 immunoconjugate Drugs 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 6
- 238000002560 therapeutic procedure Methods 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 626
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 459
- 102000036673 PRAME Human genes 0.000 claims description 307
- 108060006580 PRAME Proteins 0.000 claims description 307
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 claims description 300
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 283
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 236
- 101710117290 Aldo-keto reductase family 1 member C4 Proteins 0.000 claims description 166
- 102100035361 Cerebellar degeneration-related protein 2 Human genes 0.000 claims description 125
- 101000737796 Homo sapiens Cerebellar degeneration-related protein 2 Proteins 0.000 claims description 125
- 101100112922 Candida albicans CDR3 gene Proteins 0.000 claims description 123
- 206010028980 Neoplasm Diseases 0.000 claims description 119
- 201000011510 cancer Diseases 0.000 claims description 109
- 238000006467 substitution reaction Methods 0.000 claims description 79
- 238000000034 method Methods 0.000 claims description 53
- 238000012258 culturing Methods 0.000 claims description 31
- 150000007523 nucleic acids Chemical class 0.000 claims description 18
- 102000039446 nucleic acids Human genes 0.000 claims description 16
- 108020004707 nucleic acids Proteins 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 16
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims description 10
- 241000700605 Viruses Species 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims 6
- 239000000032 diagnostic agent Substances 0.000 claims 1
- 229940039227 diagnostic agent Drugs 0.000 claims 1
- 239000003814 drug Substances 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 238000002405 diagnostic procedure Methods 0.000 abstract 1
- 238000003556 assay Methods 0.000 description 145
- 235000001014 amino acid Nutrition 0.000 description 131
- 101000737793 Homo sapiens Cerebellar degeneration-related antigen 1 Proteins 0.000 description 113
- 102210047469 A*02:01 Human genes 0.000 description 73
- 239000012636 effector Substances 0.000 description 72
- 229940024606 amino acid Drugs 0.000 description 70
- 102000004196 processed proteins & peptides Human genes 0.000 description 69
- 150000001413 amino acids Chemical class 0.000 description 67
- 230000000694 effects Effects 0.000 description 66
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 55
- 241000282414 Homo sapiens Species 0.000 description 34
- 108060003951 Immunoglobulin Proteins 0.000 description 31
- 108091008874 T cell receptors Proteins 0.000 description 31
- 230000004913 activation Effects 0.000 description 31
- 102000018358 immunoglobulin Human genes 0.000 description 31
- 238000005406 washing Methods 0.000 description 30
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 29
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 29
- 238000001514 detection method Methods 0.000 description 28
- 108090000623 proteins and genes Proteins 0.000 description 28
- 239000012131 assay buffer Substances 0.000 description 27
- 102000004169 proteins and genes Human genes 0.000 description 27
- 239000000872 buffer Substances 0.000 description 26
- 235000018102 proteins Nutrition 0.000 description 25
- 238000010186 staining Methods 0.000 description 24
- 230000004069 differentiation Effects 0.000 description 23
- 230000003013 cytotoxicity Effects 0.000 description 21
- 231100000135 cytotoxicity Toxicity 0.000 description 21
- 238000011002 quantification Methods 0.000 description 21
- 230000010782 T cell mediated cytotoxicity Effects 0.000 description 20
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 20
- 239000002953 phosphate buffered saline Substances 0.000 description 20
- 230000006044 T cell activation Effects 0.000 description 19
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 19
- 230000035755 proliferation Effects 0.000 description 19
- 239000012634 fragment Substances 0.000 description 18
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 18
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 17
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 17
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 16
- 229920001184 polypeptide Polymers 0.000 description 16
- 238000003501 co-culture Methods 0.000 description 15
- 238000000684 flow cytometry Methods 0.000 description 15
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 14
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 14
- 238000012286 ELISA Assay Methods 0.000 description 13
- 238000004113 cell culture Methods 0.000 description 12
- 238000002844 melting Methods 0.000 description 12
- 230000008018 melting Effects 0.000 description 12
- 230000009258 tissue cross reactivity Effects 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- VDABVNMGKGUPEY-UHFFFAOYSA-N 6-carboxyfluorescein succinimidyl ester Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=C2)OC(=O)C1=CC=C2C(=O)ON1C(=O)CCC1=O VDABVNMGKGUPEY-UHFFFAOYSA-N 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- 101100046814 Homo sapiens TAPBP gene Proteins 0.000 description 10
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 10
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 10
- 102100028082 Tapasin Human genes 0.000 description 10
- 125000000539 amino acid group Chemical group 0.000 description 10
- 239000012091 fetal bovine serum Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 230000001464 adherent effect Effects 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 8
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 8
- 230000004075 alteration Effects 0.000 description 8
- 230000000295 complement effect Effects 0.000 description 8
- 238000007865 diluting Methods 0.000 description 8
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 8
- 239000011534 wash buffer Substances 0.000 description 8
- -1 DAGE Proteins 0.000 description 7
- 102000011786 HLA-A Antigens Human genes 0.000 description 7
- 108010075704 HLA-A Antigens Proteins 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 239000006143 cell culture medium Substances 0.000 description 7
- 239000007850 fluorescent dye Substances 0.000 description 7
- 210000002865 immune cell Anatomy 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 210000000581 natural killer T-cell Anatomy 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000004471 Glycine Substances 0.000 description 6
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 6
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 6
- 230000008823 permeabilization Effects 0.000 description 6
- 238000000159 protein binding assay Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
- 101001074035 Homo sapiens Zinc finger protein GLI2 Proteins 0.000 description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
- 102100035558 Zinc finger protein GLI2 Human genes 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 230000022534 cell killing Effects 0.000 description 5
- 238000010494 dissociation reaction Methods 0.000 description 5
- 230000005593 dissociations Effects 0.000 description 5
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 5
- 238000003306 harvesting Methods 0.000 description 5
- 238000011296 nano differential scanning fluorimetry Methods 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 235000004400 serine Nutrition 0.000 description 5
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 4
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 108091054437 MHC class I family Proteins 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 4
- 239000004473 Threonine Substances 0.000 description 4
- 108091008324 binding proteins Proteins 0.000 description 4
- 230000024245 cell differentiation Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000009260 cross reactivity Effects 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 235000008521 threonine Nutrition 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 235000002374 tyrosine Nutrition 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- 101100454808 Caenorhabditis elegans lgg-2 gene Proteins 0.000 description 3
- 102000001398 Granzyme Human genes 0.000 description 3
- 108060005986 Granzyme Proteins 0.000 description 3
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 3
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 3
- 101000669640 Homo sapiens Mitochondrial import inner membrane translocase subunit TIM14 Proteins 0.000 description 3
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 3
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 3
- 102100037020 Melanoma antigen preferentially expressed in tumors Human genes 0.000 description 3
- 102100039325 Mitochondrial import inner membrane translocase subunit TIM14 Human genes 0.000 description 3
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 3
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 3
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 230000001472 cytotoxic effect Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 235000014304 histidine Nutrition 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000004068 intracellular signaling Effects 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000010188 recombinant method Methods 0.000 description 3
- 210000003289 regulatory T cell Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 210000001550 testis Anatomy 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 2
- 101100454807 Caenorhabditis elegans lgg-1 gene Proteins 0.000 description 2
- 101100217502 Caenorhabditis elegans lgg-3 gene Proteins 0.000 description 2
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 2
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 235000003332 Ilex aquifolium Nutrition 0.000 description 2
- 241000209027 Ilex aquifolium Species 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 102000043129 MHC class I family Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 101150081791 PAM17 gene Proteins 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 230000006052 T cell proliferation Effects 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000012867 alanine scanning Methods 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 235000009697 arginine Nutrition 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 239000012911 assay medium Substances 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000004554 glutamine Nutrition 0.000 description 2
- 201000005787 hematologic cancer Diseases 0.000 description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 239000000252 konjac Substances 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 210000003071 memory t lymphocyte Anatomy 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 230000000869 mutational effect Effects 0.000 description 2
- 230000010807 negative regulation of binding Effects 0.000 description 2
- 108010087904 neutravidin Proteins 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000006432 protein unfolding Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 102100037904 CD9 antigen Human genes 0.000 description 1
- 102100025466 Carcinoembryonic antigen-related cell adhesion molecule 3 Human genes 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 244000148064 Enicostema verticillatum Species 0.000 description 1
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 1
- 101000914337 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 3 Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 description 1
- 101001095088 Homo sapiens Melanoma antigen preferentially expressed in tumors Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101100137533 Homo sapiens PRAME gene Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 1
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 1
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 description 1
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000012214 Immunoproteins Human genes 0.000 description 1
- 108010036650 Immunoproteins Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 208000032004 Large-Cell Anaplastic Lymphoma Diseases 0.000 description 1
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000251323 Matthiola oxyceras Species 0.000 description 1
- 101710178381 Melanoma antigen preferentially expressed in tumors Proteins 0.000 description 1
- 206010027626 Milia Diseases 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 210000004460 N cell Anatomy 0.000 description 1
- 108010001657 NK Cell Lectin-Like Receptor Subfamily K Proteins 0.000 description 1
- 102000000812 NK Cell Lectin-Like Receptor Subfamily K Human genes 0.000 description 1
- 108010004217 Natural Cytotoxicity Triggering Receptor 1 Proteins 0.000 description 1
- 102000002755 Natural Cytotoxicity Triggering Receptor 1 Human genes 0.000 description 1
- 108010004222 Natural Cytotoxicity Triggering Receptor 3 Proteins 0.000 description 1
- 102000002752 Natural Cytotoxicity Triggering Receptor 3 Human genes 0.000 description 1
- 102000007399 Nuclear hormone receptor Human genes 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 101800004191 Peptide P2 Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical class [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101150036449 SIRPA gene Proteins 0.000 description 1
- 101150044842 SKM1 gene Proteins 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 206010068771 Soft tissue neoplasm Diseases 0.000 description 1
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 description 1
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 108091005906 Type I transmembrane proteins Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 108010030694 avidin-horseradish peroxidase complex Proteins 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000005880 cancer cell killing Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 108091007930 cytoplasmic receptors Proteins 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000012645 endogenous antigen Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 210000000428 immunological synapse Anatomy 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 108010082406 peptide permease Proteins 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 206010042863 synovial sarcoma Diseases 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
Definitions
- the present invention relates generally to the field of antigen binding proteins, in particular to antibodies which bind to, or specifically bind to, the pMHC (peptide- Major Histocompatibility Complex) HLA-A*02:01/PRAME 425 ' 433 .
- the invention further relates to compositions and immunoconjugates comprising such antibodies and to methods of producing such antibodies.
- the invention also relates to methods and uses which employ such antibodies, for example in the treatment of cancer.
- HLA class I molecules Human leukocyte antigen (HLA) class I molecules are expressed on the surface of all nucleated cells presenting peptides for T-cell recognition.
- the peptides presented in HLA class I molecules are protein fragments of intracellular origin, which are degraded by an array of proteases. The protein fragments are truncated to smaller peptides and translocated into the endoplasmic reticulum (ER).
- ER endoplasmic reticulum
- the peptide-HLA class I molecule a type of peptide-MHC (pMHC) complex, is assembled from a peptide, a polymorphic heavy chain, and the monomorphic light chain p2-microglobulin (P2m).
- a peptide with adequate binding motif residues will bind into the peptide-binding groove of the HLA class I molecule, allowing the assembled molecule to leave the ER and be transported via the Golgi complex to the cell surface to display the peptides.
- PRAME PReferentially expressed Antigen in MEIanoma
- MAPE MAPE
- DAGE DAGE
- OIP4 OIP4
- PRAME is also an important modulator of retinoic acid signalling.
- PRAME expression is noted in the nucleus.
- PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. PRAME is known to be expressed in a wide range of cancers, including solid, soft tissue, hematological and metastatic tumors.
- lymphomas include Hodgkin's lymphomas, peripheral T/N cell lymphomas, anaplastic large cell lymphomas, and peripheral B cell lymphomas.
- the 9-mer peptide SLLQHLIGL corresponds to amino acids 425-433 of the full length PRAME protein. This peptide binds to HLA-A*02:01 and the peptide-HLA complex is considered a suitable target for PRAME specific T-cell receptor therapy.
- TCR T-cell receptor
- the use of recombinant TCRs for the purpose of detecting peptide presentation is challenging. TCRs have low affinity for pMHC, and soluble TCRs are intrinsically unstable and production is demanding.
- Monoclonal antibodies against pMHC targets are therefore desirable, but are difficult to generate given the small epitope of the bound peptide in the human leukocyte antigen (HLA). Given the complex structure of the epitope targets, generating monoclonal antibodies against pMHC targets with high binding specificity and acceptably low levels of off-target activity, and further with good cytotoxicity profiles at low doses, is a particular difficulty.
- HLA human leukocyte antigen
- the present invention provides one such alternative and improved therapeutic option in the form of antigen binding proteins (e.g. antibodies) directed to HLA- A*02:01/PRAME 425 ' 433 .
- antigen binding proteins e.g. antibodies
- the antibodies generated by the inventors have advantageous properties which make them ideal agents for the above-mentioned uses.
- antigen binding proteins (e.g. antibodies) of the invention have been shown to be capable of binding to HLA- A*02:01/PRAME 425-433 with high specificity, and have excellent ability to induce T-cell mediated killing of cancer cells.
- the cell killing effects are observed at very low concentrations, notably in assays comprising an effector cell to target cell ratio (E:T) of only 1 :1 , and notably in disparate cancer types.
- E:T effector cell to target cell ratio
- the cell killing effects are observed whilst the antigen binding proteins (e.g. antibodies) bind with relatively low strength to target.
- the antigen binding proteins e.g.
- the antigen binding proteins (e.g. antibodies) of the invention have also been shown to have outstanding specificity, in particular in terms of their demonstrated lack of binding to/ cross-reactivity with many HLA-A*02:01 restricted peptides with high and very high sequence identity to the target peptide PRAME 425 ' 433 .
- the antigen binding proteins e.g.
- antibodies of the invention also redirect T-cell activity preferentially against to HLA-A*02:01/PRAME 425 ' 433 positive cells, to preferentially induce activation, differentiation and proliferation of T cells directed against HLA-A*02:01/PRAME 425 ' 433 positive cells and to induce T-cell mediated cytotoxicity preferentially against HLA- A*02:01/PRAME 425 ' 433 positive cells.
- the antigen binding proteins (e.g. antibodies) of the invention have also been shown to have advantageous thermal stability. To the inventors’ knowledge, no other anti-PRAME pMHC antibodies have been disclosed to have this advantageous combination of properties, and antigen binding proteins (e.g. antibodies) with one or more, preferably all, of these properties are preferred.
- Such antigen binding proteins (e.g. antibodies) of the invention comprise a HLA-A*02:01/PRAME 425 ' 433 antigen binding domain as described herein, and can conveniently and advantageously be used for the treatment of diseases associated with HLA-A*02:01/PRAME 425 ' 433 expression, in particular for the treatment of cancer.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or
- MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113) or a sequence substantially homologous thereto; or wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein comprising at least one antigen binding domain which binds to, or specifically binds to, HLA-A*02:01/ A*02:01/PRAME 425 ' 433 , said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said VH and VL domains are as defined above.
- VH domain heavy chain variable domain
- CDRs complementarity determining regions
- VL domain light chain variable domain
- antigen binding domain means “at least a first antigen binding domain”. As discussed elsewhere herein, further (i.e. second, third) etc. antigen binding domains may be present. However, it is the “first” antigen binding domain referred to herein that binds to, or specifically binds to, HLA-A*02:01/ A*02:01/PRAME 425 ' 433 .
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 132, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 132, or a sequence substantially homologous thereto, and/or (preferably “and
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 132, or a sequence substantially homologous thereto, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 132, or a sequence substantially homologous thereto, and a VL domain that comprises the amino acid sequence of S
- the invention provides the specific antigen binding proteins (e.g. antibodies) disclosed in Table A.
- Table A provides the specific amino acid and nucleotide sequences of these antigen binding proteins (e.g. antibodies) and the regions/domains thereof, including the CDR sequences, the framework region sequences, and the VH and VL domain sequences.
- sequences of the specific antibodies of the invention disclosed in Table A i.e. those having a given SEQ ID NO., are referred to herein as “specific”, “reference”, “given” or “disclosed” sequences.
- substantially homologous sequences i.e. sequences that are “substantially homologous” to a specific sequence disclosed in Table A.
- the invention also encompasses antigen binding proteins (e.g. antibodies) that comprise i) one or more CDRs, and/or ii) one or more framework regions, and/or iii) a VH domain and/or iv) a VL domain that have a sequence substantially homologous to a specific sequence disclosed in Table A.
- Substantially homologous sequences are defined by reference to a “given”, “reference”, “disclosed” or “specific” sequence, which is a sequence disclosed in Table A, having a specific SEQ ID NO. Alternatively viewed, the substantially homologous sequences are “based on” a specific sequence of Table A.
- substantially homologous sequences applies to all aspects and embodiments of the invention described elsewhere herein, wherever the terms “substantially homologous”, “or a sequence substantially homologous thereto”, or similar terms, are used.
- the specific antigen binding protein (e.g. antibody), which provides the “specific”, “given”, “disclosed”, or “reference” sequence(s) on which the substantially homologous sequence(s) is/are based is an antibody disclosed in Table A, i.e. the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody.
- the “specific”, “given”, “disclosed” or “reference” antigen binding protein e.g.
- antibody is the 5-A05/1-C04 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody. More preferably, the “specific”, “given”, “disclosed” or “reference” antigen binding protein (e.g. antibody) is the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
- Antigen binding proteins (e.g. antibodies) of the invention comprising one or more CDR sequences that are “substantially homologous” to one or more of the specific CDRs disclosed in Table A, e.g. to SEQ ID Nos: 5 to 7 (or SEQ ID NOs: 111 to 113) and/or to SEQ ID Nos: 8 to 10, and antigen binding proteins (e.g. antibodies) of the invention comprising heavy and/or light chain variable domains that comprise an amino acid sequence that is substantially homologous to a specific heavy and/or light chain variable domain disclosed in Table A, e.g.
- substantially homologous antigen binding proteins e.g. substantially homologous antibodies
- substantially homologous antigen binding proteins are any such antigen binding protein/antibody comprising one or more CDR or variable domain sequence that is substantially homologous to the sequence of a specific CDR or variable domain sequence with a given SEQ ID NO herein.
- antigen binding proteins e.g. antibodies, containing substantially homologous sequences retain the ability to bind to HLA- A*02:01/PRAME 425 ' 433 .
- antigen binding proteins, e.g. antibodies, containing substantially homologous sequences retain one or more (preferably all) of the other properties of a specific antigen binding protein (e.g. antibody) of the invention, i.e. an antigen binding protein (e.g. antibody) disclosed in Table A, e.g.
- substantially homologous as used herein in connection with an amino acid or nucleic acid sequence includes sequences having at least 30%, 40%, 50%, 55%. 60%, 62%, 65%, 70%, 74% or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the specific amino acid or nucleic acid sequence disclosed.
- Substantially homologous sequences of the invention thus include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the specific sequences of the invention.
- substantially homologous sequences are sequences containing conservative amino acid substitutions of the specific amino acid sequences disclosed.
- the antigen binding proteins (e.g. antibodies) of the invention preferably comprise at least one heavy chain variable domain (region) that includes an amino acid sequence region of at least 60%, 62%, 65%, 70%, 74% or 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a heavy chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g.
- SEQ ID NO: 3, 132, or 142 or 144 preferably SEQ ID NO: 3 or 132, more preferably SEQ ID NO: 132; and/or (preferably “and”) at least one light chain variable domain (region) that includes an amino acid sequence region of at least 60%, 62%, 65%, 70%, 74% or 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a light chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g. SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, preferably SEQ ID NO: 4, 140 or 147, more preferably SEQ ID NO: 4 or 147, most preferably SEQ ID NO: 4.
- a specific antigen binding protein e.g. antibody
- the antigen binding proteins (e.g. antibodies) of the invention comprise at least one heavy chain variable domain that includes an amino acid sequence region of at least 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a heavy chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g.
- SEQ ID NO: 132, 142 or 144 preferably SEQ ID NO: 132; and/or (preferably “and”) at least one light chain variable domain that includes an amino acid sequence region of at least 74%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% amino acid sequence identity to the amino acid sequence of a light chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g. SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, preferably SEQ ID NO: 4, 140 or 147, more preferably SEQ ID NO: 4 or 147, most preferably SEQ ID NO: 4.
- a specific antigen binding protein e.g. antibody
- sequences that are substantially homologous to a given VH domain sequence have at least 90% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 85%, more preferably at least 95% identity) to said given sequence.
- sequences that are substantially homologous to a given VH domain sequence have at least 95% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 90%, more preferably at least 95% identity) to said given sequence.
- preferred substantially homologous antigen binding proteins contain up to 27, 26 or 25, more preferably up to 15, more preferably up to 10, e.g. only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably up to 5, for example only 1 , 2, 3, 4 or 5, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids, in the VH domain and/or the VL domain as compared to the VH and/or VL domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1- C04).
- a specific antigen binding protein e.g. antibody
- Preferred substantially homologous antigen binding proteins (e.g. antibodies) of the invention contain up to 5, for example only 1 , 2, 3, 4 or 5, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids, or no altered amino acids, in the VH domain as compared to the VH domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1- C04, i.e.
- SEQ ID NO: 132); and/or (preferably “and”) contain up to 27, more preferably up to 15, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids, or no altered amino acids, in the VL domain as compared to the VL domain of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO: 4).
- a specific antigen binding protein e.g. antibody
- Preferred substantially homologous antigen binding proteins (e.g. antibodies) of the invention contain i) up to 4, e.g. only 1, 2, 3 or 4, altered amino acids in the VH domain as compared to the VH domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO: 132), and/or ( preferably “and”) contain no altered amino acids in the VL domain as compared to the VL domain of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e.
- SEQ ID NO:4 no altered amino acids in the VH domain as compared to the VH domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO:132), and/or (preferably “and”) contain up to 27, more preferably up to 15, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1, 2 or 3, altered amino acids in the VL domain as compared to the VL domain of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO:4).
- a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO:4).
- the antigen binding proteins (e.g. antibodies) of the invention comprise at least one heavy chain variable domain and/or (preferably “and”) at least one light chain variable domain as defined above, and further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of the VH CDR1, VH CDR2, VH CDR3 of a specific antibody of the invention (disclosed in Table A, e.g. 5-A05/1-C04), or sequences substantially homologous thereto; and/or (preferably “and”) said light chain variable domain comprises three CDRs comprising the amino acid sequences of the VL CDR1, VL CDR2, VL CDR3 of a (preferably said) specific antibody of the invention (disclosed in Table A, e.g. 5-A05/1-C04), or sequences substantially homologous thereto.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of the VH CDR1, VH CDR2, VH CDR3 of a specific antibody of the invention (disclosed in Table
- Sequences substantially homologous to a given CDR sequence are preferably as described below.
- substantially homologous sequences are sequences having at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 62%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, amino acid sequence identity to the amino acid sequence of one or more of the CDR regions or one or more of the FR regions disclosed in Table A.
- a “substantially homologous” CDR sequence may be a sequence having at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 62%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a given CDR sequence described herein.
- antigen binding proteins e.g. antibodies
- antigen binding proteins e.g. antibodies having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence
- the altered amino acid residues(s) are not in a CDR region.
- antigen binding proteins e.g. antibodies
- having a VH domain that has a certain degree of sequence identity to a given VH domain sequence of a particular antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g.
- the altered (or variant) residue(s) are not in a CDR region.
- antigen binding proteins e.g. antibodies
- the altered amino acid residues(s) are in one or more framework regions.
- the altered amino acid residues(s) may be in one or more CDR regions.
- preferred substantially homologous sequences contain up to 5, e.g. only 1 , 2, 3, 4 or 5, preferably up to 4, e.g. only 1 , 2, 3, or 4, preferably up to 3, e.g. 1 , 2 or 3, more preferably up to 2, e.g. 1 or 2, more preferably only 1 altered amino acids, in one or more of the framework regions and/or one or more of the CDRs making up the sequences of the invention (disclosed in Table A).
- Such alterations might be amino acid substitutions, e.g. conserved or non-conserved amino acid substitutions, or a mixture thereof.
- a sequence substantially homologous to a given CDR sequence preferably comprises up to 4, e.g. only 1 , 2, 3, or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions) as compared to the given CDR sequence.
- sequences substantially homologous to a given VH CDR3, VL CDR1 , and/or VL CDR3 sequences herein e.g. disclosed in Table A.
- Sequences substantially homologous to a given VH CDR1 and/or VH CDR2 sequence herein preferably comprise up to 2, e.g.
- Sequences substantially homologous to a given VL CDR2 sequence herein preferably comprise only 1 altered amino acid (preferably a substitution) as compared to the given CDR sequence.
- a given starting (reference) sequence is relatively short (e.g. three amino acids in length)
- fewer amino acid substitutions may be present in sequences substantially homologous thereto as compared with the number of amino acid substitutions that might optionally be made in a sequence substantially homologous to a longer starting (reference) sequence.
- the VL CDR2 sequence of antigen binding proteins of the invention is three amino acids in length.
- a sequence substantially homologous to a starting (reference) VL CDR2 sequence in accordance with the present invention e.g.
- a starting (reference) VL CDR2 sequence which is three amino acid residues in length, preferably has only 1 or 2, more preferably only 1 altered amino acid in comparison with the starting (reference) sequence.
- a sequence substantially homologous to a starting (reference) VH CDR3 sequence in accordance with the present invention e.g. a starting (reference) VH CDR3 sequence which is twelve amino acid residues in length, preferably has up to 4, preferably up to 3, more preferably 1 or 2 altered amino acid in comparison with the starting (reference) sequence.
- the number of altered amino acids in substantially homologous sequences can be tailored to the length of a given starting CDR sequence.
- different numbers of altered amino acids can be present depending on the length of a given starting CDR sequence such as to achieve a particular % sequence identity in the CDRs, for example a sequence identity of at least 30%, 40%, 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.
- a sequence substantially homologous to a given VH CDR1 sequence is preferably a sequence containing up to 3 (e.g. only 1 , 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
- a sequence substantially homologous to a given VH CDR2 sequence is preferably a sequence containing up to 3 (e.g. only 1, 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
- a sequence substantially homologous to a given VH CDR3 sequence is preferably a sequence containing up to 4 (e.g. only 1 , 2, 3 or 4), more preferably up to 3 (e.g. only 1 , 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 , amino acid substitutions as compared to the given CDR sequence.
- a sequence substantially homologous to a given VL CDR1 sequence is preferably a sequence containing up to 4 (e.g. only 1 , 2, 3 or 4), preferably up to 3 (e.g. only 1 , 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
- a sequence substantially homologous to a given VL CDR2 sequence is preferably a sequence containing only 1 amino acid substitution as compared to the given CDR sequence.
- a sequence substantially homologous to a given VL CDR3 sequence is preferably a sequence containing up to 4 (e.g. only 1 , 2, 3 or 4), preferably up to 3 (e.g. only 1 , 2 or 3), more preferably only 1 , amino acid substitutions as compared to the given CDR sequence.
- a sequence substantially homologous to a given VH CDR1 or VH CDR2 sequence is a sequence containing up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given CDR sequence;
- a sequence substantially homologous to a given VH CDR3 sequence is a sequence containing up to 3 (e.g. only 1 , 2 or 3) amino acid substitutions as compared to the given CDR sequence;
- a sequence substantially homologous to a given VL CDR1 or VL CDR3 sequence is a sequence containing up to 4 (e.g. only 1, 2, 3 or 4) amino acid substitutions, preferably up to 3 (e.g.
- a sequence substantially homologous to a given VL CDR2 sequence is a sequence containing only 1 amino acid substitution as compared to the given CDR sequence.
- the antigen binding protein (e.g. antibody) of the invention comprises a VH CDR1 comprising the amino acid sequence of the given VH CDR1 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 111) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given VH CDR1 sequence; a VH CDR2 comprising the amino acid sequence of the given VH CDR2 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g.
- SEQ ID NO: 112, 114 or 115 or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given VH CDR2 sequence; a VH CDR3 comprising the amino acid sequence of the given VH CDR3 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 113) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g.
- VL CDR1 comprising the specific amino acid sequence of the given VL CDR1 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 8, 116, 117, 118, 119, or 120) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g. only 1 , 2, or 3), preferably up to 2 (e.g.
- VL CDR2 comprising the specific amino acid sequence of the given VL CDR2 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises only 1 amino acid substitution as compared to the given VL CDR2 sequence
- a VL CDR3 comprising the specific amino acid sequence of the given VL CDR3 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 10 or 121) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g. only 1 , 2 or 3), amino acid substitutions as compared to the given VL CDR3 sequence.
- the antigen binding protein (e.g. antibody) of the invention comprises a VH CDR1 comprising the amino acid sequence of the given VH CDR1 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 111); a VH CDR2 comprising the amino acid sequence of the given VH CDR2 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g.
- VH CDR3 comprising the amino acid sequence of the given VH CDR3 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 113); a VL CDR1 comprising the specific amino acid sequence of the given VL CDR1 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 8, 116, 117, 118, 119, or 120), or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g.
- VL CDR1 a VL CDR2 comprising the specific amino acid sequence of the given VL CDR2 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 9); and a VL CDR3 comprising the specific amino acid sequence of the given VL CDR3 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 10, or 121).
- each (i.e. all) of the substantially homologous sequences are substantially homologous to a given sequence derived from the same specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04).
- each sequence substantially homologous to a given CDR sequence comprises only 1 amino acid substitution as compared to the given CDR sequence.
- each (i.e. all) of the substantially homologous sequences are substantially homologous to a given sequence derived from the same specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A).
- Preferred specific antigen binding proteins (e.g. antibodies) of the invention (disclosed in Table A) are described elsewhere herein.
- an antigen binding protein e.g. antibody
- a substantially homologous sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence
- the three VH CDR amino acid sequences i.e. all three VH CDR sequences taken together
- the three VL CDR amino acid sequences i.e.
- the starting (or reference) antigen binding protein may have the CDR sequences of a specific antibody of the present invention shown in Table A, e.g. the 5-A05/1-C04 antibody.
- Preferred substantially homologous antigen binding proteins contain up to 12, e.g. only 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably up to 8, e.g. only 1 , 2, 3, 4, 5, 6, 7, or 8, preferably up to 7, e.g. only 1, 2, 3, 4, 5, 6, or 7, preferably up to 6, e.g. only 1, 2, 3, 4, 5 or 6, preferably up to 5, for example only 1, 2, 3, 4 or 5, preferably up to 4, for example only 1, 2, 3 or 4, preferably up to 3, e.g. only 1, 2 or 3, more preferably up to 2, e.g.
- the whole CDR complement as compared to in the whole CDR complement of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody).
- a specific antigen binding protein e.g. antibody
- the antigen binding protein contains up to 6, e.g. only 1, 2, 3, 4, 5 or 6 altered amino acids (preferably substitutions) as compared to the whole complement of the given CDR sequences.
- the 5-A05/1-C04 antibody contains up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, preferably up to 4, e.g. only 1 , 2, 3 or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, altered amino acids (preferably substitutions), more preferably no altered amino acids, in the combined CDRs (i.e. the three CDR regions) of the VL domain as compared to those of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody).
- a specific antigen binding protein e.g. antibody
- preferred substantially homologous antigen binding proteins contain i) up to 12, e.g. only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably up to 8, e.g. only 1 , 2, 3, 4, 5, 6, 7, or 8, preferably up to 7, e.g. only 1, 2, 3, 4, 5, 6, or 7 altered amino acids in the combined CDRs (i.e. the three CDR regions) of the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); and/or (preferably “and”), contain no altered amino acids in the combined CDRs (i.e.
- the three CDR regions) of the VL domain as compared to those of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); or i) no altered amino acids in the combined CDRs (i.e. the three CDR regions) of the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); and/or (preferably “and”), contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, preferably up to 4, e.g.
- preferred substantially homologous sequences contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, for example 1 , 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids, in the combined framework regions (e.g. the four framework regions), and/or the combined CDRs (e.g. the three CDR regions) making up the VL or VH domains of the antigen binding proteins (e.g. antibodies) of the invention.
- preferred substantially homologous sequences contain up to 6, e.g.
- preferred substantially homologous antigen binding proteins contain up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions), more preferably no altered amino acids, in the combined framework regions (e.g. the four framework regions), and/or the combined CDRs (e.g. the three CDR regions) making up the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A); and/or (preferably “and”), contain up to 6, e.g.
- the antigen binding protein (e.g. antibody) comprises a VH CDR1 , a VH CDR2, and a VH CDR3 comprising, respectively, the amino acid sequence of the given VH CDR1 , VH CDR2, and VH CDR3 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), i.e. rather than sequences substantially homologous thereto; and/or the antigen binding protein (e.g.
- antibody comprises a VL CDR1 , a VL CDR2, and a VL CDR3 comprising, respectively, the amino acid sequence of the given VL CDR1 , VL CDR2, and VL CDR3 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), i.e. rather than sequences substantially homologous thereto.
- a specific antigen binding protein e.g. antibody
- the variant amino acids lie only in either the VH CDRs, or only in the VL CDRs.
- antibody of the invention (shown in Table A, e.g. SEQ ID NOs: 111 , 112 and 113, respectively), or sequences substantially homologous thereto, and/or (preferably “and”) at least one light chain variable domain that includes an amino acid sequence region of at least 74%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% amino acid sequence identity to the amino acid sequence of a specific light chain variable domain of said specific antigen binding protein (e.g. antibody) of the invention (shown in Table A, e.g.
- said light chain variable domain comprises a VL CDR1 , a VL CDR2, and a VL CDR3, preferably comprising the amino acid sequences of the specific VL CDR1 , VL CDR2, and VL CDR3 of said specific antigen binding protein (e.g. antibody) of the invention (shown in Table A, e.g. SEQ ID NOs: 8, 9 and 10, respectively), or sequences substantially homologous thereto, wherein said sequences substantially homologous to a given CDR sequence are as defined anywhere else herein.
- said alterations can be with conservative or nonconservative amino acids.
- said alterations are conservative amino acid substitutions.
- Altered residues might be conserved or non-conserved amino acid substitutions, or a mixture thereof. In such embodiments, preferred alterations are conservative amino acid substitutions.
- a “conservative amino acid substitution”, as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.
- families of amino acid residues can be grouped based on hydrophobic side groups or hydrophilic side groups.
- Routine methods in the art such as alanine scanning mutagenesis and/or analysis of crystal structure of the antigen-antibody complex can be used in order to determine which amino acid residues of the CDRs do not contribute or do not contribute significantly to antigen binding and therefore are good candidates for alteration or substitution in the embodiments of the invention involving substantially homologous sequences.
- the addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent antigen binding protein (e.g. antibody) to form a new antigen binding protein (e.g. antibody), wherein said parent antigen binding protein (e.g. antibody) is one of the antigen binding proteins (e.g. antibodies) of the invention as defined elsewhere herein (e.g. disclosed in Table A), and testing the resulting new antigen binding protein (e.g. antibody) to identify antigen binding proteins (e.g. antibodies) that bind to HLA-A*02:01/PRAME 425 ' 433 in accordance with the invention can be carried out using techniques which are routine in the art. Such methods can be used to form multiple new antigen binding proteins (e.g. antibodies) that can all be tested for their ability to bind HLA- A*02:01/PRAME 425 ' 433 .
- said addition, deletion, substitution or insertion of one or more amino acids takes place in one or more of the CDR domains.
- said manipulations could conveniently be carried out by genetic engineering at the nucleic acid level wherein nucleic acid molecules encoding appropriate binding proteins and domains thereof are modified such that the amino acid sequence of the resulting expressed protein is in turn modified in the appropriate way.
- Testing the ability of one or more of the modified antigen binding proteins (e.g. antibodies) to bind to HLA-A*02:01/PRAME 425 ' 433 can be carried out by any appropriate method, which are well known and described in the art. Suitable methods are also described elsewhere herein and in the Examples section.
- New antigen binding proteins e.g. antibodies
- New antigen binding proteins produced, obtained or obtainable by these methods form a yet further aspect of the invention.
- substantially homologous also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the proteins or nucleic acid molecules of the invention in substantially the same way.
- any substantially homologous antigen binding protein e.g. antibody
- any substantially homologous antigen binding protein e.g. antibody
- Substantially homologous sequences of proteins of the invention include, without limitation, conservative amino acid substitutions, or for example alterations that do not affect the VH, VL or CDR domains of the antigen binding proteins (e.g. antibodies), e.g. antigen binding proteins (e.g. antibodies) where tag sequences, toxins or other components are added that do not contribute to the binding of antigen, or alterations to convert one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g. conversion from Fab to scFv or whole antibody or vice versa), or the conversion of an antibody molecule to a particular class or subclass of antibody molecule (e.g. the conversion of an antibody molecule to IgG or a subclass thereof, e.g. lgG2).
- the antigen binding proteins e.g. antibodies
- antigen binding proteins e.g. antibodies
- tag sequences, toxins or other components where tag sequences, toxins or other components are added that do not contribute to the
- Homology or sequence identity may be assessed by any convenient method. However, for determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used together with BLOSLIM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1 , so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment.
- Clustal W Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994.
- the Clustal W algorithm can be used together with BLOSLIM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl.
- sequences according to the present invention having at least 30%, 40%, 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, adjoin, France).
- the antigen binding proteins (e.g. antibodies) of the invention preferably comprise the specific sequences disclosed, and not sequences substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- GYTFTGYY (SEQ ID NO: 5), or GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto,
- INPNSGGT SEQ ID NO:6
- IIPIFGTA SEQ ID NO: 112
- LVVGRA SEQ ID NO: 114
- MQVGPTRHPLWA SEQ ID NO: 115
- ARDRWELLGSSDDY SEQ ID NO:7
- ARDGYNYGQFDY SEQ ID NO: 113
- said light chain variable domain comprises:
- VL variable light
- QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto,
- variable light VL CDR2 that comprises the amino acid sequence of
- AAS SEQ ID NO:9, or a sequence substantially homologous thereto, and
- QQANSFPFT (SEQ ID NO:121), or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- GGTFSSYA SEQ ID NO: 111
- ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto,
- variable light VL CDR2 that comprises the amino acid sequence of
- AAS SEQ ID NO:9, or a sequence substantially homologous thereto, and (f) a VL CDR3 that comprises the amino acid sequence of
- QQYNRLPLT SEQ ID NO: 10
- QQANSFPFT SEQ ID NO:121
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- GGTFSSYA SEQ ID NO: 111
- ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- QGVSNW (SEQ ID NO:8), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
- variable light VL CDR2 that comprises the amino acid sequence of
- AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and (f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10), or or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- GGTFSSYA SEQ ID NO: 111
- MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto;
- ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- variable light VL CDR2 that comprises the amino acid sequence of
- AAS SEQ ID NO:9, or a sequence substantially homologous thereto;
- QQYNRLPLT (SEQ ID NO: 10), or or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- GGTFSSYA SEQ ID NO: 111
- ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto, preferably
- variable light VL CDR2 that comprises the amino acid sequence of
- AAS SEQ ID NO:9, or a sequence substantially homologous thereto;
- QQANSFPFT (SEQ ID NO:121), or a sequence substantially homologous thereto, preferably
- QQYNRLPLT (SEQ ID NO: 10), or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- GGTFSSYA SEQ ID NO: 111
- MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, preferably
- MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto;
- ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- QGVSNW (SEQ ID NO:8), or a sequence substantially homologous thereto;
- variable light VL CDR2 that comprises the amino acid sequence of
- AAS SEQ ID NO:9, or a sequence substantially homologous thereto;
- QQYNRLPLT (SEQ ID NO: 10), or or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain heavy chain variable domain
- VL domain a light chain variable domain that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain heavy chain variable domain
- VL domain a light chain variable domain that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT ; (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) or b) EFVSSW AAS QQYNRLPLT ;
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain heavy chain variable domain
- VL domain a light chain variable domain that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain heavy chain variable domain
- VH CDR1 VH CDR2 VH CDR3 a) GGTFSSYA IIPIFGTA ARDGYNYGQFDY (SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) and/or (preferably “and”) a light chain variable domain (VL domain) that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto: VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT ; (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) or b) EFVSSW AAS QQYNRLPLT ; (SEQ ID NO: 119) (SEQ ID NO: 9) (SEQ ID NO: 10) or c) QGISSW AAS QQYNRLPLT (SEQ ID NO: 120) (SEQ ID NO: 9)
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain heavy chain variable domain
- VL domain a light chain variable domain that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 that comprise, respectively, the amino acid sequences of the VH CDR1 , VH CDR2 and VH CDR3 of a specific antibody of the invention (disclosed in Table A), or sequences substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises a variable light (VL) CDR1 , a VL CDR2 and a VL
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of the VH domain of a specific antibody of the invention (disclosed in Table A) or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of the VL domain of a (preferably said) specific antibody of the invention (disclosed in Table A).
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of the VH domain of a specific antibody of the invention (disclosed in Table A) or
- the specific antibody of the invention may be the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody; preferably the 5-A05/1-C04 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody; more preferably, the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of a specific antibody of the invention (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said specific antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said specific antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA-A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of a specific antibody of the invention (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said specific antibody, or sequences substantially homologous thereto; and/or (preferably “and “
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the 5-A05 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the 5-A05 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the 5- A05/1-C04 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the 5- A05/1-C04 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the PAM6 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the PAM6 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the PAM8 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the PAM8 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the PAM 17 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the PAM 17 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the PAM 18 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the PAM 18 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the PAM22 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the PAM22 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the PAM23 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the PAM23 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the NGS55 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the NGS55 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME 425 ' 433 , and comprises i) the three VH CDR sequences of the NGS17 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- HLA- A*02:01/PRAME 425 ' 433 comprises i) the three VH CDR sequences of the NGS17 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the V
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises: (a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114) or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequences is separately a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3) amino acid substitutions compared to the given CDR sequence.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence
- VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3, 4 or 5 amino acid substitutions compared to the given CDR sequence; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 amino acid substitution compared to the given CDR sequence, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises: the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence; the amino acid sequence of LVVGRA (SEQ ID NO: 114) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence, or the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence; and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1, 2, or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence, and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 amino acid substitution compared to the given CDR sequence
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises: the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence; the amino acid sequence of LVVGRA (SEQ ID NO: 114) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence; or the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence; and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2, or 3) amino acid substitutions compared to the given CDR sequence, and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- QGVSNW SEQ ID NO:8
- DWISGW SEQ ID NO:116
- AFISSW SEQ ID NO: 117
- EFISQW SEQ ID NO: 118
- EFVSSW SEQ ID NO: 119
- QGISSW SEQ ID NO: 120
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 amino acid substitution compared to the given CDR sequence, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10) or QQANSFPFT (SEQ ID NO: 121), a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 (preferably 1 or 2) amino acid substitutions compared to the given CDR sequence.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, wherein said heavy chain variable domain comprises: (a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5),
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6)
- VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7); and/or (preferably “and”) wherein said light chain variable domain comprises: (d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8),
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9)
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6)
- VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7); and wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9)
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, said heavy chain variable domain comprises:
- VH variable heavy
- GGTFSSYA SEQ ID NO:111
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114) or MQVGPTRHPLWA (SEQ ID NO: 115)
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113); and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9)
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112)
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113); and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9)
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises: (a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of LVVGRA (SEQ ID NO:114) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO:115) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112) or a sequence substantially homologous thereto, and
- VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
- the substantially homologous sequences may be as described anywhere else herein, and are preferably as follows: a sequence substantially homologous to a given VH CDR1 sequence (i.e. a VH CDR1 is preferably a sequence containing up to 2 (e.g. only 1 or 2), preferably only 1 amino acid substitutions as compared to the given CDR sequence; a sequence substantially homologous to a given VH CDR2 sequence is preferably a sequence containing up to 2 (e.g.
- a sequence substantially homologous to a given VH CDR3 sequence is preferably a sequence containing up to 3 (e.g. only 1 , 2, or 3), preferably up to 2 (e.g. only 1 or 2), preferably only 1 amino acid substitutions as compared to the given CDR sequence;
- a sequence substantially homologous to a given VL CDR1 sequence is preferably a sequence containing up to 2 (e.g.
- a sequence substantially homologous to a given VL CDR2 sequence is preferably a sequence containing only 1 amino acid substitution as compared to the given CDR sequence
- a sequence substantially homologous to a given VL CDR3 sequence is preferably a sequence containing up to 3 (e.g. only 1 , 2 or 3), more preferably only 1 or 2, more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
- each of the sequences substantially homologous to a given CDR sequence comprises only 1 amino acid substitution as compared to the given CDR sequence.
- the antigen binding protein comprises the given VL CDR2 sequence (i.e. rather than a sequence substantially homologous thereto).
- the heavy chain variable domain comprises the given CDR sequences (i.e. rather than sequences substantially homologous thereto), and/or the light chain variable domain comprises the given CDR sequences (i.e. rather than sequences substantially homologous thereto).
- the antigen binding protein (e.g. antibody) contains up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, preferably up to 5, for example only 1 , 2, 3, 4 or 5, preferably up to 4, for example only 1 , 2, 3 or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions) as compared to the whole complement of the given CDR sequences.
- CDR sequences of certain antibodies of the invention are set forth herein in Table A.
- CDR sequences of antibodies of the invention may be CDR sequences in the VH domains and VL domains of antibodies of the invention as identified using any suitable method (or tool), for example as identified using the well-known ImMunoGeneTics information system method, i.e. the IMGT numbering scheme (e.g. Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imqt.org), e.g. as shown in Table A, or as identified according to the well-known methods of Kabat (e.g. Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed.
- IMGT numbering scheme e.g. Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imqt.org
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3, 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3, 132, 142 or 144, or
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 140, or 147, or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto, and/or
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, or 147, or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain and/or (preferably “and”) a VL domain comprising the following amino acid sequences or sequences substantially homologous thereto: VH domain VL domain i) SEQ ID NO:3 SEQ ID NO:4 ; or ii) SEQ ID NO:132 SEQ ID NO:4 ; or iii) SEQ ID NO:132 SEQ ID NO:134 ; or iv) SEQ ID NO: 132 SEQ ID NO:136 ; or v) SEQ ID NO: 132 SEQ ID NO:138 ; or vi) SEQ ID NO: 132 SEQ ID NQ:140 ; or vii) SEQ ID NO:142 SEQ ID NO:4 ; or viii
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising: i) a VH domain comprising SEQ ID NO: 132 or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, or a sequence substantially homologous thereto; or ii) a VH domain comprising SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain and/or (preferably “and”) a VL domain comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain VL domain i) SEQ ID NO: 132 SEQ ID NO:4 ; or ii) SEQ ID NO:132 SEQ ID NQ:140 ; or iii) SEQ ID NO:142 SEQ ID NO:4 ; or iv) SEQ ID NO:144 SEQ ID NO:4 ; or v) SEQ ID NO:132 SEQ ID NO:147
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising: i) a VH domain comprising SEQ ID NO: 132 or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, 140, or 147, or a sequence substantially homologous thereto; or ii) a VH domain comprising SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain and/or (preferably “and”) a VL domain comprising the following amino acid sequences or sequences substantially homologous thereto:
- VH domain VL domain i) SEQ ID NO: 132 SEQ ID NO: 4 ; or ii) SEQ ID NO:142 SEQ ID NO:4 ; or iii) SEQ ID NO:144 SEQ ID NO:4 ; or iv) SEQ ID NO: 132 SEQ ID NO:147
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising: i) a VH domain comprising SEQ ID NO: 132 or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4 or 147, or a sequence substantially homologous thereto; or ii) a VH domain comprising SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 140 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 142 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 142 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 144 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 144 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 147 or a sequence substantially homologous thereto.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ
- sequences that are substantially homologous to a given VH domain sequence have at least 90% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 85%, more preferably at least 95% identity) to said given sequence.
- sequences that are substantially homologous to a given VH domain sequence have at least 95% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 90%, more preferably at least 95% identity) to said given sequence.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 78%, 80%, 85%, 90%, 95% or 98%) and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 85%, 90%, 95% or 98%).
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:132, 142 or 144 (preferably SEQ ID NO: 132), or a sequence having at least 74% sequence identity thereto (e.g.
- VL domain that comprises the amino acid sequence of SEQ ID NO:4, 134, 136, 138, 140, 147 or 150 (preferably SEQ ID NO: 4), or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 80%, 85%, 90%, 95% or 98%).
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:132, 142 or 144 (preferably SEQ ID NO: 132), or a sequence having at least 95% sequence identity thereto and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO:4, 140, or 147 (preferably SEQ ID NO: 4), or a sequence having at least 85% sequence identity thereto (e.g. at least 95%).
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs, preferably comprising the amino acid sequences of SEQ ID NO:5, 6 and 7, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs, preferably comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:132, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO: 119, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 142, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 114 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 144, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 115 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 147, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NQ:120, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:5, 6 and 7; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10.
- the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:132, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113; and/or (preferably “and”) i) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10; or ii) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto (e.g.
- said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ I D NO: 119, 9 and 10; or iii) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 147, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO: 120, 9 and 10.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113; or ii) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 142, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 114 and 113; or iii) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 144, or a sequence having at least 80% sequence identity thereto (e.g.
- said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 115 and 113; and/or (preferably “and”) i) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10.
- sequences indicated as having sequence identity to a sequence with a given SEQ ID NO: can have at least 60%, 65%, 70% or 75% identity to the sequence with the given SEQ ID NO.
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4.
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, preferably 142 or 144, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, preferably 142 or 144, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4.
- the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 140 or 147, preferably SEQ ID NO: 4 or 147.
- an antigen binding protein for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 140 or 147, preferably SEQ ID NO: 4 or 147.
- a preferred antigen binding protein of the invention is or comprises an antibody defined herein (Table A) selected from the group consisting of the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody.
- Table A selected from the group consisting of the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody.
- a preferred antigen binding protein of the invention is or comprises an antibody defined herein (in Table A) selected from the group consisting of the 5- A05/1-C04 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
- a particularly preferred antigen binding protein of the invention is or comprises the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody defined herein (in Table A).
- An antigen binding protein according to any aspect of the present invention and disclosure may be defined as a binding protein comprising an antigen-binding domain obtained or derived from an antibody, or based on an antigen binding domain of an antibody.
- light and heavy chain variable domains i.e. light and heavy chain variable regions
- light and heavy chain variable regions as described herein are those obtained or derived from an antibody, or based on an antigen binding domain of an antibody.
- antigen binding proteins for example antibodies, or antigen binding proteins comprising antibodies or the antigen binding domain of an antibody, which bind to (or specifically recognise or specifically bind to) HLA-A*02:01/PRAME 425 ' 433 .
- Preferred antigen binding proteins of the invention are antibodies.
- embodiments as described herein which relate to antibodies apply equally, mutatis mutandis, to other types of antigen binding proteins, or vice versa.
- other antigen binding proteins can comprise the antibodies of the invention or can comprise the antigen binding domains of the antibodies of the invention, e.g.
- VL and VH domain of the antibodies of the invention a domain typically comprising the three VH or the three VL CDR regions (CDR1 , CDR2 and CDR3) and the four VH or the four VL framework (“FR”) regions (FR1 , FR2, FR3 and FR4)).
- Appropriate types of antigen binding protein which could be used in the invention are known in the art.
- immunoglobulin based polypeptides are used, which generally comprise CDR regions (and optionally FR regions or an immunoglobulin based scaffold), such that the CDR regions (and optionally FR regions) of the antibodies of the invention can be grafted onto an appropriate scaffold or framework, e.g. an immunoglobulin scaffold.
- the antigen binding domains or the antibodies of the invention can be incorporated into any appropriate antigen binding fragment or antibody containing format, e.g. can be incorporated into a chimeric antigen receptor (CAR) format or a CAR-T cell format.
- CAR chimeric antigen receptor
- the antigen binding domain that binds to (or specifically binds to) HLA- A*02:01/PRAME 425-433 is not from, or does not correspond to, a T-cell receptor (TCR).
- TCR T-cell receptor
- antigen binding proteins in accordance with the present invention do not include TCRs as an antigen binding domain specific for HLA-A*02:01/PRAME 425 ' 433 .
- the antigen binding proteins of the present invention are not TCRs, i.e. do not comprise a T-cell receptor a-chain, and/or preferably (“and”) do not comprise a T-cell receptor p-chain.
- the antigen binding protein i.e. the protein having an antigen binding domain
- the antigen binding protein is an antibody or an antigen binding fragment thereof (an antibody fragment).
- antibody is used as shorthand to refer to “antibody or an antigen binding fragment thereof” unless otherwise clear from context.
- antibody and "immunoglobulin”, as used herein, refer broadly to any immunological binding agent that comprises an antigen binding domain (e.g. a human antigen binding domain), including polyclonal and monoclonal antibodies.
- an antigen binding domain e.g. a human antigen binding domain
- whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM and the antibodies of the invention may be in any one of these classes.
- subclasses or isotypes such as lgG1 , lgG2, lgG3, lgG4, and the like.
- the heavy-chain constant domains that correspond to the difference classes of immunoglobulins are termed a, 5, s, y and p, respectively.
- the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
- the "light chains” of mammalian antibodies are assigned to one of two clearly distinct types: kappa (K) and lambda ( ), based on the amino acid sequences of their constant domains and some amino acids in the framework regions of their variable domains.
- heavy chain complementarity determining region refers to regions of hypervariability within the heavy chain variable region (VH domain) of an antibody molecule.
- the heavy chain variable region (domain) has three CDRs termed heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus.
- the heavy chain variable region (domain) also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.
- VH domain refers to the variable region of a heavy chain of an antibody molecule.
- light chain complementarity determining region refers to regions of hypervariability within the light chain variable region (VL domain) of an antibody molecule.
- the light chain variable region (domain) has three CDRs termed light chain CDR1 , light chain CDR2 and light chain CDR3 from the amino terminus to the carboxy terminus.
- the light chain variable region (domain) also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.
- L domain refers to the variable region of a light chain of an antibody molecule.
- the immunological binding reagents encompassed by the term “antibody” includes or extends to all antibodies, antigen binding fragments thereof and antibody “formats”, including whole antibodies, dimeric, trimeric and multimeric antibodies; bispecific antibodies; trispecific antibodies; multispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof.
- antibody format and “antibody construct” are used interchangeably herein. In the field, and herein, the term “antibody format” encompasses both antibody fragments, whole antibodies and multimeric antibodies.
- antibody fragment refers to fragments of biological relevance, e.g. fragments that comprise the above mentioned antigen binding domain, i.e. contribute to antigen binding, e.g. form part of the antigen binding domain. Certain preferred fragments comprise a heavy chain variable region (VH domain) and a light chain variable region (VL domain) of the antibodies of the invention.
- VH domain heavy chain variable region
- VL domain light chain variable region
- Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art.
- antibody is thus used to refer to any antibody-like molecule that has an immunoglobulin (Ig) antigen binding domain, and this term includes antibody fragments and formats that comprise an antigen binding domain including but not limited to Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, scFv/Fc KIH , linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively, including scFv/Fab-Fc, and scFv/Fab-Fc KIH ); sc-diabody; single chain bispecific diabody (scDb); kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager
- the antigen binding protein (e.g. antibody) of the present invention may be in a single chain format, e.g. a single chain antibody format. In other embodiments, the antigen binding protein (e.g. antibody) of the present invention may be in a multichain format.
- Antibody formats, and their preparation, is within the general competencies of the person of ordinary skill in the antibody field.
- the antigen binding protein (e.g. antibody) of the present invention comprises all or a portion of a heavy chain constant region, such as an lgG1, lgG2, lgG3, lgG4, lgA1 , lgA2, IgE, IgM, or IgD constant region.
- a heavy chain constant region such as an lgG1, lgG2, lgG3, lgG4, lgA1 , lgA2, IgE, IgM, or IgD constant region.
- the heavy chain constant region is an IgG heavy chain constant region or a portion thereof.
- I gG 1 and lgG4 are examples of appropriate formats for the antibodies of the invention.
- Antibodies of the invention may comprise or contain human heavy-chain constant regions.
- antigen binding protein (e.g. antibody) of the invention can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof.
- Antigen binding proteins (e.g. antibodies) of the invention may comprise or contain human light-chain constant regions.
- full length heavy chain sequences of the antigen binding proteins (e.g. antibodies) of the invention comprise an amino acid sequence comprising or consisting of SEQ ID NOs: 109, 153, 164 or 153, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%)).
- full length light chain sequences of the antigen binding proteins (e.g. antibodies) of the invention comprise an amino acid sequence comprising or consisting of SEQ ID NOs: 110 or 171 , or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%)).
- the CDR, VH and/or VL sequences in said full heavy and light chain sequences are as defined anywhere else herein.
- the antigen binding proteins (e.g. antibodies) of the invention may comprise: a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 109, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), and in which said heavy chain comprises a heavy chain variable domain that comprises three CDRs comprising the amino acid sequences of SEQ ID NO:5, 6 and 7, or sequences substantially homologous thereto, as defined elsewhere herein; and/or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 110, or a sequence substantially homologous thereto (e.g.
- said light chain comprises a light chain variable domain that comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
- antigen binding proteins e.g. antibodies
- such antigen binding proteins are typically referred to herein as “full length” antibodies or “whole” antibodies. In some embodiments such full length or whole antibodies are provided.
- the antigen binding proteins e.g. antibodies
- the antigen binding proteins can be produced naturally or can be wholly or partially synthetically produced.
- the antigen binding protein may be from any appropriate source, for example recombinant sources and/or produced in transgenic animals or transgenic plants, or in eggs using the IgY technology.
- the antigen binding protein (e.g. antibody) molecules can be produced in vitro or in vivo.
- the antigen binding domains of the antigen binding proteins (e.g. antibodies) of the invention generally comprise an antibody light chain variable region (domain) (VL) that comprises three CDR domains and an antibody heavy chain variable region (domain) (VH) that comprises three CDR domains.
- VL antibody light chain variable region
- VH antibody heavy chain variable region
- camelid VHH antibodies and other single domain antibodies comprising VH domains alone show that these domains can bind to antigen with acceptably high affinities.
- three CDRs (or even a single CDR) can effectively bind antigen and form an antigen binding domain.
- antigen binding domains in the antigen binding proteins (e.g. antibodies) of the invention might comprise six CDR regions (three from a light chain and three from a heavy chain), antigen binding proteins (e.g. antibodies) with antigen binding domains with fewer than six CDR regions (e.g. 3 CDR regions) are encompassed by the invention.
- Antigen binding proteins (e.g. antibodies) with antigen binding domains with CDRs from only the heavy chain or light chain are also contemplated.
- Preferred light chain CDR regions for use in conjunction with the specified heavy chain CDR regions to form the antigen binding domain are described elsewhere herein.
- other light chain variable regions (domains) that comprise three CDRs for use in conjunction with the heavy chain variable regions (domains) of the invention are also contemplated.
- Appropriate light chain variable regions (domains) which can be used in combination with the heavy chain variable regions (domains) of the invention and which give rise to an antibody which binds to HLA-A*02:01/PRAME 425 ' 433 in accordance with the invention can be readily identified by a person skilled in the art.
- a heavy chain variable region (domain) of the invention can be combined with a single light chain variable region (domain) or a repertoire of light chain variable regions (domains) and the resulting antigen binding proteins (e.g. antibodies) tested for binding to HLA-A*02:01/PRAME 425 ' 433 .
- antigen binding proteins e.g. antibodies
- antigen binding protein-based constructs e.g. antibody-based constructs and fragments
- Diabodies in particular, are further described in EP404097 and WO 93/11161 ; whereas linear antibodies are further described in the art.
- an antigen binding protein (e.g. antibody) of the invention that binds to HLA-A*02:01/PRAME 425 ' 433 may be referred to elsewhere herein simply as a PRAME pMHC antigen binding protein, or a PRAME antigen binding protein, e.g. a PRAME pMHC antibody, or a PRAME antibody.
- the present invention provides antigen binding proteins (e.g. antibodies), for example isolated antigen binding proteins (e.g. isolated antibodies), which bind to (or specifically recognise or specifically bind to) HLA- A*02:01/PRAME 425 ' 433 .
- This is the “target” antigen of the antigen binding proteins (e.g. antibodies) of the invention.
- the antigen binding proteins (e.g. antibodies) of the invention are described as having a “specificity” for this target antigen. Due to their binding to (or specifically recognising or specifically binding to) a H LA-restricted peptide, the antigen binding proteins (e.g. antibodies) of the invention are termed “TCR-like” antigen binding proteins (e.g. TCR-like antibodies).
- the antigen binding proteins, e.g. antibodies, of the invention comprise at least one antigen binding domain that binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , i.e. it is this antigen binding domain that confers onto the antigen binding proteins of the invention the ability to bind to (or specifically recognise or specifically bind to) this antigen.
- the antigen binding proteins (e.g. antibodies) of the invention thus comprise a “first” antigen binding domain, with a “first” binding specificity, also termed “specificity A”, which is for HLA- A*02:01/PRAME 425 ' 433 .
- the presence of an antigen binding domain described herein as a “first” antigen binding domain does not imply that the presence of one or more further antigen binding domains is essential; such further antigen binding domains are optional.
- the heavy chain variable domain and the light chain variable domain can also each be described as having a “specificity” for the antigen to which the antigen binding domain binds.
- the antigen binding proteins e.g. antibodies, of the invention, comprise at least one antigen binding domain that comprises a heavy chain variable domain with specificity for HLA- A*02:01/PRAME 425 ' 433 , and a light chain variable domain with specificity for HLA- A*02:01/PRAME 425 ' 433 .
- specificity for HLA- A*02:01/PRAME 425 ' 433 is termed the “first” specificity, or “specificity A”.
- HLA-A*02 (also termed “HLA-A2”, “HLA-A02”, and “HLA-A*2”) is a human class I major histocompatibility complex (MHC) allele group at the HLA-A locus.
- HLA-A*02 is a human leukocyte antigen serotype within the HLA-A serotype group.
- the HLA-A*02 protein (encoded by the respective HLA gene) constitutes the a chain of the respective class I MHC (major histocompatibility complex) protein, which further comprises a p2 microglobulin subunit, also termed the “P chain”, which is encoded by the p2 microglobulin (P2M) locus.
- HLA-A*02:01 also referred to as HLA-A02:01 , HLA-A0201 , or HLA-A*02.01
- HLA-A*02:01 HLA-A*02:01
- HLA-A*02:01 HLA-A*02:01
- HLA-A*02:01 I PD Accession: HLA00005 and HLA00006
- HLA-A*02:01 encoded at these loci is set forth herein in SEQ ID NO: 21.
- This protein comprises a N-terminal leader peptide, an extracellular domain (ECD), a transmembrane domain and a C-terminal intracellular domain.
- ECD extracellular domain
- transmembrane domain e.g. a transmembrane domain
- C-terminal intracellular domain e.g. antibodies
- the antigen binding proteins (e.g. antibodies) of the invention may bind to functionally equivalent pHLA-A2 complexes in which PRAME 425 ' 433 is displayed, and in which the HLA-A2 comprises an amino acid sequence substantially homologous to SEQ ID NO: 21.
- amino acid sequence of human p2 microglobulin is set forth herein in SEQ ID NO: 23.
- HLA-A*02:01 can present peptides that are fragments of intracellular proteins, including PRAME-derived peptides, i.e. peptides derived from PRAME proteins.
- PRAME stands for Preferentially Expressed Antigen in Melanoma.
- the PRAME protein is a Cancer Testis Antigen (CTA).
- PRAME refers to any native PRAME from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated.
- the term encompasses “full-length,” unprocessed PRAME as well as any form of PRAME that results from processing in the cell.
- the term also encompasses naturally occurring variants of PRAME, e.g., splice variants or allelic variants.
- PRAME is human PRAME, which is described in UniProt (www.uniprot.org) accession no. P78395.
- An amino acid sequence of human PRAME is set forth in SEQ ID NO: 20 herein.
- Recombinant human PRAME is commercially available.
- the antigen binding proteins (e.g. antibodies) of the present invention bind to (or specifically bind to) the HLA-A*02:01 restricted PRAME derived peptide PRAME 425 ' 433 .
- PRAME 425 ' 433 is meant the PRAME derived peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 19; found at positions 425-433 of the PRAME protein of SEQ ID NO: 20).
- HLA-A*02:01/PRAME 425 ' 433 refers to a complex of PRAME 425 ' 433 presented I displayed on the class I MHC molecule comprising HLA-A*02:01 , i.e. HLA-A*02:01 restricted PRAME 425 ' 433 .
- HLA-A*02:01/PRAME 425 ' 433 means a HLA-A*02:01 molecule that is presenting (or “loaded” with) the PRAME 425 ' 433 peptide.
- HLA-A*02:01/PRAME 425 ' 433 means a HLA-A*02:01 - peptide complex (pMHC) in which the PRAME 425 ' 433 epitope is presented in the antigen binding groove (or accommodated in the antigen binding groove) of the MHC.
- pMHC HLA-A*02:01 - peptide complex
- HLA-A*02:01/PRAME 425 ' 433 to which the antigen binding proteins (e.g. antibodies) of the invention can bind may comprise recombinant PRAME 425 ' 433 , e.g. a recombinant human PRAME 425 ' 433 , or a native or natural form of PRAME 425 ' 433 , for example PRAME 425 ' 433 when presented via HLA- A*02:01 on the cell surface, e.g. on the surface of a cancer cell.
- the antigen binding proteins (e.g. antibodies) of the invention do not bind to (or do not cross-react with), e.g. do not significantly bind to (or do not significantly cross-react with) the HLA-A*02:01 molecule itself/ alone (i.e. the MHC molecule itself), i.e. in the absence of a presented PRAME 425 ' 433 peptide, i.e. without the PRAME 425 ' 433 peptide presented or complexed thereto, i.e. to an unloaded HLA- A*02:01 molecule.
- the antigen binding proteins (e.g. antibodies) of the invention do not bind to (or do not cross-react with), e.g. do not significantly bind to (or do not significantly cross-react with) the soluble form of PRAME 425 ' 433 , i.e. they do not bind (or do not significantly bind) to PRAME 425 ' 433 unless it is presented by a HLA-A*02:01 complex, i.e. unless it is HLA-A*02:01 restricted.
- the antigen binding proteins (e.g. antibodies) of the invention typically bind to, or specifically bind to, the PRAME 425 ' 433 epitope (or peptide), solely (or strictly) in the context of the MHC, i.e. HLA-A*02:01.
- a convenient and appropriate method for assessing binding would include in vitro binding assays such as ELISA assays to assess binding of antigen binding proteins (e.g. antibodies) to immobilised antigen, such as immobilised forms of HLA- A*02:01/PRAME 425 ' 433 as described elsewhere herein.
- ELISA assays to assess binding of antigen binding proteins (e.g. antibodies) to immobilised antigen, such as immobilised forms of HLA- A*02:01/PRAME 425 ' 433 as described elsewhere herein.
- antigen binding proteins (e.g. antibodies) of the present invention can bind to HLA-A*02:01/PRAME 425 ' 433 in an ELISA assay.
- ELISA assays The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of an antibody to bind to HLA- A*02:01/PRAME 425 ' 433 in such an assay. Suitable assays are discussed elsewhere herein. Particularly preferred ELISA assays are described in the Examples section herein
- antigen binding proteins (e.g. antibodies) of the present invention bind to HLA-A*02:01/PRAME 425 ' 433 in (as determined in) a Surface Plasmon Resonance (SPR) assay (e.g. a BIACore assay, e.g. using a BIAcore S200 instrument).
- SPR Surface Plasmon Resonance
- BIACore assay e.g. using a BIAcore S200 instrument
- antibodies (or binding proteins) of the invention are able to bind to HLA-A*02:01/PRAME 425 ' 433 in an SPR assay, or in an ELISA assay, preferably both.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding.
- an antigen binding protein (e.g. antibody) of the invention binds to, or specifically binds to, the HLA-A*02:01/PRAME 425 ' 433 complex when said complex is present on cells, e.g. cancer cells.
- the antigen binding proteins (e.g. antibodies) of the invention are capable of binding to HLA-A*02:01/PRAME 425 ' 433 positive cells, i.e. HLA-A*02:01 positive, PRAME 425 ' 433 positive cells.
- Such cells are preferred “target cells” herein; target cells being cells displaying an antigen to which the antibodies bind, or specifically bind.
- cancer cells which includes tumour cells and blood cancer cells.
- Cancer cells may be any HLA-A*02:01/PRAME 425 ' 433 positive cancer cell, i.e. any cancer cell on the surface of which is displayed HLA- A*02:01/PRAME 425 ' 433 .
- the cancer cells are selected from the group consisting of lung cancer cells (e.g. NCI-H1703 cells), melanoma cells (e.g. A-375 cells), and monocytes (e.g. THP-1 cells).
- lung cancer cells e.g. NCI-H1703 cells
- melanoma cells e.g. A-375 cells
- monocytes e.g. THP-1 cells
- the cancer cells are cancer cells in or from a subject suffering from cancer.
- the cancer is lung cancer, skin cancer or leukaemia.
- Cancer cells may be cancer cell line cells, which may be used in the assays described herein.
- the cells may be T2 cells, which are well-known and widely used in the field.
- T2 cells can be induced (“pulsed”) to display MHC class I molecules complexed with exogenously administered peptides.
- T2 cells are deficient in a peptide transporter involved in endogenous antigen processing (TAP) and therefore have markedly reduced ability to display MHC class I molecules complexed with endogenous peptides.
- T2 cells e.g. antibodies
- the antigen binding proteins e.g. antibodies
- the antigen binding proteins are capable of binding to T2 cells induced to display exogenously administered PRAME 425 ' 433 in a HLA-A*02:01 restricted manner.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding.
- the antigen binding proteins (e.g. antibodies) of the invention comprise a first antigen binding domain which binds to (or specifically binds to) HLA-A*02:01/PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention may comprise further antigen binding domains.
- the number of antigen binding domains possessed by an antigen binding protein (e.g. antibody) determines its valency.
- the antigen binding proteins (e.g. antibodies) of the invention comprise a second antigen binding domain (i.e. are bivalent), and optionally a third antigen binding domain (i.e. are trivalent), and optionally further antigen binding domains (i.e. are multivalent).
- any further antigen binding domain(s) may incorporate, singly or in combination, any of the features described herein in relation to the first antigen binding domain.
- the antigen binding proteins (e.g. antibodies) of the invention may be bispecific, trispecific or multispecific.
- the term “-specific” or "- specificity" in the context of bispecific, trispecific, multispecific, etc. as used herein refers to the ability of a domain (or antigen binding domain) to recognize a particular target antigen and refers to the capability of binding that target antigen. Bispecific antibodies are therefore capable of recognizing and binding to (or specifically binding to) two different target antigens.
- bispecific antigen binding proteins e.g. antibodies
- bispecific antigen binding proteins of the invention comprise a “second antigen binding domain” having a “second antigen binding specificity” (or simply a “second specificity” or “specificity B”) which is for an antigen other than HLA-A*02:01/PRAME 425 ' 433 .
- Trispecific antigen binding proteins e.g.
- antibodies of the invention comprise a “third antigen binding domain” having a “third antigen binding specificity” (or simply a “third specificity”, or “specificity C”), which is for an antigen other than HLA-A*02:01/PRAME 425 ' 433 and which is different from the specificity of the second antigen binding domain (i.e. the second antigen binding specificity).
- Multispecific antigen binding proteins e.g. antibodies
- Multispecific antigen binding proteins comprise four or more antigen binding domains each of which have a different antigen binding specificity.
- the bivalent, trivalent, multivalent, bispecific, trispecific and multispecific antigen binding proteins (e.g. antibodies) of the invention may comprise antigen binding domains fused to each other, optionally via peptide linkers.
- the production of bivalent, trivalent, multivalent, bispecific, trispecific and multispecific antigen binding proteins (e.g. antibodies), the selection of (e.g. length and sequence of) peptide linkers, and the orientation of domains and peptide linkers, are well-known in the field, and would be within the competencies of the person of ordinary skill in the art.
- the first and second antigen binding domains may be fused to each other, optionally via a peptide linker.
- the first and the second antigen binding domain may be fused in any orientation, for instance (i) the C-terminus of the first antigen binding domain may be fused, optionally via a peptide linker, to the N-terminus of the second antigen binding domain, or (ii) the C-terminus of the second antigen binding domain may be fused, optionally via a peptide linker, to the N-terminus of the first antigen binding domain.
- variable light (VL) and variable heavy (VH) domains from the first (A) and second (B) antigen binding domains may be fused (optionally via peptide linkers) in any sequence/ orientation (i.e. order), provided that upon proper folding of the resulting polypeptide, two functional antigen binding domains are formed.
- antigen binding proteins e.g. antibodies
- selection of (e.g. length and sequence of) peptide linkers, and the orientation of domains and peptide linkers are well-known in the field, and would be within the competencies of the person of ordinary skill in the art.
- Preferred orientations are, in the N to C direction, i) VLA-VHB-VLB-VHA, ii) VLB-VHA-VLA-VHB; iii) VHA-VLB-VHB-VLA and iv) VHB-VLA-VHA-VLB, most preferred are i) VLA-VHB-VLB-VHA, and ii) VLB-VHA-VLA-VHB, particularly i) VLA- VHB-VLB-VHA.
- the antigen binding protein (e.g. antibody) is at least bispecific, e.g. is a bispecific antibody.
- the antigen binding protein (e.g. antibody) of the invention preferably comprises a second antigen binding domain with a second specificity, i.e. which binds to, or specifically binds to, a second antigen, i.e. to an antigen other than HLA-A*02:01/PRAME 425 ' 433 .
- the (at least) bispecific antigen binding protein (e.g. antibody) of the present invention may be of any antibody fragment, format or construct described herein, including single chain formats and multichain formats.
- Preferred antigen binding proteins (e.g. antibodies) of the invention are in a T- cell engager format, i.e. are T-cell engagers.
- T-cell engagers This is a well-understood term in the art, which refers to antigen binding proteins (e.g. antibodies) that bind to (or specifically bind to) HLA-A*02:01/PRAME 425 ' 433 and to an antigen present on T cells (T lymphocytes) via different antigen binding domains.
- TCE T-cell engagers
- TCEs are also termed T-cell engaging antibodies (or antigen binding proteins). TCEs physically recruit T cells to target cells (e.g.
- T-cell antigen referred to herein as simply a T-cell antigen.
- a T-cell surface antigen is an antigen present on the surface of T cells.
- a T-cell specific antigen is an antigen present in or on T-cells specifically. This dual binding and recruitment of T cells to target cells leads to T-cell activation, proliferation and T-cell mediated target cell killing.
- T-cell engagers may be trispecific or multispecific, but they are at least bispecific, as described above.
- the antigen binding protein (e,g. antibody) of the invention further comprises a second antigen binding domain that binds to (or specifically binds to) a T-cell surface antigen.
- the T-cell surface antigen is preferably a T-cell specific antigen, particularly CD3, most particularly CD3E.
- the second antigen binding domain may be specific for another T-cell surface antigen, e.g. the T-cell receptor (i.e. a polypeptide comprised in the T-cell receptor).
- the T-cell surface antigen, e.g. CD3 is a human T-cell antigen, e.g. human CD3.
- Such (at least) bispecific antibodies trigger T-cell activation in a target specific manner, i.e. they redirect T cell activity (e.g. T-cell mediated lysis) against target cells (e.g. cancer cells and tumours) displaying the HLA-A*02:01/PRAME 425 ' 433 antigen.
- T cell activity e.g. T-cell mediated lysis
- target cells e.g. cancer cells and tumours
- Antibodies against suitable T-cell surface antigens are numerous and widely available, for instance OKT3 and LICHT1.
- the second antigen binding domain which is specific for a T-cell surface antigen may be the antigen binding domain of any such suitable antibody against a T-cell surface antigen, preferably CD3.
- the skilled person will readily be able to identify suitable antibodies and binding domains for their purposes.
- a preferred anti-CD3 antibody in this regard is LICHT1, the heavy chain and light chain variable domain amino acid sequences of which are well-known (SEQ ID NOs: 24 and 25 herein, respectively). These VH and VL domain sequences represent preferred VH and VL domains of the second antigen binding domain of the (at least) bispecific antigen binding proteins (e.g. antibodies) of the present invention.
- the antigen binding proteins (e.g. antibodies) of the invention preferably comprise a second antigen binding domain that binds to (or specifically binds to) CD3, said antigen binding domain comprising a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:24, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto), and/or (preferably “and”) a light chain variable region that comprises the amino acid sequence of SEQ ID NO:25, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto).
- a second antigen binding domain that binds to (or specifically binds to) CD3, said antigen binding domain comprising a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:24, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto), and/or (preferably “and”) a light chain variable region that comprises the amino acid sequence of SEQ ID NO:25, or a sequence substantially
- antigen binding proteins e.g. antibodies
- T-cell engager format i.e. that comprise a second antigen binding domain that is specific for a T-cell surface antigen
- redirect T cells displaying said T-cell surface antigen to the site of the target antigen HLA-A*02:01/PRAME 425 ' 433 e.g. to cancer cells or tumours, and induce activation, differentiation, proliferation of T-cells directed to said target antigen, and thus T-cell mediated cytotoxicity against said target cells.
- effector T cells may be CD8 + T cells, CD4 + T cells, CD4 + CD8 + T cells, regulatory T cells, gammadelta T cells, memory T cells, natural killer T cells (NKTs) or any combination thereof.
- the T cells may be comprised within an effector cell population, which may also comprise other effector cells.
- Effective cell is a term well-known in the art to mean any immune cell that can effect or enhance an immune response.
- Preferred effector cells are peripheral blood mononuclear cells (PBMCs), and pan-T cells derived therefrom.
- PBMCs are a mixed composition of various effector cell types including lymphocytes (T cells, B cells, and NK cells) and monocytes. These cells can be extracted from whole blood or buffy coat samples, e.g.
- Pan T-cells are a population of CD3+ cells that be isolated from PBMCs. Obtaining PBMCs, pan-T cells and other effector cell populations is within the competencies of the person of ordinary skill in the art, and suitable methods are provided in the Examples herein.
- CD3 is a preferred T cell surface antigen, and is a T cell specific antigen, i.e. is displayed specifically on T cells, including on CD8 + T cells, CD4 + T cells, CD4 + CD8 + T cells, regulatory T cells, gamma-delta T cells, memory T Cells, and natural killer T cells (NKTs).
- T cells including on CD8 + T cells, CD4 + T cells, CD4 + CD8 + T cells, regulatory T cells, gamma-delta T cells, memory T Cells, and natural killer T cells (NKTs).
- the second antigen binding domain may bind to (or specifically bind to) a T-cell surface antigen specific to the desired subset of T cells.
- the second antigen binding domain may bind to (or specifically bind to) a “public” T-cell receptor (i.e. polypeptides thereof), i.e. TCRs of a sequence known to be shared by a large number of individuals within a population.
- a “public” T-cell receptor i.e. polypeptides thereof
- TCRs of a sequence known to be shared by a large number of individuals within a population.
- Antigen binding proteins e.g. antibodies
- Such a public T-cell receptors, and the sequences of their TCRs, are well documented in the field.
- Such public TCRs typically arise in response to a common infectious agent, e.g. a virus.
- preferred public TCR to which the second antigen binding domain of the antigen binding proteins (e.g. antibodies) binds (or specifically binds) are virus specific public TCRs, preferably human virus specific public TCRs.
- gamma-delta T cells may be redirected to the site of the target antigen HLA-A*02:01/PRAME 425 ' 433 , e.g. to cancer cells or tumours, and activated, in embodiments wherein the second antigen binding domain of the antigen binding proteins (e.g. antibodies) of the invention binds to (or specifically binds to) an antigen present on the surface of the gamma-delta T cells.
- the second antigen binding domain may be bind to (or specifically bind to) the public Vy9V52 TCR, preferably to human TCRs.
- NKT cells may be redirected to the site of the target antigen HLA-A*02:01/PRAME 425 ' 433 , e.g. to cancer cells or tumours, and activated, in embodiments wherein the second antigen binding domain of the antigen binding proteins (e.g. antibodies) of the invention binds to (or specifically binds to) an antigen present on the surface of the NKT cells.
- the second antigen binding domain may bind to (or specifically bind to) the public Va24 TCR (Va14 in mice), preferably to human TCRs.
- non-T cells may be redirected to the site of the target antigen HLA-A*02:01/PRAME 425 ' 433 antigen, e.g. to cancer cells or tumours, and activated, in embodiments wherein the second antigen binding domain of the antigen binding proteins (e.g. antibodies) of the invention binds to (or specifically binds to) an antigen present on the surface of the effector cells.
- the second antigen binding domain may bind to (or specifically bind to) CD16, NKG2D, NKp30, or NKp46 receptors, in which case natural killer (NK) cells may be redirected and activated.
- the second antigen binding domain may be bind to (e.g. specifically bind to) SIRPa, in which case macrophages may be recruited and activated.
- the antigen binding protein (e.g. antibody) of the invention is bispecific, e.g. is a bispecific antibody, which comprises a third, and optionally further, antigen binding domain(s).
- the second antigen binding domain binds to, or specifically binds to, a second antigen, i.e. to an antigen other than HLA-A*02:01/PRAME 425 ' 433 , preferably as described elsewhere herein
- the third antigen binding domain binds to, or specifically binds to HLA- A*02:01/PRAME 425 ' 433 .
- the third antigen binding domain may incorporate, singly or in combination, any of the features described herein in relation to the first antigen binding domain.
- the third antigen domain is identical to the first antigen binding domain. Further antigen binding domains may bind to, i.e. specifically bind to HLA-A*02:01/PRAME 425 ' 433 , or to the same antigen as the second binding domain.
- the antigen binding protein (e.g. antibody) of the invention is trispecific, e.g. is a trispecific antibody.
- the antigen binding protein (e.g. antibody) of the invention preferably comprises a second antigen binding domain which binds to, or specifically binds to, a second antigen, preferably as described elsewhere herein, and a third antigen binding domain which binds to, or specifically binds to, a third antigen, wherein said first, second and third antigens are different from each other.
- the trispecific antigen binding proteins (e.g. antibodies) of the invention are preferably also T-cell engagers (as defined and described above).
- the bispecific, trispecific or multispecific antigen binding protein may be of any known format, including one selected from the group consisting of single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a bispecific T-cell engager (BiTE; tandem di-scFv), a tandem tri-scFv, a tribody (Fab-(scFv)2) or bibody (Fab- (scFv)1), triabody, scDb-scFv, bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc- scFv fusion, di-diabody, DVD-lg, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG- s
- the antigen binding proteins (e.g. antibodies) of the present invention are preferably T-cell engaging antibodies (or antigen binding proteins), also termed “T-cell engagers” (TCEs). These are (at least) bispecific molecules, and in a preferred embodiment are T-cell engaging bispecific antigen binding proteins (e.g. antibodies).
- the antigen binding protein (e.g. antibody) of the invention is a single-chain T-cell engaging bispecific antigen binding protein (e.g. antibody).
- the second antigen binding preferably binds to (or specifically binds to) CD3.
- the antigen binding domains in such bispecific molecules are as defined elsewhere herein.
- the antigen binding protein of the invention is a single-chain bispecific diabody, i.e. a bispecific antibody in the single-chain bispecific diabody (scDb) format.
- the scDb has the capability of binding to (or specifically binding to) both HLA-A*02:01/PRAME 425 ' 433 via a first antigen binding domain, as described above, and to a T-cell surface antigen, preferably CD3, via a second antigen binding domain as described above.
- the antigen binding domains in such bispecific molecules are as defined elsewhere herein.
- the scDb format has a more compact form in comparison to other bispecific antibody formats and allows the formation of immunocytolytic synapses.
- the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME 425 ' 433 , wherein said VHA comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 that comprise, respectively, the amino acid sequences of the VH CDR1 , VH CDR2 and VH CDR3 of a specific antibody of the invention (disclosed in Table A), or sequences substantially homologous thereto; ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises a variable light (VL) CDR1, a VL CDR2 and a VL CDR3 that comprise, respectively, the amino acid sequences of the VL CDR1 , VL CDR2
- the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME 425 ' 433 , wherein said VHA comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto;
- VLA immunoglobulin with said first specificity
- VL variable light
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto
- VHB second heavy chain variable domain of an immunoglobulin with a second specificity
- VLB second light chain variable domain of an immunoglobulin with said second specificity
- the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME 425 ' 433 , wherein said VHA comprises:
- VH variable heavy
- VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115) or a sequence substantially homologous thereto, and
- VLA immunoglobulin with said first specificity
- VL variable light CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO:117), EFISQW (SEQ ID NO:118), EFVSSW (SEQ ID NO:119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
- VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto
- VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO:10), or QQANSFPFT (SEQ ID NO: 121), or a sequence substantially homologous thereto
- VHB second heavy chain variable domain of an immunoglobulin with a second specificity
- VLB second light chain variable domain of an immunoglobulin with said second specificity
- VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker.
- Preferred CDRs and substantially homologous sequences are as described anywhere else herein.
- the present invention provides a single-chain bispecific diabody comprising: iii) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME 425 ' 433 , wherein said VHA comprises the amino acid sequence of the VH domain of a specific antibody of the invention (disclosed in Table A) or a sequence substantially homologous thereto; iv) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises the amino acid sequence of the VL domain of a (preferably said) specific antibody of the invention (disclosed in Table A); v) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and vi) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); where
- the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME 425 ' 433 , wherein said VHA comprises the amino acid sequence of SEQ ID NO: 3 or a sequence substantially homologous thereto; ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto; iii) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and iv) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single
- VH and VL domains and substantially homologous sequences are as described anywhere else herein.
- the VLA, VHB, VLB and VHA domains are connected within (i.e. in) a single polypeptide chain, i.e. are fused, i.e. connected linearly, via peptide linkers. There are thus three peptide linkers connecting the four domains.
- the domains may be connected in any order/ orientation, provided that upon proper folding of the resulting polypeptide, two functional antigen binding domains are formed.
- Preferred orders/ orientations are, in the N to C direction: i) VLA-VHB-VLB- VHA, ii) VLB-VHA-VLA-VHB; iii) VHA-VLB-VHB-VLA and iv) VHB-VLA-VHA-VLB, most preferred are i) VLA-VHB-VLB-VHA, and ii) VLB-VHA-VLA-VHB, particularly i) VLA-VHB-VLB-VHA.
- the antigen binding proteins of the invention comprise at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs.
- VH domain heavy chain variable domain
- VL domain light chain variable domain
- the scDbs of the invention comprise two such antigen binding domains: one comprising VHA and VLA, and one comprising VHB and VLB.
- first heavy and light chain variable domains including the CDR and domain sequences, and the substantially homologous sequences
- second heavy and light chain variable domains including the CDR and domain sequences, and the substantially homologous sequences
- the scDbs comprise a peptide linker between each of: i) the VLA and the VHB domains (or vice versa, depending on which orientation of domains is present); and ii) the VLB and the VHA domains (or vice versa, depending on which orientation of domains is present); and iii) the VHB and the VLB domains (or vice versa, depending on which orientation of domains is present); or iv) the VHA and VLA domains (or vice versa) depending on which orientation of domains is present.
- the sDbs comprise both linker i) and linker ii) and either linker iii) or linker iv), depending on the orientation of domains therein.
- the peptide linker between the VLA and the VHB domains (or vice versa) (linker (i)) and the peptide linker between the VLB and the VHA domains (or vice versa) (linker (ii)), is relatively short, e.g. 3 to 7 amino acids, e.g. 4 to 7 amino acids or 3 to 5 amino acids, i.e. 3, 4 or preferably 5 amino acids. These are termed the “scDb short linkers” herein.
- the peptide linker between the VHB and the VLB domains (or vice versa) (linker (iii)) or in other arrangements between the VHA and VLA domains (or vice versa) (linker (iv)), is relatively longer, e.g. 12 to 21, preferably 13-19, more preferably 14 to 17 amino acids, e.g. 14, 15, 16 or 17 amino acids. This is termed the “scDb short linker” herein.
- the peptide linker between the VHB and the VLB domains (or vice versa) (linker (iii)) or in other arrangements between the VHA and VLA domains (or vice versa) (linker (iv)), is 2.5 to 3.5 times longer, e.g. about 3 times longer, than the peptide linker between the VLA and the VHB domains (or vice versa) (linker (i)), and the peptide linker between the VLB and the VHA domains (or vice versa) (linker (ii)).
- (ii) may be of identical length and/or (preferably “and”) amino acid sequence.
- amino acid sequences of the various peptide linkers are not particularly limited; suitable linker sequences could be identified and prepared by the person of ordinary skill in the art, e.g. with reference to Vdlkel et al., (2001) Protein Engineering, Design and Selection, Volume 14, Issue 10, Pages 815-823, and any suitable linker sequence may be used.
- the peptide linker sequences comprise only hydrophilic amino acids (e.g. arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine) and aliphatic amino acids (e.g. alanine, glycine, isoleucine, leucine, proline, and valine).
- hydrophilic amino acids e.g. arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine
- aliphatic amino acids e.g. alanine, glycine, isoleucine, leucine, proline, and valine.
- the peptide linker sequences comprise only amino acids selected from the group consisting of threonine, asparagine, serine, aspartic acid, glycine and alanine.
- said peptide linker between the VLA and the VHB domains (or vice versa) (linker (i)) and between the VLB and the VHA domains (or vice versa) (linker (ii)) consists of four Glycine amino acid residues and one Serine amino acid residue (GGGGS, SEQ ID NO: 26), and the peptide linker between the VHB and the VLB domains (or vice versa) (linker (iii)), or between the VHA and VLA domains (or vice versa) (linker (iv)) consists of three contiguous sequences consisting each of four Glycine amino acid residues and one Serine amino acid residues (GGGGS, SEQ ID NO: 26), i.e. GGGGSGGGGSGGGGS (SEQ ID NO: 27).
- the antigen binding proteins are chimeric antigen receptors (CARs).
- CARs chimeric antigen receptors
- the term “chimeric antigen receptor” or “CAR” refers to a receptor that is capable of activating an immune cell in response to antigen binding.
- CARs are recombinant membrane spanning molecules and are advantageously expressed on immune cells. Their structure typically comprises (i) an extracellular domain (“ectodomain” or “antibody domain”), (ii) a transmembrane domain and (iii) a cytoplasmic domain (endodomain or intracellular signalling domain).
- the ectodomain (i.e., antibody domain) comprises an antigen binding domain that binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , as defined elsewhere herein.
- the ectodomain comprises an antigen binding protein in the scFv format, but other antibody fragments/formats may also be used.
- a spacer sequence generally connects the ectodomain and the transmembrane domain, which in turn is connected to an endodomain.
- the receptors cluster and an activation signal is transmitted to the immune cell which results in initiation of an immune response against the cell on which the target antigen (HLA-A*02:01/PRAME 425 ' 433 ) is displayed.
- First generation CARs have a simply structured endodomain comprising CD3- zeta.
- a co- stimulatory domain was added in the second-generation CARs; and third generation CARs include two or more costimulatory domain.
- Said co-stimulatory domains may be selected from the group consisting of CD28, 0X40 and/or 4-1 BB.
- CD3-zeta Apart from CD3-zeta, other ITAM- containing domains have been explored including the Fc receptor for IgE-y domain.
- the invention provides a chimeric antigen receptor (CAR) that specifically recognizes HLA-A*02:01/PRAME 425 ' 433 , comprising: i) an ectodomain that comprises an antigen binding domain which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 , as defined anywhere elsewhere herein; ii) a transmembrane domain; and iii) an intracellular signalling domain.
- CAR chimeric antigen receptor
- the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domains of a type I transmembrane protein, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
- the intracellular signalling domain is selected from the group consisting of cytoplasmic signalling domains of a human CD3 zeta chain, FcyRIII, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
- ITAM immunoreceptor tyrosine-based activation motif
- Suitable components e.g. domains, of CARs, and means for their inclusion are well-known in the field, and any suitable component or domain may be included in the CARs of the present invention.
- the invention provides immune cells engineered to express CARs comprising the antigen binding proteins described herein.
- Suitable immune cells include, without being limited to, T cells, Natural Killer T (NKT) cells, natural killer (NK) cells, human embryonic stem cells, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS).
- T cells may be a cytotoxic T lymphocyte (CTL), a regulatory T lymphocyte, an inflammatory T- lymphocytes, or a helper T-lymphocyte or a gamma-delta T cell.
- the T cell may be a CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells.
- T cells expressing a CAR are termed CAR-T cells and in embodiments, immune cells engineered to express CARs comprising the antigen binding proteins described herein are CAR-T cells.
- Example 1 herein describes the examination of the fine-specificity of an antibody of the invention, 5-A05, towards the HLA-A*02:01 presented PRAME 425 ' 433 peptide.
- Positional alanine (Ala) scanning was performed.
- An array of PRAME 425 ' 433 variant peptides were produced, in each of which one residue of the PRAME 425 ' 433 sequence was mutated to alanine.
- the variant peptides sequences are shown in Table C.
- the binding of 5-A05 to the Ala-mutated peptides was assessed using pHLA phage capture ELISA, as described in Example 1 , relative to the binding to HLA-A*02:01 presented PRAME 425 ' 433 .
- clone 5-A05 exhibited restricted positional binding dependency towards 2 of the 4 solvent exposed residues - namely the amino acids at positions 5 and 7 of PRAME 425 ' 433 (SEQ ID NO: 19).
- the central binding mode of 5-A05 indicates a high specificity for the particular peptide sequence of PRAME 425 ' 433 in the HLA-A*02:01/PRAME 425 ' 433 complex.
- the antigen binding proteins (e.g. antibodies) of the invention bind to (i.e. are capable of binding to) the amino acid at position 5 or/and (preferably “and”) to the amino acid at position 7 of the HLA-A*02:01 restricted peptide PRAME 425 ' 433 (SEQ ID NO: 19).
- the antigen binding proteins (e.g. antibodies) of the invention bind to (i.e. are capable of binding to) the amino acids at positions 5, 7 and 8 of the HLA-A*02:01 restricted peptide PRAME 425 ' 433 (SEQ ID NO: 19).
- the antigen binding proteins (e.g. antibodies) of the invention preferentially bind to (or selectively bind to) HLA-A*02:01/PRAME 425 ' 433 as compared to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 35 and/or (preferably “and”) as compared to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37 (P5 and P7 of Table C).
- the antigen binding proteins (e.g. antibodies) of the invention exhibit binding to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 35 and/or (preferably “and”) to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37 (P5 and P7 of Table C), which is at most 25%, e.g. at most 20%, of the exhibited binding of the same antigen binding protein (e.g. antibody) to HLA-A*02:01/PRAME 425 ' 433
- the antigen binding proteins e.g.
- antibodies of the invention exhibit a decrease of at least 75%, e.g. at least 80%, in binding to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 35 and/or (preferably “and”) to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37 (P5 and P7 of Table C), as compared to binding to HLA-A*02:01/PRAME 425 - 433
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding.
- binding is exhibited when the antigen binding protein is an antibody in the scFv format, preferably wherein the scFV is present on the surface of a phage particle.
- ASSAY 1 Assay for binding specificity (ELISA)
- binding of an antigen binding protein e.g. antibody
- H LA-restricted peptide pH LA
- suitable methods e.g. an ELISA assay such as an ELISA assay in which the pHLA is coated on ELISA plates/wells.
- binding of an antigen binding protein (e.g. antibody) of the invention to H LA-restricted peptide (pHLA) may be as determined by (or as assessed by) an ELISA assay, e.g. an ELISA assay that comprises:
- a secondary antibody having (e.g. conjugated to) a detectable label (e.g. horse radish peroxidase, “HRP”), e.g. anti-M13-HRP (e.g. stock solution diluted 1 :5000 in 100 pl blocking buffer (e.g. PBSTM)/well) for scFv-phage, or protL (e.g. diluted 1 :10,000 in 100 pl blocking buffer (e.g. PBSTM)/well) for scDb or IgG;
- HRP horse radish peroxidase
- anti-M13-HRP e.g. stock solution diluted 1 :5000 in 100 pl blocking buffer (e.g. PBSTM)/well) for scFv-phage
- protL e.g. diluted 1 :10,000 in 100 pl blocking buffer (e.g. PBSTM)/well) for scDb or IgG
- HRP horse radish peroxidase
- Detecting (and quantifying) the detectable label For example, if HRP (horseradish peroxidase) is used as the detectable label, then a HRP substrate (e.g. TMB substrate (3,3',5,5'-Tetramethylbenzidine)) may be added to the wells and incubated (e.g. for 10 min at room (or ambient) temperature (e.g. 25°C), and the reaction may then be stopped (e.g. with 100pl/well of 1M HCI) and absorbance measured, e.g. at 450nm, e.g. using a microplate reader.
- HRP substrate e.g. TMB substrate (3,3',5,5'-Tetramethylbenzidine
- H LA-restricted peptide a preferred ELISA assay for assessing the ability of an antigen binding protein (e.g. antibody) to bind to a H LA-restricted peptide (pH LA) is described in the Examples section herein.
- an antigen binding protein e.g. antibody
- the antigen binding protein may be an antibody in any format.
- the antibody is in the scDb, IgG or scFv format. If the scFv format is used, then the scFv is present on the surface of a phage particle. If phage particles are used, then preferably 10 9 phages/well are used in step (g) of the above method.
- Example 3 herein describes the assessment of the specificity with which an antibody of the invention, 5-A05 in the scDb format, redirects T-cell activity against cells displaying the target antigen HLA-A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 restricted peptides comprising (or consisting of) sequences of human genomic origin identified as having high levels of sequence similarity to PRAME 425 ' 433 (termed “risk peptides”). The identified human peptides are shown in Table F. Such HLA-A2 restricted peptides represent a potential crossreactivity risk, should antigen binding proteins (e.g. antibodies) directed to HLA- A*02:01/PRAME 425 ' 433 also bind to them and induce T-cell activation and redirection against the cells displaying them.
- antigen binding proteins e.g. antibodies
- a panel of genomic risk peptides was prepared (Table F), and exogenously added to a culture of T2 cells which were pulsed to induce HLA-A*02:01 presentation of said peptides.
- a Jurkat-NFAT activation assay was performed as described in Example 3. This is a well-known and widely used assay in the field, which comprises the use of Jurkat T cell line (TCR/CD3 “Effector Cells”) that expresses a luciferase reporter driven by a Nuclear factor of activated T-cells-response element (NFAT- RE). When the Jurkat T cells are engaged with an anti-CD3 antibody, their T-cell receptor (TCR) transduces intracellular signals resulting in NFAT-RE-mediated luminescence.
- TCR T-cell receptor
- the assay indicates the ability of a T-cell engaging antigen binding protein (e.g. antibody) with a first antigen binding domain specific for a target antigen, and a second antigen binding domain specific to a T-cell surface antigen (e.g. CD3), to redirect T cell activity in a specific manner against cells displaying the target antigen.
- a T-cell engaging antigen binding protein e.g. antibody
- a second antigen binding domain specific to a T-cell surface antigen e.g. CD3
- Figures 7A and 7B demonstrate that the assay is a valid proxy for T-cell mediated killing, i.e. the level of T-cell activation seen via NFAT-RE-mediated luminescence, correlates with the level of target cell killing.
- Figure 8A shows the extent to which 5-A05 induced Jurkat T-cell activation/ redirection against pHLA restricted risk peptides relative to HLA-A*02:01/PRAME 425 ' 433 and relative to nonpulsed T2 cells (i.e. cells lacking any pHLA restricted exogenous peptide).
- 5-A05 induced T-cell activation (redirected T-cell activity) with remarkable specificity against cells displaying HLA-A*02:01/PRAME 425 ' 433 over cells displaying all of the HLA-A*02:01 restricted risk peptides.
- Jurkat activation was only observed when T2 cells were pulsed with PRAME 425 ' 433 peptide and SACS and TRGV10-2 peptides; the level of activation observed with all (each) of the other risk peptides was comparable to non-pulsed T2 cells, despite these risk peptides sharing high sequence similarity with PRAME 425 ' 433 .
- Example 3 herein also describes the assessment of the specificity with which the antibodies of the invention, 5-A05/1-C04, PAM22, PAM23 and NGS55, in the scDb format, redirect T-cell activity against cells displaying the target antigen HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 restricted peptides comprising (or consisting of) sequences of human genomic origin identified as having high levels of sequence similarity to PRAME 425 ' 433 (termed “PRAME 425 ' 433 related degenerate risk peptides”, or simply “risk peptides”). The identified human peptides are shown in Table K.
- HLA-A2 restricted peptides represent a potential cross-reactivity risk, should antigen binding proteins (e.g. antibodies) directed to HLA-A*02:01/PRAME 425 ' 433 also bind to them and induce T- cell activation and redirection against the cells displaying them.
- antigen binding proteins e.g. antibodies
- a panel of PRAME 425 ' 433 related degenerate risk peptides was prepared (Table K), and exogenously added to a culture of T2 cells which were pulsed to induce HLA-A*02:01 presentation of said peptides.
- a Jurkat-NFAT activation assay was performed as described in Example 3. The assay indicates the ability of a T- cell engaging antigen binding protein (e.g. antibody) with a first antigen binding domain specific for a target antigen, and a second antigen binding domain specific to a T-cell surface antigen (e.g. CD3), to redirect T cell activity in a specific manner against cells displaying the target antigen.
- a T- cell engaging antigen binding protein e.g. antibody
- a second antigen binding domain specific to a T-cell surface antigen e.g. CD3
- Figures 11A to 11 D show the extent to which 5-A05/1-C04, PAM22, PAM23 and NGS55 induced Jurkat T-cell activation/ redirection against pHLA restricted PRAME 425 ' 433 related degenerate risk peptides relative to HLA-A*02:01/ PRAME 425 ' 433 and relative to non-pulsed T2 cells (i.e. cells lacking any pH LA restricted exogenous peptide).
- antigen binding proteins e.g. antibodies
- the antigen binding proteins of the invention e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 and NGS55 antibodies, in terms of redirecting potent and relevant T-cell activity specifically against HLA-A*02:01/PRAME 425 ' 433 positive cells.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T- cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect (i.e. are capable of preferentially (or selectively) redirecting) T-cell activity against cells displaying HLA-A*02:01/PRAME 425 ' 433 (i.e. HLA-A*02:01/PRAME 425 ' 433 positive cells).
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against cells displaying HLA-A*02:01/PRAME 425 ' 433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against cells displaying HLA-A*02:01/PRAME 425 ' 433 as compared to against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of i) SEQ ID NOs: 40 to 105 (genomic risk peptides), and/or ii) SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME 425-433 related degenerate risk peptides).
- a T-cell engager format e.g. in the scDb format
- the term “redirect T-cell activity” against cells displaying a given (target) antigen is meant that the T-cell engaging antigen binding protein (e.g. antibody) forms an immunological synapse by binding to the T-cell antigen (e.g. CD3) and to the given target antigen displayed on target cells, leading to recruitment of T cells to the target cells and activation of T-cell function against said target cells.
- the term means induce (i.e., promote, enhance or increase) T-cell activity against the cells displaying the target antigen. Induce need not mean that the starting activity level is zero, i.e. it may mean “induce further”.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), redirect T-cell activity against pulsed T2 cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of i) SEQ ID NOs: 40 to 105 (genomic risk peptides), and/or ii) SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME 425 ' 433 related degenerate risk peptides), to an extent that is not different, e.g.
- Suitable negative control cells may be non-pulsed T2 cells, i.e. T2 cells lacking any pH LA-restricted exogenous peptide.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), redirect T-cell activity against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of i) SEQ ID NOs: 40 to 105 (genomic risk peptides), and/or ii) SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME 425-433 related degenerate risk peptides), to an extent that is not different, e.g.
- Suitable negative control cell cells may be cells displaying neither said HLA-A*02:01 risk peptides, nor HLA-A*02:01/PRAME 425-433 , i.e. cells against which merely background levels of redirected T-cell activity are observed.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), i) redirect T-cell activity against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of SEQ ID NOs: 40 to 105 (genomic risk peptides) to an extent that is at most 25% of the T-cell activity redirected by the same antigen binding protein (e.g.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, i) exhibit a decrease of at least 75% in T-cell activity redirected against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of SEQ ID NOs: 40 to 105 (genomic risk peptides) as compared to the T-cell activity redirected by the same antigen binding protein (e.g.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such redirection of T-cell activity.
- the antigen binding protein is any T-cell engager format, preferably in a bispecific format.
- the antibody is in the scDb format.
- such redirection of T cell activity is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME 425 ' 433 (“target cells”) is about 1 :1.
- target cells preferably such redirection of T cell activity is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME 425 ' 433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of T cell activity.
- detection and quantification is as described below.
- Example 3 herein also describes, again using a Jurkat-NFAT activation assay, that 5-A05 effectively redirects T-cell activity against cancer cells displaying HLA-A*02:01/PRAME 425 ' 433 but not against cancer cells displaying HLA-A*02:01 lacking the PRAME 425 ' 433 peptide ( Figure 9B).
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect (i.e. are capable of preferentially (or selectively) redirecting) T- cell activity against cells displaying HLA-A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against cells displaying HLA-A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA- A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- a T-cell engager format e.g. in the scDb format
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against HLA- A*02:01/PRAME 425 ' 433 positive cells as compared to against HLA-A*02:01 positive, PRAME 425 ' 433 negative cells.
- a T-cell engager format e.g. in the scDb format
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such redirection of T-cell activity.
- the antigen binding protein is any T-cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- such redirection of T cell activity is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME 425 ' 433 (“target cells”) is about 1 :1.
- target cells preferably such redirection of T cell activity is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME 425 ' 433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of T cell activity.
- detection and quantification is as described below.
- ASSAY 2 Assay for redirection of T-cell activity (Jurkat)
- an antigen binding protein e.g. antibody
- a T-cell engager format to redirect T-cell activity against cells displaying a H LA-restricted peptide (pHLA)
- pHLA H LA-restricted peptide
- Jurkat assays may comprise the co-culturing of Jurkat NFAT reporter cells representing effector cells (“E”), target cells (“T”) (e.g. peptide pulsed T2 cells (described elsewhere herein) or cancer cells e.g. cancer cell line cells), preferably at an E:T ratio of about 1 :1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently applying the cultured cells to wells comprising a luciferase substrate and detecting luminescence.
- E effector cells
- T target cells
- cancer cells e.g. cancer cell line cells
- an antigen binding protein e.g. antibody
- a Jurkat activation assay that comprises:
- target cells e.g. T2 cells, or cancer cells e.g. cancer cell line cells
- cell culture medium e.g. RPMI1640 supplemented with 10% FBS (Fetal Bovine Serum) and 5U/ml PS (Penicillin-Streptomycin);
- FBS Fetal Bovine Serum
- 5U/ml PS Penicillin-Streptomycin
- Harvesting said target cells e.g. by transferring suspension cells to a Falcon tube (e.g. of 50ml volume), or by removing cell culture supernatant from adherent cells (cancer cells may be adherent cells), washing the adherent cells once with sterile 1xPBS, incubating with an appropriate amount of Trypsin (e.g.
- step (e) Seeding said target cells in the wells of cell culture plates (e.g. white 96 well plates*) in cell culture medium (e.g. at 50000 cancer cells/well the day before coculturing (step (q)) to allow for monolayer formation of adherent cells, or 25000 T2 cells/well on the day of co-culture);
- cell culture medium e.g. at 50000 cancer cells/well the day before coculturing (step (q)
- step (f) If cancer cells are seeded in step (e), then incubating said seeded adherent cancer cells e.g. for 18h at 37°C in a humidified incubator to allow for monolayer formation;
- step (l) Diluting the antigen binding protein (e.g. antibody) to be tested in assay medium (e.g. RPMI1640 supplemented with 10% FBS and 5U/ml PS), for example at a concentration range of 0.2nM to 200nM; (m) Adding the antigen binding protein (e.g. antibody) to be tested to the coculture of cells of step (k) (e.g. at a final concentration range from 0.02nM to 20nM);
- assay medium e.g. RPMI1640 supplemented with 10% FBS and 5U/ml PS
- H LA-restricted peptides e.g. PRAME 425 ' 433 or other peptides
- assay medium e.g. RPMI1640 supplemented with 10% FBS and 5U/ml PS
- concentration range 2.4nM to 200pM (if cancer cells are used as target cells, this step is not performed)
- T2 cells are used as target cells, transfer (e.g. about 50 pl) of peptide dilutions (e.g. in duplicates or triplicates) to a 96-well cell culture plate (i.e. wells of the plate) e.g. at final concentration in the range of 600pM to 50pM (if cancer cells are used as target cells, this step is not performed)
- luciferase substrate e.g. D-Luciferin, Monopotassium Salt (Promega)
- luciferase substrate e.g. D-Luciferin, Monopotassium Salt (Promega)
- step (s) Adding diluted luciferase substrate (e.g. D-Luciferin Monopotassium Salt (Promega)) from step (r), e.g. at a ratio of 1 :3 luciferase substrate to cell co-culture (e.g. by adding 50pl of luciferase substrate to the 150 pl of co-culture from step (q)); and
- diluted luciferase substrate e.g. D-Luciferin Monopotassium Salt (Promega)
- Step (t) typically requires the co-culture and D-Luciferin to be present in the wells of a white cell culture plate in order for the luminescence to be recorded, so it is preferable that cells are seeded in white cell culture plates in step (e). However, this is not essential; step (e) may alternatively comprise seeding said target cells in the wells of cell culture plates that are not white. If so, then step (q) further comprises pelleting the co-culture plates (e.g. at 300g for 5min), discarding excess cell culture supernatant, e.g. 100pl; and re-suspending pelleted cells in remaining cell culture supernatant (e.g 100pl).
- step (q) further comprises pelleting the co-culture plates (e.g. at 300g for 5min), discarding excess cell culture supernatant, e.g. 100pl; and re-suspending pelleted cells in remaining cell culture supernatant (e.g 100pl).
- step (s) an aliquot of the diluted luciferase substrate from step (r) and an aliquot of the resuspended cells would be added at a ratio of 1 :3 luciferase substrate to cell co-culture in the wells of a white cell culture plate (e.g. a white 96-well plate), e.g. 25pl per well of luciferase substrate and 75pl per well of cell co-culture, prior to the performance of the luminescence recordal step (t).
- a white cell culture plate e.g. a white 96-well plate
- the antigen binding protein may be an antibody in any T-cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T- cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA-A*02:01/PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), are capable of preferentially (or selectively) inducing activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA-A*02:01/PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto.
- a T-cell engager format e.g. in the scDb format
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- a T-cell engager format e.g. in the scDb format
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against HLA- A*02:01/PRAME 425 ' 433 positive cells as compared to against HLA-A*02:01 positive, PRAME 425 ' 433 negative cells.
- the HLA-A*02:01/PRAME 425 ' 433 displaying, or positive or negative cells referred to herein, may be any cell type including the preferred target and cancer cell types disclosed elsewhere herein.
- the T-cells may be as defined anywhere else herein, and are preferably CD4 + T cells or CD8 + T cells.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such inducement of T-cell activation, differentiation, or proliferation.
- induce means promote, i.e. enhance or increase the stated property or activity. Induce need not mean that the starting level of the property or activity is zero, i.e. it may mean “induce further”.
- the antigen binding protein is any T-cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- such induction of activation, differentiation or proliferation of T cells is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME 425 ' 433 (“target cells”) is about 1 :1.
- target cells preferably such induction of activation, differentiation or proliferation of T cells is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME 425 ' 433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of T cell activation, differentiation or proliferation.
- detection and quantification is as described below.
- ASSAY 3 Assay for induced T-cell activation/ differentiation
- an antigen binding protein e.g. antibody
- target cells can be assessed by any appropriate means, and the skilled person is familiar with suitable methods, for example coculturing the target cells (“T”), and effector cells (“E”) comprising T cells, such as PBMCs, preferably at an E:T ratio of about 1 :1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting and quantifying markers of T-cell activation or differentiation.
- Suitable T-cell activation/ differentiation markers are well-known, and include CD25 and CD71 (both markers of T-cell activation), and Granzyme B (a marker of T- cell differentiation). Markers can be detected by any suitable means, including using fluorescently-labelled antibodies against said markers, with subsequent quantification thereof, e.g. by flow cytometry techniques, which are well-known. If desired, signal specifically from T cells, or specifically from CD8 + or specifically from CD4 + T cells in particular can be assessed by also performing detection using fluorescently labelled antibodies against CD3, CD8 + and CD4 + , respectively.
- Target cells may be T2 cells pulsed with exogenously applied PRAME 425 ' 433 peptide.
- control cells are non-pulsed T2 cells, and T2 cells pulsed with an unrelated peptide.
- target cells may be HLA- A*02:01/PRAME 425 ' 433 positive cancer cells.
- control cells are HLA- A*02:01/PRAME 425 ' 433 negative cancer cells.
- T cells In assays of the ability of a T-cell engager to induce T-cell activity, proliferation, differentiation and/or cytotoxicity, it is possible to use T cells, or a subset of T cells as the effector cells (e.g. PBMCs or pan T-cells isolated from PBMCs).
- T cells or a subset of T cells as the effector cells (e.g. PBMCs or pan T-cells isolated from PBMCs).
- the apparent efficacy of an antibody can be artificially inflated through use of a high E:T ratio in the co-culturing steps.
- the E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity against the target cells.
- E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested. Therefore, it is preferred that an E:T ratio of 1 :1 is used.
- a suitable assay may comprise
- T assay plate target cells
- E effector cells
- T cells e.g. peripheral blood mononuclear cells (PBMCs)
- PBMCs peripheral blood mononuclear cells
- staining may be performed with anti-human fluorescently-labelled antibodies against one or more biomarkers of T-cell activity, e.g. CD25 (e.g. APC-CD25 (clone BC96)) or CD71 (e.g. PercpCy5.5-CD71 (clone CY1G4)); and/or (ii) staining may be performed with anti-human fluorescently-labelled antibodies against one or more biomarkers of T-cell differentiation, e.g.
- biomarkers of T-cell activity e.g. CD25 (e.g. APC-CD25 (clone BC96)) or CD71 (e.g. PercpCy5.5-CD71 (clone CY1G4)
- staining may be performed with anti-human fluorescently-labelled antibodies against one or more biomarkers of T-cell differentiation, e.g.
- Granzyme B e.g. PE-Granzyme B (clone QA18A28)
- PE-Granzyme B clone QA18A28
- T2 cells are pulsed with the relevant peptide prior to step (a), e.g. by resuspension in assay buffer containing the peptide (e.g. about 50pM of peptide) and incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide.
- assay buffer containing the peptide e.g. about 50pM of peptide
- such an assay comprises the use of peptide pulsed T2 cells or cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs as effector cells, and the assay comprises:
- T2 cells are resuspended in assay buffer (e.g. RPMI/10% FBS/1% P/S) containing peptide (e.g. about 50pM of peptide), which may be PRAME 425 ' 433 or a comparator peptide, incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide (if cancer cells are used as target cells, this step is not performed);
- assay buffer e.g. RPMI/10% FBS/1% P/S
- PBMCs Preparing PBMCs, e.g. by thawing frozen PBMCs obtained from liquid nitrogen storage (e.g. in a 37°C water bath), and transferring said PBMCs drop wise into pre-warmed assay buffer, centrifuging and resuspending the PBMCs in fresh assay buffer, counting and resuspending the PBMCs at a desired concentration;
- (k) Resuspending cells in staining buffer e.g. for about 30 minutes in the dark at about 4°C, said staining buffer containing fluorescently labelled antibodies against T cell antigens (e.g. CD3, CD4, CD8), and against markers of T-cell activation (e.g. CD25, CD71);
- T cell antigens e.g. CD3, CD4, CD8
- markers of T-cell activation e.g. CD25, CD71
- fixation buffer e.g. BD cytofix, for e.g. about 20 mins, at about 4°C in the dark (the fixation buffer preserves the light-scattering characteristics and fluorescence intensities of the anti-bound T cells);
- Permeabilizing cells by i) Centrifuging cells and discarding supernatant, and resuspending cells in a permeabilization buffer (e.g. eBiosciences Perm/Wash (1x)) e.g. for about 10 minutes at about 4°C in the dark; and ii) centrifuging cells, washing and discarding supernatant, and resuspending cells in permeabilization buffer (e.g. eBiosciences Fixation/permeabilization buffer) e.g. for about 30 minutes, at about 4°C in the dark;
- a permeabilization buffer e.g. eBiosciences Perm/Wash (1x)
- permeabilization buffer e.g. eBiosciences Fixation/permeabilization buffer
- fluorescence (of cells) is from differently labelled antibodies against different ? cell antigens and activation/differentiation markers.
- the different fluorescence signals can be distinguished using standard flow cytometry techniques, thereby permitting detection of particular activation/differentiation markers of interest, either from all T cells and/or from particular T cell types of interest.
- the antigen binding protein may be an antibody in any bispecific antibody format.
- the antibody is in the scDb format.
- ASSAY 4 Assay for T-cell proliferation
- target cells T-cell engager format
- T T-cell engager format
- target cells T-cell engager format
- T co-culturing the target cells
- E fluorescently-labelled effector cells
- T e.g. cancer cells (e.g. cancer cell line cells), or peptide pulsed T2 cells
- T co-culturing the target cells
- E fluorescently-labelled effector cells
- T cells such as CFSE-labelled PBMCs, preferably at an E:T ratio of about 1:1
- E fluorescently-labelled effector cells
- signal from specifically T cells, or CD8 + or CD4 + T cells in particular can be assessed by also performing detection using fluorescently labelled antibodies against CD3, CD8 + and CD4 + , respectively.
- a suitable assay may comprise:
- T assay plate target cells
- E effector cells comprising T cells
- a fluorescent label e.g. Carboxyfluorescein succinimidyl ester (CFSE)
- CFSE Carboxyfluorescein succinimidyl ester
- T2 cells are pulsed with the relevant peptide prior to step (a), e.g. by resuspension in assay buffer containing the peptide (e.g. about 50pM of peptide) and incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide.
- assay buffer containing the peptide e.g. about 50pM of peptide
- such an assay comprises the use of peptide pulsed T2 cells or cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs as effector cells, and the assay comprises
- T2 cells are resuspended in assay buffer (e.g. RPMI/10% FBS/1% P/S) containing peptide (e.g. about 50pM of peptide), which may be PRAME 425 ' 433 or a comparator peptide, incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide (if cancer cells are used as target cells, then this step is not performed);
- assay buffer e.g. RPMI/10% FBS/1% P/S
- PBMCs Preparing PBMCs, e.g. by thawing frozen PBMCs obtained from liquid nitrogen storage (e.g. in a 37°C water bath), and transferring said PBMCs drop wise into pre-warmed assay buffer, centrifuging and resuspending the PBMCs in fresh assay buffer;
- PBS e.g. 1ml Dulbecco's phosphate-buffered saline (DPBS)
- CFSE e.g. about 0.5pM CFSE
- assay buffer e.g. about 9ml of assay buffer
- fixation buffer e.g. BD cytofix, e.g. for about 20 mins at about 4°C in the dark (the fixation buffer preserves the light-scattering characteristics and fluorescence intensities of the antibound T cells);
- fluorescence (of cells) is from differently labelled antibodies against different T cell antigens, and from CFSE.
- the different fluorescence can be distinguished using standard flow cytometry techniques, thereby permitting detection of proliferation signal either from all T cells and/or from particular T cell types of interest.
- fluorescence from cells is from differently labelled antibodies against different T cell antigens and activation/differentiation markers.
- the different fluorescence signals can be distinguished using standard flow cytometry techniques, thereby permitting detection of proliferation either from all T cells and/or from particular T cell types of interest.
- the antigen binding protein may be an antibody in any T- cell engager format.
- the antibody is in the scDb format.
- Example 4 herein also describes that a 7-Aminoactinomycin D (7-AAD) viability staining assay was used to assess the ability of antibodies of the invention to specifically induce T-cell mediated cytotoxicity of cells displaying the HLA- A*02:01/PRAME 425 ' 433 antigen, as compared to cells displaying HLA-A*02:01 absent the PRAM E 425 ' 433 peptide complexed thereto.
- 7-AAD 7-Aminoactinomycin D
- T target cells
- E CFSE-labelled effector cells
- T cells e.g. pan-T cells isolated from PBMCs
- 5-A05 CFSE-labelled effector cells
- the assay was used to assess the capacity of 5-A05 to induce T- cell mediated cytotoxicity of HLA-A*02:01/PRAME 425 ' 433 positive and negative cancer cells ( Figure 10A and 10B).
- a 1 :1 ratio of effector cells to target cells was again used.
- 5-A05 induced T-cell mediated target cell killing only when co-cultured with HLA-A*02:01/PRAME 425 ' 433 positive cells cancer cell lines.
- No T-cell mediated cytotoxicity resulted against cells lacking HLA-A*02:01/PRAME 425 ' 433 , i.e. HLA-A*02:01/PRAME 425 ' 433 negative cancer cells.
- Cytotoxicity against HLA- A*02:01/PRAME 425 ' 433 positive tumor cell lines representing both solid tumors (10A) and liquid tumors (10B) was demonstrated.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T- cell engager format (e.g. in the scDb format), are capable of preferentially (or selectively) inducing T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- a T-cell engager format e.g. in the scDb format
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against HLA- A*02:01/PRAME 425 ' 433 positive cells as compared to against HLA-A*02:01 positive, PRAME 425 ' 433 negative cells.
- a T-cell engager format e.g. in the scDb format
- preferentially (or selectively) induce T-cell mediated cytotoxicity against HLA- A*02:01/PRAME 425 ' 433 positive cells as compared to against HLA-A*02:01 positive, PRAME 425 ' 433 negative cells.
- the HLA-A*02:01/PRAME 425 ' 433 displaying, or positive or negative cells referred to herein, may be any cell type including the preferred target and cancer cell types disclosed elsewhere herein.
- the T-cells may be as defined anywhere else herein, and are preferably CD4 + T cells or CD8 + T cells.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such inducement of T-cell mediated cytotoxicity.
- induce means promote, i.e. enhance or increase the stated property or activity. Induce need not mean that the starting level of the property or activity is zero, i.e. it may mean “induce further”.
- T-cell mediated cytotoxicity is exhibited when the antigen binding protein is an antibody in any T-cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- T-cell mediated cytotoxicity is exhibited when the ratio of T- cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME 425 ' 433 (“target cells”) is about 1 :1.
- target cells HLA-A*02:01/PRAME 425 ' 433
- T positive target cells
- E effector cells comprising T cells
- E E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of a marker of cell death.
- detection and quantification is as described below.
- ASSAY 5 Assay for T-cell mediated cytotoxicity
- an antigen binding protein e.g. antibody
- target cells can be assessed by any appropriate means and the skilled person is familiar with suitable methods, e.g. co-culturing CFSE-labelled target cells (“T”), and effector cells (“E”) comprising T cells, such as PBMCs or preferably pan-T-cells derived therefrom, preferably at an E:T ratio of about 1 :1, together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting and quantifying a marker of cytotoxicity.
- T co-culturing CFSE-labelled target cells
- E effector cells
- T cells such as PBMCs or preferably pan-T-cells derived therefrom, preferably at an E:T ratio of about 1 :1
- detection and quantification may be performed by staining for non-viable cells (e.g. with 7-amino actinomycin D (7-AAD)) and detecting said stain, e.g. via flow cytometry.
- a suitable assay may comprise:
- T CFSE-labelled target cells
- E effector cells comprising T cells
- E effector cells comprising T cells
- PBMCs PBMCs or preferably pan-T-cells derived therefrom, e.g. as described above, at an E:T ratio of about 1 :1
- the antigen binding protein e.g. antibody
- co-culturing e.g. for about 72h at about 37°C;
- T2 cells are pulsed with the relevant peptide prior to step (a), e.g. by resuspension in assay buffer containing the peptide (e.g. 50pM of peptide) and incubating e.g. for 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide.
- assay buffer containing the peptide e.g. 50pM of peptide
- such an assay comprises the use of peptide pulsed T2 cells or cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs or pan- T cells as effector cells, and the assay comprises: (a) If peptide pulsed T2 cells are used as target cells, then T2 cells are resuspended in assay buffer (e.g. RPMI/10% FBS/1% P/S) containing peptide (e.g. about 50pM of peptide), which may be PRAME 425 ' 433 or a comparator peptide, incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide (if cancer cells are used as target cells then this step is not performed);
- assay buffer e.g. RPMI/10% FBS/1% P/S
- peptide e.g. about 50pM of peptid
- PBS e.g. 1ml Dulbecco's phosphate-buffered saline (DPBS)
- CFSE e.g. about 0.5pM CFSE
- assay buffer e.g. about 9ml of assay buffer
- PBMCs are used as effector cells, preparing PBMCs, e.g. by thawing frozen PBMCs obtained from liquid nitrogen storage (e.g. in a 37°C water bath), and transferring said PBMCs drop wise into pre-warmed assay buffer, centrifuging and resuspending the PBMCs in fresh assay buffer, counting and resuspending the PBMCs at a desired concentration; or
- pan-T cells are used as effector cells, as is preferable, preparing pan-T cells from PBMCs (e.g. prepared as in step (e) wherein counting and resuspending the PBMCs at a desired concentration in not necessary), using any known protocol, e.g. using the EasySepTM Human T cell isolation kit /protocol, e.g. said kit /protocol described in the Examples herein;
- fluorescence from cells is from CFSE and 7-AAD labelled cells.
- the different fluorescence signals can be distinguished using standard flow cytometry techniques, thereby permitting detection of proliferation either from all T cells and/or from particular T cell types of interest detection of dead cell signal (via 7- AAD fluorescence) specifically from target cells (via CFSE fluorescence).
- the antigen binding protein may be an antibody in any T- cell engager antibody format, preferably a bispecific format.
- the antibody is in the scDb format.
- an antigen binding protein e.g. antibody
- Example 5 The T-cell mediated cytotoxicity assay described above (Assay 5) was performed in Example 4 (results shown in Figure 10), which allowed for the determination of the EC50 value for many antibodies of the invention disclosed in Table A, 5-A05, which was assayed over a concentration range of 0.01-1000ng/ml.
- the parameter “ECso“ is the concentration of the antigen binding protein (e.g. antibody) of the invention that is necessary to achieve half of the maximum possible effect, which in the present case is half of the observed T-cell mediated cytotoxicity of target cells.
- the exemplified 5-A05 antibody of the invention in the scDb format shows ( Figure 10A & 10B, and Example 4): an EC50 (for cytotoxicity) of 14.2 pM as measured in the cancer cell line NCI- H1703; an EC50 (for cytotoxicity) of 22.2 pM as measured in the cancer cell line A375; an EC50 (for cytotoxicity) of 13.2 pM as measured in the cancer cell line
- THP1 THP1 ; and an EC50 (for cytotoxicity) of 6.9 pM as measured in the cancer cell line SKM1.
- Example 4 further demonstrates EC50 values for many of the antibodies of the invention disclosed in Table A in the scDb format, as measured in the cancer cell line NCI-H1703, with EC50 values ranging from 3.8 pM to 32.8 pM, and most below 25 pM.
- Such cytotoxicity values in the low (single digit or low double digit) picomolar range are markedly advantageous.
- Example 4 further demonstrates EC50 values for many of the antibodies of the invention disclosed in Table A in the scDb format, as measured in the cancer cell line THP-1 , with EC50 values ranging from 6.1 pM to 17.7 pM. Such cytotoxicity values in the low (single digit or low double digit) picomolar range are markedly advantageous.
- the apparent activity/efficacy of the antigen binding protein (e.g. antibody) can be artificially inflated through use of a high E:T ratio in the step(s) of co-culturing the effector cells comprising T cells (“E”) and the target cells (“T”).
- the E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity against the target cells. E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested.
- EC50 values exhibited by the antigen binding proteins (e.g. antibodies) of the invention are exhibited in assays in which an E:T ratio of 1 :1 is used. Lower (i.e. better) EC50 values could be achieved if a higher E:T ratio was used, e.g. 10:1.
- the present inventors Using the same assay protocol in each case, i.e. in which an E:T ratio of 1 :1 was used, the present inventors have demonstrated that the antigen binding proteins (e.g. antibodies) of the invention have marked cytotoxicity, exhibiting EC50 values in the picomolar range as measured in disparate cancer cell types.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit EC50 values in the picomolar or femtomolar range as measured in cancer cells.
- antigen binding proteins (e.g. antibodies) of the present invention for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 of less than 1O' 10 M as measured in cancer cells, e.g. less than 5 x 10' 11 M, 2.5 x 10' 11 M, 10' 11 M, 10' 12 M, 10' 13 M, or 10' 14 M as measured in cancer cells.
- antigen binding proteins (e.g. antibodies) of the present invention for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 of less than 5 x 10' 11 M, 4 x 10' 11 M, 3 x 10' 11 M, 2.5 x 10’ 11 M, 2 x 10 -11 M, 1 .5 x 10' 11 or 1 x 10' 11 M, preferably less than 2.5 x 10’ 11 M, 2 x 10- 11 M, 1.5 X 10- 11 or 1 X 10’ 11 M, more preferably less than 1 x 10’ 11 M, 9 x 10' 12 , 8 x 10' 12 M, 7 x 10' 12 M, 6 x 10' 12 M, or 5 x 10' 12 M, e.g. less than 4 x 10' 12 M, e.g. less than 3 x 10' 12 M, less than 2 x 10' 12 M or less than 1 x 10' 12
- the EC50 value is as measured in HLA-A*02:01/PRAME 425 ' 433 positive cancer cells, e.g. cancer cell line cells, preferably NCI-H1703 cells, A375 cells, THP1 cells or SKMI cells, preferably NCI-H1703 cells, A375 cells, or THPI cells, more preferably NCI-H1703 cells or THPI cells, most preferably NCI-H1703 cells.
- cancer cell line cells preferably NCI-H1703 cells, A375 cells, THP1 cells or SKMI cells, preferably NCI-H1703 cells, A375 cells, or THPI cells, more preferably NCI-H1703 cells or THPI cells, most preferably NCI-H1703 cells.
- such EC50 values are as determined in an assay comprising the co-culturing of cancer cells (target cells “T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of a marker of cell death.
- detection and quantification is performed by staining for non-viable cells (e.g. with 7-amino actinomycin D (7-AAD)) and detecting said stain, e.g. via flow cytometry.
- the effector cells may be as defined elsewhere herein.
- Preferred effector cells are PBMCs, or pan-T cells derived therefrom.
- the step of co-culturing the target and effector cells is performed for about 72 hours, at about 37°C.
- the antigen binding protein may be an antibody in any T- cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- a preferred assay for determining EC50 values is set out in Assay 5 above, and in the Examples.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such EC50 values.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against HLA-A*02:01/PRAME 425 ' 433 cancer cells, preferably with the above-mentioned EC50 values.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against one or more of NCI-H1703 cells, A375 cells, and THP1 cells, preferably with the above-mentioned EC50 values.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against NCI-H1703 cells, preferably with the above-mentioned EC50 values.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against NCI-H1703 cells, and one or more of A375 cells, and THP1 cells, preferably with the above-mentioned EC50 values.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against NCI-H1703 cells, and THP1 cells, preferably with the above-mentioned EC50 values.
- such EC50 values are as determined in an assay comprising the co-culturing of cancer cells (target cells “T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of a marker of cell death.
- detection and quantification is performed by staining for non-viable cells (e.g. with 7-amino actinomycin D (7-AAD)) and detecting said stain, e.g. via flow cytometry.
- the effector cells may be as defined elsewhere herein.
- Preferred effector cells are PBMCs, or pan-T cells derived therefrom.
- the step of co-culturing the target and effector cells is performed for about 72 hours, at about 37°C.
- the antigen binding protein may be an antibody in any T- cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- a preferred assay for determining EC50 values is set out in Assay 5 above, and in the Examples.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such EC50 values.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, may have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of about 1200 nM, 1000 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300nM, 250nM, 200nM, 150nM, 100nM, 50nM, 10, 5, 4, 3, 2, or 1nM, 500pM, 400pM, 300pM, 200pM, 100pM, 50pM, 20pM, 10pM, or 5pM.
- a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of about 1200 nM, 1000 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300nM, 250nM, 200nM,
- antigen binding proteins e.g. antibodies
- a binding affinity for HLA-A*02:01/PRAME 425 ' 433 e.g. have a KD (equilibrium dissociation constant) in the range of 2000 nM or less, 1800 nM or less, 1600 nM or less, 1400 nM or less, preferably 1200 nM or less, 1000 nM or less, 800 nM or less, 600 nM or less, or 500 nM or less, or 300 nM or less for example when determined in an SPR assay.
- antigen binding proteins e.g.
- antibodies) of the invention for example when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of less than 2000 nM, less than 1800 nM, less than 1600 nM, less than 1400 nM, preferably less than 1200 nM, less than 1000 nM, less than 800 nM, less than 600 nM, less than 500nM, less than 400nM, less than 375nM, less than 350nM, or less than 300nM.
- the antigen binding proteins (e.g. antibodies) of the invention for example when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of less than 300nM, less than 250nM, 200nM, 150nM, 100nM, 50nM, 10, 5, 4, 3, 2, or 1 nM, or less than 500pM, less than 400pM, less than 300pM, 200pM, 100pM, 50pM, 20pM, 10pM, or 5pM.
- a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of less than 300nM, less than 250nM, 200nM, 150nM, 100nM, 50nM, 10, 5, 4, 3, 2, or 1 nM, or less than 500pM, less than 400pM, less than 300pM, 200pM, 100
- the present antibodies demonstrate advantageous cytotoxicity against target-positive cancer cells, despite having relatively weak target binding affinity (in the nanomolar or high picomolar range, rather than in the mid- or low picomolar range).
- this relatively weak target binding affinity is advantageous.
- antibodies that display a weaker binding affinity are actually advantageous in terms of their safety profile, since an antibody with a weaker affinity for the target is less likely to bind strongly to off-target molecules of a similar structure.
- Highly cytotoxic antibodies often bind to their targets with high affinity, but antibodies that display similar cytotoxicity with a lower affinity would be desirable given the balance of efficacy and safety
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of 1 to 1200 nM, 1 to 1000 nM, 1 to 500nM, 1 to 400nM, 1 to 375nM, or 1 to 350nM, more preferably 1 to 300nM.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of 1 to 300 nM, 1 to 250 nM, or 1 to 200 nM.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of 3 to 1200 nM, 3 to 1000 nM, 3 to 500nM, 3 to 400nM, 3 to 375nM, or 3 to 350nM, more preferably 3 to 300nM.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of 3 to 300 nM, 3 to 250 nM, or 3 to 200 nM.
- the antigen binding proteins (e.g. antibodies) of the invention when in the Fab format have a binding affinity for HLA-A*02:01/PRAME 425 ' 433 that is or corresponds to a KD of 1 to 5000 nM, e.g. 50 to 4700 nM, or 100 to 4700 nM, preferably 50 to 300 nM, or 100 to 250 nM.
- the exemplified 5-A05 antibody of the invention shows a binding affinity of 254 nM in the scDb format (Example 2)
- the 5-A05/1-C04 antibody shows a binding affinity of 155nM in the scDb format (Example 2).
- exemplified antibodies of the invention in the Fab format show the following binding affinities: 558 nM (5-A05), 4656 nM (5-A05/1-C04), 113 nM (PAM22) and 231 nM (NGS17) (Example 2).
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding affinity.
- KD binding affinity
- a Surface Plasmon Resonance assay e.g. a BIAcore assay
- kinetic parameters are determined.
- Suitable SPR assays are known in the art and for example may involve immobilising HLA-A*02:01/PRAME 425 ' 433 on a solid support and applying the antigen binding protein (e.g. antibody) to be tested. Suitable assays are discussed elsewhere herein. Particularly preferred SPR assays are described in the Examples section herein.
- HLA-A*02:01/PRAME 425 ' 433 is captured (or immobilised) on a solid support (e.g. a sensor chip) and various concentrations (e.g. a dilution series, e.g. a doubling dilution series) of the antigen binding protein (e.g. antibody) to be tested is then injected.
- a solid support e.g. a sensor chip
- concentrations and response units (Rll) are generally selected at a range and level, respectively, such that the chip is not saturated and which allow robust fitting, e.g. robust 1 :1 fitting, by the SPR/Biacore software.
- concentrations and flow- rates for injection, together with appropriate RU Units are described in the Examples section.
- Suitable association periods and dissociation periods to be used in an SPR assay are known to a skilled person, for example, a preferred association period in the SPR assay is 2 minutes and a preferred dissociation period in the SPR assay is 2 minutes (in a single cycle analysis). In certain embodiments, all measurements may be performed at 25°C in 20mM PBS, pH7.4, 2.7mM KCI, 137 mM NaCI.
- Kinetic parameters may be determined or calculated by any suitable model or software, for example by fitting the sensorgram experimental data assuming a 1 :1 interaction, in other words using a 1 :1 binding model, for example using Single Cycle Kinetics software. Particularly preferred SPR assays are described in the Examples section herein. Preferably a single cycle analysis is used.
- binding affinity (KD) of an antigen binding protein (e.g. antibody) of the invention to H LA-restricted peptide (pHLA), e.g. HLA- A*02:01/PRAME 425 ' 433 may be as determined by (or as assessed by) an SPR assay, e.g. an SPR assay that comprises:
- NeutrAvidin e.g. 10 pg/mL in 10 mM sodium acetate, pH 5.0
- a solid support e.g. a sensor chip, such as a CM3 series S sensor chip
- amine coupling e.g. to 500 response units (RU);
- pHLA e.g. soluble, recombinant, biotinylated pHLA
- a solid support e.g. to 20-100 RU
- Example 2 herein describes the assessment of the thermal stability of an antibody of the invention, 5-A05, in the scDb format.
- the melting temperature of 5-A05 measured by thermal unfolding and defined as the temperature at which 50% of the molecules are unfolded, was 66.7°C. This advantageous stability was surprising.
- the antigen binding Fab unit of the wellperforming therapeutic antibody Trastuzumab was included as control, in a format (Fab) which normally has higher thermostability due to the CH1-CL pair, yet 5-A05 (in scDb) format had a similar melting temperature.
- the antigen binding proteins (e.g. antibodies) of the invention when in an scDb format, have a melting temperature of at least 60°C, preferably at least 62°C, more preferably at least 64°C, more preferably at least 66°C, e.g. 60°C to 80°C, 62°C to 80°C, 64°C to 80°C, or 66°C to 80°C, or 60°C to 75°C, 62°C to 75°C, 64°C to 75°C, or 66°C to 75°C, more preferably 63°C to 71 °C, 64°C to 70°C, 65°C to 69°C, or 66°C to 68°C.
- a melting temperature of at least 60°C, preferably at least 62°C, more preferably at least 64°C, more preferably at least 66°C, e.g. 60°C to 80°C, 62°C to 80°C, 64°C to 80°C,
- the exemplified 5-A05 antibody of the invention shows a melting temperature of 66.7°C in the scDb format (Example 2, Figure 6) and the 5-A05 antibody shows a melting temperature of 67.9°C in the scDb format (Example 2).
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such a melting temperature.
- the melting temperature is the temperature at which 50% of the molecules are unfolded.
- the melting temperature is determined in a nano differential scanning fluorimetry (nanoDSF) assay, which is a well-known assay in the field, in which tryptophan or tyrosine fluorescence is used to monitor protein unfolding, and the ratio of the fluorescence intensities at 350 nm and 330 nm is suitable to detect any changes in protein structure due to protein unfolding.
- nanoDSF nano differential scanning fluorimetry
- the melting temperature of an antigen binding protein (e.g. antibody) of the invention to H LA-restricted peptide (pHLA), e.g. HLA- A*02:01/PRAME 425 ' 433 may be as determined by (or as assessed by) a nanoDSF method, e.g. a nanoDSF method that comprises:
- Cytotoxic T cells release cytokines such as IFN-y, which induce the increased expression of MHC class I and other molecules involved in peptide loading in cancer cells, which in turn increases the chance that cancer cells will be recognized as target cells for cytotoxic attack.
- IFN-y also activates macrophages, recruiting them to target sites both as effector cells and as antigen-presenting cells.
- the ability of an antigen binding protein, particularly at low concentrations, to induce the release of IFN-y from T-cells when co-cultured with HLA-A*02:01/PRAME 425 ' 433 positive target cells would therefore be advantageous, and demonstrative of the cytotoxic and therapeutic activity of the molecules.
- the antigen binding proteins (e.g. antibodies) of the invention when in a T- cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of interferon gamma (IFNy) from T cells directed against cells displaying H LA-A*02: 01 /PRAM E 425 ’ 433 .
- IFNy interferon gamma
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), are capable of preferentially (or selectively) inducing release of IFNy from T cells directed against cells displaying HLA-A*02:01/PRAME 425 - 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy from T cells directed against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto.
- a T-cell engager format e.g. in the scDb format
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy of T cells directed against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy from T cells directed against cells displaying HLA- A*02:01/PRAME 425 ' 433 as compared to against cells displaying HLA-A*02:01 without the PRAME 425 ' 433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME 425 ' 433 .
- a T-cell engager format e.g. in the scDb format
- the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy from T cells directed against HLA-A*02:01/PRAME 425 ' 433 positive cells as compared to against HLA- A*02:01 positive, PRAME 425 ' 433 negative cells.
- a T-cell engager format e.g. in the scDb format
- the HLA-A*02:01/PRAME 425 ' 433 displaying, or positive or negative cells referred to herein, may be any cell type including the preferred target and cancer cell types disclosed elsewhere herein.
- the T-cells may be as defined anywhere else herein, and are preferably CD4 + T cells or CD8 + T cells.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such inducement of T-cell activation, differentiation, or proliferation.
- induce means promote, i.e. enhance or increase the stated property or activity. Induce need not mean that the starting level of the property or activity is zero, i.e. it may mean “induce further”.
- the antigen binding protein is any T-cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- such induction of release of IFNy from T cells is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA- A*02:01/PRAME 425 ' 433 (“target cells”) is about 1 :1.
- target cells preferably such induction of release of IFNy from T cells is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME 425 ' 433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of IFNy.
- detection and quantification is as described below.
- ASSAY 8 Assay for induced T-cell release of IFNy
- an antigen binding protein e.g. antibody
- target cells can be assessed by any appropriate means and the skilled person is familiar with suitable methods, e.g. co-culturing target cells (“T”), and effector cells (“E”) comprising T cells, such as PBMCs, preferably at an E:T ratio of about 1 :1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting and quantifying IFNy release from PBMCs.
- T co-culturing target cells
- E effector cells
- T cells co-culturing target cells
- E effector cells
- Target cells may be T2 cells pulsed with exogenously applied PRAME 425 ' 433 peptide.
- control cells are non-pulsed T2 cells, and T2 cells pulsed with an unrelated peptide.
- target cells may be HLA- A*02:01/PRAME 425 ' 433 positive cancer cells.
- control cells are HLA- A*02:01/PRAME 425 ' 433 negative cancer cells.
- T cells or a subset of T cells as the effector cells (e.g. PBMCs or pan T-cells isolated from PBMCs).
- the apparent efficacy of an antibody can be artificially inflated through use of a high E:T ratio in the co-culturing steps.
- the E:T ratio is simply the ratio of the number of effector cells to the number of target cells.
- the use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity (e.g. IFNy release) against the target cells.
- E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested. Therefore, it is preferred that an E:T ratio of 1 :1 is used.
- Assay kits for the detection and quantification of IFN- y are well-known and widely available, for instance the ELISA Max Set from BioLegend ®, and any suitable assay or kit may be used.
- a suitable assay may comprise (a) Co-culturing in wells of an assay plate HLA-A*02:01/PRAME 425 ' 433 positive target cells (“T”), e.g. cancer cells (e.g. cancer cell line cells), as described above, and effector cells comprising T cells (“E”), e.g. PBMCs as described above, at an E:T ratio of about 1 :1 , and the antigen binding protein (e.g. antibody) to be tested, e.g. at a single concentration (e.g. about 1 nM), or over a concentration range (e.g. of about 0.1-10,000pM), and co-culturing e.g. for about 24h at about 37°C;
- such an assay comprises the use of HLA- A*02:01/PRAME 425 ' 433 positive cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs as effector cells, and the assay comprises:
- wash buffer e.g. PBS with 0.05% Tween 20
- wash buffer e.g. PBS with 0.05% Tween 20
- wash buffer e.g. PBS with 0.05% Tween 20
- step (f) Adding 10OpI of supernatant obtained in step (a) (e.g. for about 2 hrs) at room (or ambient) temperature (e.g. 25°C);
- wash buffer e.g. PBS with 0.05% Tween 20
- wash buffer e.g. PBS with 0.05% Tween 20
- wash buffer e.g. PBS with 0.05% Tween 20
- T-cell I FNy release assay 8 was performed in Example 5 (results shown in Table J), which allowed for the determination of an EC50 value for the antibodies of the invention disclosed in Table A.
- the parameter “ECso“ is the concentration of the antigen binding protein (e.g. antibody) of the invention that is necessary to achieve half of the maximum possible effect, which in the present case is half of the observed maximal amount of IFNy released from T-cells when co-cultured with HLA-A*02:01/PRAME 425 ' 433 positive target cells.
- Example 5 (Table J) demonstrates EC50 values (for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME 425 ' 433 positive cancer cells) for each of the antibodies of the invention disclosed in Table A, with EC50 values ranging from 35 pM to 202 pM, and most below 150 pM. Such values are markedly advantageous.
- the present inventors have demonstrated that the antibodies of the invention, advantageously, have marked ability to induce release of I FNy from T-cells when cocultured with HLA-A*02:01/PRAME 425 ' 433 positive target (cancer) cells.
- the apparent activity/efficacy of the antigen binding protein (e.g. antibody) can be artificially inflated through use of a high E:T ratio in the step(s) of co-culturing the effector cells comprising T cells (“E”) and the target cells (“T”).
- the E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity against the target cells. E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested.
- EC50 values for IFNy release from T-cells exhibited by the antigen binding proteins (e.g. antibodies) of the invention are exhibited in assays in which an E:T ratio of 1 :1 is used. Lower (i.e. better) EC50 values could be achieved if a higher E:T ratio was used, e.g. 10:1.
- the present inventors have demonstrated that the antigen binding proteins (e.g. antibodies) of the invention achieve marked IFNy release, exhibiting EC50 values in the picomolar range.
- the antigen binding proteins (e.g. antibodies) of the invention exhibit EC50 values for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME 425 ' 433 positive target (cancer) cells in the picomolar range.
- antigen binding proteins (e.g. antibodies) of the present invention for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME 425 ' 433 positive target (cancer) cells of less than 5 x 1O' 10 M, e.g. less than 1 x 10 10 , 5 x 10' 11 M, 2.5 x 10’ 11 M, 10’ 11 M, 10’ 12 M, 10’ 13 M, or 10’ 14 M.
- antigen binding proteins (e.g. antibodies) of the present invention for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 for IFNy release from T-cells in the presence of HLA- A*02:01/PRAME 425 ' 433 positive target (cancer) cells of less than 5 x 10' 1 ° M, less than 3 x 10' 1 ° M, less than 2.5 x 10' 1 ° M, less than 2 x 10' 1 ° M, less than 1.5 x 10' 1 ° M or less than 1 x 10' 1 ° M.
- a bispecific format e.g. the scDb format
- the EC50 value is as measured in HLA-A*02:01/PRAME 425 ' 433 positive cancer cells, e.g. cancer cell line cells, preferably THP-1 cells or NCI-H1703 cells, preferably THP-1 cells.
- cancer cell line cells e.g. THP-1 cells or NCI-H1703 cells, preferably THP-1 cells.
- such EC50 values are as determined in an assay comprising the co-culturing of cancer cells (target cells “T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably over a concentration range, with subsequent detection and quantification of IFNy.
- target cells target cells
- E effector cells comprising T cells
- E antigen binding protein
- detection and quantification is performed by ELISA.
- the effector cells may be as defined elsewhere herein.
- Preferred effector cells are PBMCs, or pan-T cells derived therefrom.
- the step of co-culturing the target and effector cells is performed for about 72 hours, at about 37°C.
- the antigen binding protein may be an antibody in any T- cell engager format, preferably a bispecific format.
- the antibody is in the scDb format.
- a preferred assay for determining said EC50 values is set out in Assay 8 above, and in the Examples.
- Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such EC50 values for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME 425 ' 433 positive target (cancer) cells.
- E effector cells
- T target cells
- E:T ratio used in such assays is no greater than about 1:1, i.e. no more than about 1 :1.
- the number of effector cells is not greater than, or is not significantly greater than, the number of target cells.
- E:T ratios lower than 1 : 1 may be used, however ratios of about 1 :1, e.g. 1 :1 are preferred. It is within the competencies of the person of ordinary skill in the art to count cell densities in cell cultures, dilute cells to a desired density and provide a suitable volume of cells to provide a given number of cells for an assay. In this way, desired E:T ratios can be straightforwardly provided.
- antigen binding protein e.g. antibody
- antigen binding protein e.g. antibody
- assays for their assessment there are disclosed parameters/ conditions with the term “about” applied to a specific value, e.g. “about 72 hours”, “about 20°C”, “about 100 ng/ml”, “about 1:1”, etc.
- the person of ordinary skill in the art will appreciate that minor modifications of parameters/conditions can be tolerated, and particular parameters/conditions can be readily selected by the skilled person for their particular purposes.
- disclosures are also disclosures of said specific value in particular, i.e. without the term “about” applied thereto.
- any substantially homologous antigen binding protein should retain the ability to bind to, or specifically bind to, the same epitope of the antigen as recognized by the antigen binding protein (e.g. antibody) in question, for example, the same epitope recognized by the CDR domains of the invention, or the antigen binding domains of the invention, or the VH and VL domains of the invention, as described herein, e.g. bind to the same epitope as an antibody of the invention shown in Table A, e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody.
- any substantially homologous antigen binding protein e.g. antibody
- any substantially homologous antigen binding protein e.g. antibody
- Binding to the same epitope can, for example be tested, e.g. by using epitope mapping assays, e.g. by analysis of the crystal structure of the antigen-antibody complex, or by mutational studies of individual residues (e.g. using alanine scanning and/or deep mutational scanning, DMS, for example yeast display in combination with DMS, see for example as described in Sierocki et al., 2021 , PLoS Negl Trop Dis.,15(3):e0009231 ; see also Van Blarcom et al., 2015, JMB, 427:6(B):1513-1534 and Medina-Cucurella and Whitehead, 2018, Methods Mol. Biol., 1764:101-121) and any of the above such analyses to determine the epitope can be used.
- epitope mapping assays e.g. by analysis of the crystal structure of the antigen-antibody complex, or by mutational studies of individual residues (e.g. using alanine scanning and/
- antibodies which bind to the same epitope as one or more of the various antibodies of the invention form yet further aspects of the invention.
- binding assays can be used to test whether "substantially homologous" antigen binding proteins (e.g. antibodies) have the same binding specificities as the antigen binding proteins of the invention, for example, binding assays such as competition assays or ELISA assays, e.g. as described elsewhere herein.
- binding assays such as competition assays or ELISA assays, e.g. as described elsewhere herein.
- Surface Plasmon Resonance (e.g. BIAcore) assays could also readily be used to establish whether "substantially homologous" antigen binding proteins can bind to the relevant antigen.
- BIAcore Surface Plasmon Resonance
- a competition binding assay can be used to test whether "substantially homologous" antigen binding proteins (e.g. antibodies) retain the ability to bind to, or specifically bind to, substantially the same epitope of the relevant antigen as recognized by the antigen binding proteins (e.g. antibodies) of the invention (e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody), or have the ability to compete with one or more of the various antigen binding proteins (e.g. antibodies) of the invention.
- the method described below is only one example of a suitable competition assay. The skilled person will be aware of other suitable methods and variations.
- An exemplary competition assay involves assessing the binding of various effective concentrations of an antigen binding protein (e.g. antibody) of the invention to the relevant antigen in the presence of varying concentrations of a test antigen binding protein (e.g. a substantially homologous antigen binding protein e.g. antibody). The amount of inhibition of binding induced by the test antigen binding protein can then be assessed.
- a test antigen binding protein that shows increased competition with an antigen binding protein of the invention at increasing concentrations i.e. increasing concentrations of the test antigen binding protein result in a corresponding reduction in the amount of antigen binding protein of the invention binding to the relevant antigen
- the test antigen binding protein significantly reduces the amount of antigen binding protein of the invention that binds to the relevant antigen.
- the test antigen binding protein reduces the amount of antigen binding protein of the invention that binds to the relevant antigen by at least about 95%.
- ELISA assays may be used for assessing inhibition of binding in such a competition assay but other suitable techniques would be well known to a person skilled in the art.
- substantially homologous antigen binding proteins e.g. antibodies
- antigen binding proteins e.g. antibodies
- substantially the same (or the same) epitope of the relevant antigen as recognized by antigen binding proteins (e.g. antibodies) of the invention e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody
- antigen binding proteins e.g. antibodies of the invention
- the various antigen binding proteins (e.g. antibodies) of the invention e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody
- antigen binding proteins e.g. antibodies which have the ability to bind (or specifically bind) to the same (or substantially the same) epitope of HLA- A*02:01/PRAME 425-433 as recognized by the antibody of the invention (e.g. 5-A05, 5- A05/1-C04, PAM22, PAM23 or NGS55 of Table A) are further embodiments of the present invention.
- antigen binding proteins e.g. antibodies
- Such antigen binding proteins e.g. antibodies
- Such antigen binding proteins which have the ability to compete with one or more of the antibodies of the invention (e.g. with 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 shown in Table A) for binding to HLA- A*02:01/PRAME 425 ' 433 are further embodiments of the present invention.
- an antigen binding protein e.g. antibody
- HLA-A*02:01/PRAME 425 ' 433 which binds to (or specifically binds to) HLA-A*02:01/PRAME 425 ' 433 and which has the ability to bind to the same (or substantially the same) epitope for binding to HLA-A*02:01/PRAME 425 - 433 as: the 5-A05 antibody (Table A), i.e. an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO:3, as described herein, or the ability to bind to the same (or substantially the same) epitope as an antibody comprising the same CDRs as the 5-A05 antibody (Table A), i.e.
- antigen binding proteins e.g. antibodies
- antigen binding proteins have the ability to compete with one or more of the antibodies of the invention (e.g. 5-A05, 5- A05/1-C04, PAM22, PAM23 or NGS55 (Table A)) for binding to HLA- A*02:01/PRAME 425 ' 433 .
- Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.
- Competing antigen binding protein refers to antigen binding proteins (e.g. antibodies) that bind to about, substantially or essentially the same, or even the same, epitope as a “reference antigen binding protein” (e.g. antibody). Competing antigen binding proteins are thus able to effectively compete with a reference antigen binding protein for binding to the relevant antigen.
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein can bind to the same epitope as the reference antigen binding protein (e.g. antibody).
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein e.g. antibody
- the competing antigen binding protein
- reference antigen binding proteins are antigen binding proteins which can bind to the relevant antigen in accordance with the invention.
- reference antigen binding proteins e.g. antibodies
- a reference antigen binding protein which binds to, or specifically binds to, HLA-A*02:01/PRAME 425 ' 433 may comprise a VL domain and a VH domain of the 5-A05 antibody (i.e. comprise a VL domain of SEQ ID NO:4 and a VH domain of SEQ ID NO:3), or of any other antibody disclosed in Table A.
- Reference antigen binding proteins are preferably antibodies.
- a preferred reference antigen binding protein may be the 5-A05 antibody as defined herein (Table A).
- competing antigen binding proteins e.g. antibodies
- reference antibodies such as the 5-A05 antibody and the other antibodies of the invention disclosed in Table A have been provided.
- competing antigen binding proteins e.g. antibodies
- a reference antigen binding protein e.g. antibody
- epitope mapping can be performed using standard techniques, if desired.
- the CDRs of the antigen binding proteins (e.g. antibodies) of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and/or effective engineered antibodies.
- appropriate framework regions such as those found in naturally occurring antibodies and/or effective engineered antibodies.
- the VH, VL and individual CDR sequences of the invention are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding, herein HLA-A*02:01/PRAME 425 ' 433 binding.
- Such framework sequences or regions may correspond to naturally occurring framework regions, FR1 , FR2, FR3 and/or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions.
- framework regions are well known and documented in the art and any of these may be used.
- Exemplary sequences for framework regions are one or more of the framework regions making up the VH, and/or VL domains of the antibodies of the invention, e.g. one or more of the framework regions of antibodies as disclosed in Table A (e.g., 5-A05, 5-A05/1- C04, PAM6, PAM8, PAM 17, PAM 18, PAM22, PAM23, NGS55, or NGS17), or framework regions substantially homologous thereto, and in particular framework regions that allow the maintenance of antigen specificity, for example framework regions that result in substantially the same or the same 3D structure of the antibody.
- Table A e.g., 5-A05, 5-A05/1- C04, PAM6, PAM8, PAM 17, PAM 18, PAM22, PAM23, NGS55, or NGS17
- framework regions substantially homologous thereto e.g., 5-A05, 5-A05/1- C04, PAM6, PAM8, PAM 17, P
- the antigen binding protein (e.g. antibody) comprises a VH domain that comprises a VH FR1 , a VH FR2, a VH FR3 and a VH FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VL domain that comprises a VL FR1, a VL FR2, a VL FR3 and a VL FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
- the antigen binding protein (e.g. antibody) comprises a VH domain that comprises a VH FR1 , a VH FR2, a VH FR3 and a VH FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
- VL domain that comprises a VL FR1, a VL FR2, a VL FR3 and a VL FR4 comprising the following amino acid sequences or sequences substantially homologous thereto: VL FR1 VL FR2 VL FR3 VL FR4 a) SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 ; or
- the antigen binding protein comprises one or more, preferably all, of the framework regions making up the VH, and/or (preferably “and”) VL domains of a specific antibody of the invention disclosed in Table A, or framework regions substantially homologous thereto.
- all four of the variable heavy chain e.g. a) SEQ ID NOs:11 , 12, 13 and 14; or b) SEQ ID NOs: 122, 123, 124 and 14
- variable light chain e.g.
- the antigen binding protein (e.g. antibody) and nucleic acid molecules of the invention are generally "isolated” or “purified” molecules insofar as they are distinguished from any such components that may be present in situ within a human or animal body or a tissue sample derived from a human or animal body.
- the sequences may, however, correspond to or be substantially homologous to sequences as found in a human or animal body.
- the term "isolated” or “purified” as used herein in reference to nucleic acid molecules or sequences and proteins or polypeptides, e.g. antigen binding proteins (e.g. antibodies) refers to such molecules when isolated from, purified from, or substantially free of their natural environment, e.g. isolated from or purified from the human or animal body (if indeed they occur naturally), or refers to such molecules when produced by a technical process, i.e. includes recombinant and synthetically produced molecules.
- the term "isolated” or “purified” typically refers to a protein substantially free of cellular material or other proteins from the source from which it is derived. In some embodiments, particularly where the protein is to be administered to humans or animals, such isolated or purified proteins are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. It can be noted that in embodiments the antigen binding proteins (e.g.
- antibodies etc., of the invention do not occur in nature and are, in that respect, manmade constructs in that they do not correspond to molecules that occur naturally.
- preferred antigen binding proteins e.g. antibodies
- preferred antigen binding proteins can be engineered or recombinantly produced.
- the antigen binding proteins (e.g. antibodies) etc, of the invention are non-native or not naturally occurring.
- proteins and polypeptides of the invention such as the heavy and light chain CDRs, the heavy and light chain variable regions (domains), antigen binding proteins, antibodies and antibody fragments, may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods.
- the antibodies of the invention are human antibodies, more preferably fully human antibodies.
- human antibodies generally have at least two potential advantages for use in human therapy. First, the human immune system should not recognize the antibody as foreign. Second, the half-life in the human circulation will be similar to naturally occurring human antibodies, allowing smaller and less frequent doses to be given.
- human as used herein in connection with antibody molecules and binding proteins first refers to antibodies and binding proteins having variable regions e.g., VH, VL, CDR or FR regions) and, optionally, constant antibody regions, isolated or derived from a human repertoire or derived from or corresponding to sequences found in humans or a human repertoire, e.g., in the human germline or somatic cells.
- variable regions e.g., VH, VL, CDR or FR regions
- constant antibody regions isolated or derived from a human repertoire or derived from or corresponding to sequences found in humans or a human repertoire, e.g., in the human germline or somatic cells.
- the "human” antibodies and binding proteins of the invention further include amino acid residues not encoded by human sequences, e.g., mutations introduced by random or site directed mutations in vitro, for example mutations introduced by in vitro cloning or PCR, for example in an affinity maturation method.
- mutations are mutations that involve conservative substitutions or other mutations in a small number of residues of the antibody or binding protein, e.g., in up to 6, 5, 4, 3, 2 or 1 of the residues of the antibody or binding protein, preferably e.g., in up to 4, 3, 2 or 1 of the residues making up one or more of the CDRs of the antibody or binding protein.
- Certain examples of such "human” antibodies include antibodies and variable regions that have been subjected to standard modification techniques to reduce the amount of potentially immunogenic sites.
- the "human” antibodies of the invention include sequences derived from and related to sequences found in humans, but which may not naturally exist within the human antibody germline repertoire in vivo.
- the human antibodies and binding proteins of the present invention include proteins comprising human consensus sequences identified from human sequences, or sequences substantially homologous to human sequences.
- human antibodies and binding proteins of the present invention are not limited to combinations of VH, VL. CDR or FR regions that are themselves found in combination in human antibody molecules.
- the human antibodies and binding proteins of the invention can include or correspond to combinations of such regions that do not necessarily exist naturally in humans (e.g. are not naturally occurring antibodies).
- the human antibodies will be fully human antibodies.
- “Fully human” antibodies are antibodies comprising "human” variable region domains and/or CDRs, as defined above, without substantial non-human antibody sequences or without any non-human antibody sequences.
- antibodies comprising human variable region domains "without substantial non-human antibody sequences” are antibodies and/or domains in which only up to 6, 5, 4, 3, 2 or 1 amino acids are amino acids that are not encoded by human antibody sequences.
- Antibodies comprising human CDRs "without substantial non- human antibody sequences” are antibodies, and/or CDRs in which only up to 4, 3, 2 or 1 amino acids are amino acids that are not encoded by human antibody sequences.
- “fully human” antibodies are distinguished from “humanized” antibodies, which are based on substantially non-human variable region domains, e.g., mouse variable region domains, in which certain amino acids have been changed to better correspond with the amino acids typically present in human antibodies.
- the "fully human” antibodies of the invention may be human variable region domains and/or CDRs without any other substantial antibody sequences, such as being single chain antibodies.
- the "fully human” antibodies of the invention may be human variable region domains and/or CDRs integral with or operatively attached to one or more human antibody constant regions.
- Certain preferred fully human antibodies are IgG antibodies with the full complement of IgG constant regions.
- “human” antibodies of the invention will be part-human chimeric antibodies.
- Part-human chimeric antibodies are antibodies comprising "human" variable region domains and/or CDRs operatively attached to, or grafted onto, a constant region of a non-human species, such as rat or mouse.
- Such part-human chimeric antibodies may be used, for example, in pre- clinical studies, wherein the constant region will preferably be of the same species of animal used in the pre-clinical testing.
- These part-human chimeric antibodies may also be used, for example, in ex vivo diagnostics, wherein the constant region of the non-human species may provide additional options for antibody detection.
- Antibodies of the present invention may also be CDR grafted antibodies.
- Such antibodies are antibodies comprising the CDR sequences (e.g. 3 VH CDRs and/or 3 VL CDRs) of an antibody of the invention grafted into a framework region that is different from the framework region with which the CDRs are associated in the VL and/or VH domains described herein.
- Preferred heavy and light chain variable region framework sequences are set forth in the sequence tables herein.
- Nucleic acid molecules e.g. one or more nucleic acid molecules
- antigen binding proteins e.g. antibodies
- immunoconjugates of the present invention as defined herein or parts or fragments thereof, or nucleic acid molecules substantially homologous thereto, form yet further aspects of the invention.
- Preferred nucleic acid molecules are those encoding an antigen binding protein (e.g. antibody) of the present invention as described elsewhere herein which can bind to HLA-A*02:01/PRAME 425 ' 433 , e.g. an antigen binding protein (e.g. antibody) of the invention with CDR and optionally FR and other regions as defined in Table A, or antibodies with sequences substantially homologous thereto.
- an antigen binding protein e.g. antibody
- an antigen binding protein e.g. antibody of the invention with CDR and optionally FR and other regions as defined in Table A, or antibodies with sequences substantially homologous thereto.
- Preferred nucleic acid molecules are those encoding a VH region of an antigen binding protein (e.g. antibody) of the present invention.
- Other preferred nucleic acid molecules are those encoding a VL region of an antigen binding protein (e.g. antibody) of the present invention.
- Other preferred nucleic acid molecules are those encoding a VH region of an antigen binding protein (e.g. antibody) of the present invention and a VL region of an antigen binding protein (e.g. antibody) of the present invention.
- preferred nucleic acid molecules are those encoding a VL region of SEQ ID NO:4 (such as SEQ ID NO:2), of SEQ ID NO: 134 (such as SEQ ID NO:133), of SEQ ID NO:136 (such as SEQ ID NO:135), of SEQ ID NO:138 (such as SEQ ID NO:137), of SEQ ID NO:139), of SEQ ID NO:147 (such as SEQ ID NO:146), or of SEQ ID NQ:150 (such as SEQ ID NO:149); and/or a VH region of SEQ ID NO:3 (such as SEQ ID NO:1), of SEQ ID NO:132 (such as SEQ ID NO:131 , 145 or 148), of SEQ ID NO:142 (such as SEQ ID NO:141), or of SEQ ID NO:144 (such as SEQ ID NO:143).
- Preferred nucleic acid molecules are those encoding an antigen binding protein (e.g. antibody) of the
- nucleic acid molecules are those having a nucleic acid sequence that is substantially homologous to the specific nucleic acid sequences defined herein, for example having at least 80% sequence identity to specific nucleic acid sequences defined herein.
- nucleic acid sequence or “nucleic acid molecule” as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof.
- the nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases.
- modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.
- the nucleic acid molecules may be double stranded or single stranded.
- the nucleic acid molecules may be wholly or partially synthetic or recombinant.
- antigen binding proteins e.g. antibodies
- polypeptides of the invention such as the light and heavy CDRs, the light and heavy chain variable regions (domains), antibodies, antibody fragments, and immunoconjugates
- the antigen binding proteins e.g. antibodies
- polypeptides of the invention such as the light and heavy CDRs, the light and heavy chain variable regions (domains), antibodies, antibody fragments, and immunoconjugates
- the light and heavy CDRs e.g. antibodies
- the light and heavy chain variable regions domains
- antibodies e.g. antibodies
- immunoconjugates may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods.
- Nucleic acid fragments encoding the light and heavy chain variable regions (domains) of the antigen binding proteins (e.g. antibodies) of the invention can be derived or produced by any appropriate method, e.g. by cloning or synthesis.
- nucleic acid fragments encoding the light and heavy chain variable regions (domains) of the antigen binding proteins (e.g. antibodies) of the invention can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region fragments into full length antigen binding protein (e.g. antibody) molecules with appropriate constant region domains, or into particular formats of antibody fragment discussed elsewhere herein, e.g. Fab fragments, scFv fragments, scDb formats, etc.
- the nucleic acid fragments encoding the antigen binding protein (e.g. antibody) molecules of the invention are generally incorporated into one or more appropriate expression vectors in order to facilitate production of the antigen binding proteins (e.g. antibodies) of the invention.
- Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used.
- the expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.
- the invention therefore contemplates a recombinant expression vector containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.
- Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes and are well known in the art. Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
- the recombinant expression vectors of the invention may also contain a selectable marker gene that facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
- the recombinant expression vectors may also contain genes that encode a fusion moiety that provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification (for example appropriate C-terminal or N-terminal "tags" to enable purification and/or identification may be present, e.g., His tags or myc tags).
- the antigen binding proteins (e.g. antibodies) of the invention do not comprise a C- terminal or N-terminal tag, e.g. a His-tag.
- Recombinant expression vectors can be introduced into host cells to produce a transformed host cell.
- transformed with is intended to encompass introduction of nucleic acid ⁇ e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sam brook et a/., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.
- Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells, as will be well known to a person skilled in the art.
- the proteins of the invention may be expressed in yeast cells or mammalian cells, for example HEK or CHO cells. Cell-free expression systems might also be used.
- promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.
- proteins of the invention may also be expressed in nonhuman transgenic animals such as, rats, rabbits, sheep and pigs.
- the proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.
- N-terminal or C-terminal fusion proteins comprising the antigen binding proteins (e.g. antibodies) of the invention conjugated to other molecules, such as proteins, may be prepared by fusing through recombinant techniques.
- the resultant fusion proteins contain an antigen binding protein (e.g. antibody) of the invention fused to the selected protein or marker protein, or tag protein as described herein.
- the antigen binding proteins (e.g. antibodies) of the invention may also be conjugated to other proteins by known techniques.
- the proteins may be coupled using heterobifunctional thiol-containing linkers as described in WO 90/10457, N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5 thioacetate.
- a yet further aspect provides an expression construct or expression vector or expression system (e.g. a viral or bacterial or other expression construct, vector or system), e.g. one or more expression constructs or expression vectors, comprising one or more of the nucleic acid fragments or segments or molecules of the invention.
- the expression constructs or vectors or systems are recombinant.
- said constructs or vectors or systems further comprise the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.
- a yet further aspect provides a host cell (e.g. a mammalian or bacterial or yeast host cell) or virus, e.g. one or more host cells or viruses, comprising one or more expression constructs or expression vectors of the invention.
- host cells e.g. a mammalian or bacterial or yeast host cell
- viruses e.g. one or more host cells or viruses, comprising one or more of the nucleic acid molecules of the invention.
- a host cell e.g. a mammalian host cell or bacterial host cell, or yeast host cell
- virus expressing an antigen binding protein (e.g. antibody) of the invention forms a yet further aspect.
- a yet further aspect of the invention provides a method of producing (or manufacturing) a protein (e.g. antibody) of the present invention comprising a step of culturing the host cells of the invention.
- Preferred methods comprise the steps of (i) culturing a host cell comprising one or more of the recombinant expression vectors or one or more of the nucleic acid sequences of the invention under conditions suitable for the expression of the encoded antigen binding protein (e.g. antibody); and optionally (ii) isolating or obtaining the antigen binding protein (e.g. antibody) from the host cell or from the growth medium/supernatant.
- Such methods of production (or manufacture) may also comprise a step of purification of the antigen binding protein (e.g. antibody) product and/or formulating the antigen binding protein (e.g. antibody) into a composition including at least one additional component, such as a pharmaceutically acceptable carrier or excipient.
- the antigen binding protein (e.g. antibody) of the invention is made up of more than one polypeptide chain (e.g. whole antibodies), then all the polypeptides are preferably expressed in the host cell, either from the same or a different expression vector, so that the complete proteins, e.g. antibody proteins of the invention, can assemble in the host cell and be isolated or purified therefrom.
- One therapeutic (or diagnostic) approach is the use of antibodies that can target specific antigens expressed on cancer cells, that are not expressed or are expressed at a lower level on normal cells.
- These target antigens of which HLA- A*02:01/PRAME 425 ' 433 is an example, can be exploited using antibodies to specifically kill antigen-bearing cancer cells by a variety of mechanisms including by delivering immuno- or radio labelled conjugates that, when delivered to the antigenbearing cell, specifically kill the target cell.
- Such targeting can also be used for diagnosis.
- the invention thus also provides a range of conjugated antigen binding proteins (e.g. antibodies), i.e. “immunoconjugates”, in which the antigen binding protein (e.g. antibody) of the invention is operatively attached to at least one other therapeutic or diagnostic agent.
- the term "immunoconjugate” is broadly used to define the operative association of the antibody (or binding protein) with another effective agent (e.g. therapeutic or diagnostic agent) and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical "conjugation”. Fusion proteins, e.g. recombinant fusion proteins, are particularly contemplated. So long as the delivery or targeting agent (the anti- HLA- A*02:01/PRAME 425 ' 433 component) is able to bind to the target and the therapeutic or diagnostic agent is sufficiently functional upon delivery, the mode of attachment will be suitable.
- the delivery or targeting agent the anti- HLA- A*02:01/PRAME 425 ' 433 component
- antibodies (or binding proteins) of the invention are part of an immunotoxin or are used (e.g. used therapeutically) as part of immunotoxins wherein the antibody is itself operatively associated or combined with a toxic agent (e.g. a chemotherapeutic agent or toxin or a radioactive material such as a radiotracer, e.g. for use in radioimmunotherapy).
- a toxic agent e.g. a chemotherapeutic agent or toxin or a radioactive material such as a radiotracer, e.g. for use in radioimmunotherapy.
- the operative attachment includes all forms of direct and indirect attachment as described herein and known in the art.
- chemotherapeutic agents or toxins are well known and described in the art, for example cytotoxic proteins derived from bacteria or plants can be used.
- the toxin should be capable of killing target cells once it has been taken up into said cells.
- preferred immunoconjugates of the invention are immunotoxins comprising an antibody of the invention linked or otherwise conjugated to a toxin (also sometimes referred to as antibody drug conjugates, ADCs).
- active ingredients such as radionuclides, toxins (for example, the diphtheria toxin), or other cytostatic agents can be bonded (conjugated) or otherwise linked to the corresponding antibodies.
- antibodies of the invention are used (e.g. used therapeutically) in their "naked" unconjugated form.
- An immunoconjugate comprising a therapeutic agent, or a carrier comprising a therapeutic agent may be used in therapy.
- An immunoconjugate comprising a diagnostic agent, or a carrier comprising a diagnostic agent may be used in in vivo and/or in vitro diagnostic methods.
- antigen binding proteins e.g. antibodies
- antigen binding proteins of the invention are used (e.g. used therapeutically) in their "naked" unconjugated form.
- compositions comprising at least a first antigen binding protein (e.g. antibody) or immunoconjugate of the invention, or at least a first nucleic acid molecule or expression vector of the invention, or at least a first host cell of the invention, constitute further aspects of the present invention.
- Formulations (compositions) comprising one or more antigen binding proteins (e.g. antibodies), etc., of the invention, optionally in admixture with a suitable diluent, carrier or excipient constitute a preferred embodiment of the present invention.
- Such formulations may be for pharmaceutical use, and thus compositions of the invention are preferably pharmaceutically acceptable or otherwise acceptable for administration to human or non-human animals, but in particular humans. Suitable diluents, excipients and carriers are known to the skilled man.
- compositions may additionally comprise further active ingredients (e.g. as described elsewhere herein) in the context of co-administration regimens or combined regimens.
- compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral (e.g. intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular), topical or rectal administration, or for mucosal delivery, and any of these modes of administration, or indeed any other appropriate mode of administration, can be used.
- compositions according to the invention are presented in a form suitable for intravenous administration.
- compositions according to the invention are presented in a form suitable for intraperitoneal (i.p.) administration.
- compositions according to the invention are presented in a form suitable for injection directly into a tumour (intratumoural).
- the active compounds e.g. the antigen binding proteins (e.g. antibodies) of the invention
- the active compounds may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, exosomes, powders, capsules or sustained release forms.
- Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms.
- Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions may then be filled into injection vials or ampoules.
- preservation agents such as p-hydroxybenzoates, or stabilizers, such as EDTA.
- EDTA stabilizers
- Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers, either with an aerosol propellant or provided with means for manual compression.
- compositions (formulations) of the present invention are preferably administered parenterally.
- Intravenous administration is preferred.
- administration is intraperitoneal (i.p.) administration.
- administration is by injection into a tumour.
- Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe.
- parenteral administration can be performed by means of an infusion pump.
- a further option is a composition which may be a powder or a liquid for the administration of the antibody in the form of a nasal or pulmonal spray.
- the antigen binding proteins (e.g. antibodies) of the invention can also be administered transdermally, e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally.
- Suitable dosage units can be determined by a person skilled in the art.
- the invention provides a method of binding HLA- A*02:01/PRAME 425 ' 433 , comprising contacting a composition comprising HLA- A*02:01/PRAME 425 ' 433 with an antigen binding protein (e.g. antibody) of the invention, or an immunoconjugate thereof.
- an antigen binding protein e.g. antibody
- the invention provides a method of detecting HLA- A*02:01/PRAME 425 ' 433 , comprising contacting a composition suspected of containing HLA-A*02:01/PRAME 425 ' 433 with an antigen binding protein (e.g. antibody) of the invention, or an immunoconjugate thereof, under conditions effective to allow the formation of HLA-A*02:01/PRAME 425 ' 433 /antigen binding protein complexes (e.g. HLA-A*02:01/PRAME 425-433 /antibody complexes) and detecting the complexes so formed.
- an antigen binding protein e.g. antibody
- the composition suspected of containing HLA- A*02:01/PRAME 425 ' 433 may have been obtained from a subject, e.g. may be a subject sample e.g. blood sample or biopsy.
- Yet further aspects are methods of diagnosis or imaging of a subject comprising the administration of an appropriate amount of an antigen binding protein (e.g. antibody) of the invention as defined herein to the subject and detecting the presence and/or amount and/or the location of the antigen binding protein (e.g. antibody) of the invention in the subject.
- an antigen binding protein e.g. antibody
- Appropriate diseases to be imaged or diagnosed in accordance with the present invention are cancers, e.g. as described elsewhere herein in connection with disease treatments.
- the invention provides a method of diagnosing cancer in a subject comprising the step of: (a) contacting a test sample taken from said subject with one or more of the antigen binding proteins (e.g. antibodies) of the invention.
- the antigen binding proteins e.g. antibodies
- the invention provides a method of diagnosing cancer in a subject comprising the steps of:
- antigen binding protein-antigen complex e.g. antibody-antigen complex
- said contacting step is carried out under conditions that permit the formation of an antigen binding protein-antigen complex (e.g. antibody-antigen complex).
- an antigen binding protein-antigen complex e.g. antibody-antigen complex
- test sample for example biopsy cells, tissues or organs suspected of being affected by disease, or histological sections.
- the subject is as defined elsewhere herein, and is preferably a human subject.
- the subject may be a subject at risk of developing cancer or at risk of the occurrence of cancer, e.g. a healthy subject or a subject not displaying any symptoms of cancer or any other appropriate “at risk” subject.
- the subject is a subject having, or suspected of having (or developing), or potentially having (or developing) cancer.
- the presence of any amount of antigen binding protein-antigen complex (e.g. antibody-antigen complex) in the test sample would be indicative of the presence of disease.
- the amount of antigen binding protein-antigen complex (e.g. antibodyantigen complex) in the test sample is greater than, preferably significantly greater than, the amount found in an appropriate control e.g. control sample. More preferably, the significantly greater levels are statistically significant, preferably with a probability value of ⁇ 0.1 , preferably ⁇ 0.05. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used.
- control controls or samples could be readily chosen by a person skilled in the art, for example, in the case of diagnosis of a particular disease, an appropriate control would be a sample from a subject that did not have that disease.
- Appropriate control "values" could also be readily determined without running a control "sample” in every test, e.g. by reference to the range for normal subjects known in the art.
- the antigen binding proteins (e.g. antibodies) of the invention may be labeled with a detectable marker such as a radioisotope, such as 3 H, 14 C, 32 P, 35 S, 123 l, 125 l, 131 1; a radioactive emitter (e.g.
- a, p or y emitters a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, luciferin or Europium; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion; or a chemical moiety such as biotin which may be detected by binding to a specific cognate detectable moiety, e.g. labelled avidin/streptavidin.
- an antigen binding protein e.g. an antibody
- detectable markers allow the presence, amount or location of antigen binding protein-antigen complexes (e.g. antibody-antigen complexes) in the test sample to be examined.
- Preferred detectable markers for in vivo use include an X-ray detectable compound, such as bismuth (III), gold (III), lanthanum (III) or lead (II); a radioactive ion, such as copper 67 , gallium 67 , gallium 68 , indium 111 , indium 113 , iodine 123 , iodine 125 , iodine 131 , mercury 197 , mercury 203 , rhenium 186 , rhenium 188 , rubidium 97 , rubidium 103 , technetium 99m or yttrium 90 ; a nuclear magnetic spin-resonance isotope, such as cobalt (II), copper (II), chromium (III), dysprosium (III), erbium (III), gadolinium (III), holmium (III), iron (II), iron (III), manganese (II), neodymium (III),
- the invention also includes diagnostic or imaging agents comprising the antigen binding proteins (e.g. antibodies) of the invention attached to a label or detectable marker that produces a detectable signal, directly or indirectly.
- diagnostic or imaging agents comprising the antigen binding proteins (e.g. antibodies) of the invention attached to a label or detectable marker that produces a detectable signal, directly or indirectly.
- Appropriate labels or detectable markers are described elsewhere herein.
- the method of diagnosing cancer is an in vitro method.
- the method of diagnosing cancer is an in vivo method.
- the present invention provides a method for screening for cancer in a subject.
- antigen binding proteins e.g. antibodies
- antigen binding proteins e.g. antibodies
- diagnostic methods of the invention are provided which further comprise a step of treating cancer by therapy, e.g. using an antigen binding protein (e.g. antibody) of the present invention.
- an antigen binding protein e.g. antibody
- an additional step of treating the cancer by therapy or surgery can be performed.
- a further aspect of the present invention provides the antigen binding proteins (e.g. antibodies) or immunoconjugates of the invention for use in therapy, in particular for use in the treatment or prevention of cancer.
- the nucleic acid molecules, expression vectors, host cells or viruses of the invention can also be used in the therapeutic methods described herein.
- treatment is meant the treatment of any medical condition.
- Such treatment may be prophylactic (i.e. preventative), curative (or treatment intended to be curative), or palliative (i.e. treatment designed merely to limit, relieve or improve the symptoms of a condition).
- the cancer is typically in a subject, i.e. a subject suffering therefrom.
- the cancer is HLA-A*02:01/PRAME 425 ' 433 positive.
- the term “cancer” encompasses cancerous cells of any type, including both tumours (e.g. solid tumours) and hematological or blood cancers.
- cancer cells may be tumour cells, or may be blood cancer cells.
- the present invention provides immunoconjugates of the invention for use in therapy, in particular for use in the treatment or prevention of cancer.
- the present invention provides antigen binding proteins (e.g. antibodies) or immunoconjugates of the invention, for use in killing cancer cells presenting/displaying HLA-A*02:01/PRAME 425 - 433 , i.e. HLA-A*02:01/PRAME 425 - 433 positive cancer cells.
- Said killing is preferably T-cell mediated killing. Such killing may also play a role in the therapeutic treatments as discussed herein.
Abstract
An antigen binding protein (e.g. antibody) comprising an antigen binding domain that binds to HLA-A*02:01/PRAME425-433, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs. Antigen binding protein-based (e.g. antibody-based) compositions, immunoconjugates, therapeutic and diagnostic methods, and kits are also provided.
Description
Antigen Binding Proteins
The present invention relates generally to the field of antigen binding proteins, in particular to antibodies which bind to, or specifically bind to, the pMHC (peptide- Major Histocompatibility Complex) HLA-A*02:01/PRAME425'433. The invention further relates to compositions and immunoconjugates comprising such antibodies and to methods of producing such antibodies. The invention also relates to methods and uses which employ such antibodies, for example in the treatment of cancer.
The treatment of cancer is still one of the biggest unmet medical needs to date. While there have been advances in cancer therapy during the last decades, cancer remains one of the leading causes of death. As populations in industrialized countries are benefitting from longer average life expectancies, the urgency for improved or new cancer therapies is increasing.
Human leukocyte antigen (HLA) class I molecules are expressed on the surface of all nucleated cells presenting peptides for T-cell recognition. The peptides presented in HLA class I molecules are protein fragments of intracellular origin, which are degraded by an array of proteases. The protein fragments are truncated to smaller peptides and translocated into the endoplasmic reticulum (ER). In the ER, the peptide-HLA class I molecule (pHLA), a type of peptide-MHC (pMHC) complex, is assembled from a peptide, a polymorphic heavy chain, and the monomorphic light chain p2-microglobulin (P2m). A peptide with adequate binding motif residues will bind into the peptide-binding groove of the HLA class I molecule, allowing the assembled molecule to leave the ER and be transported via the Golgi complex to the cell surface to display the peptides.
PRAME (PReferentially expressed Antigen in MEIanoma), also known as MAPE, DAGE, and OIP4, was originally observed as a melanoma antigen. PRAME is also an important modulator of retinoic acid signalling. PRAME expression is noted in the nucleus. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. PRAME is known to be expressed in a wide range of cancers, including solid, soft tissue, hematological and metastatic tumors. These include, but are not limited to, breast, ovarian, prostate, melanoma, pancreatic, renal, liver, and colorectal cancers, synovial sarcomas and lymphomas including Hodgkin's lymphomas, peripheral T/N cell lymphomas, anaplastic large cell lymphomas, and peripheral B cell lymphomas.
The 9-mer peptide SLLQHLIGL (SEQ ID NO: 19) corresponds to amino acids 425-433 of the full length PRAME protein. This peptide binds to HLA-A*02:01 and the peptide-HLA complex is considered a suitable target for PRAME specific T-cell receptor therapy.
Although the T-cell receptor (TCR) is the endogenous binding partner for pMHC, the use of recombinant TCRs for the purpose of detecting peptide presentation is challenging. TCRs have low affinity for pMHC, and soluble TCRs are intrinsically unstable and production is demanding. Monoclonal antibodies against pMHC targets are therefore desirable, but are difficult to generate given the small epitope of the bound peptide in the human leukocyte antigen (HLA). Given the complex structure of the epitope targets, generating monoclonal antibodies against pMHC targets with high binding specificity and acceptably low levels of off-target activity, and further with good cytotoxicity profiles at low doses, is a particular difficulty.
What are needed in the art are new, preferably improved, agents, such as antibodies, that target PRAME pMHC, which will be useful in the treatment, prophylaxis and diagnosis of cancer.
The present invention provides one such alternative and improved therapeutic option in the form of antigen binding proteins (e.g. antibodies) directed to HLA- A*02:01/PRAME425'433. The antibodies generated by the inventors have advantageous properties which make them ideal agents for the above-mentioned uses.
As will be described in more detail elsewhere herein, antigen binding proteins (e.g. antibodies) of the invention have been shown to be capable of binding to HLA- A*02:01/PRAME425-433with high specificity, and have excellent ability to induce T-cell mediated killing of cancer cells. Advantageously, the cell killing effects are observed at very low concentrations, notably in assays comprising an effector cell to target cell ratio (E:T) of only 1 :1 , and notably in disparate cancer types. Advantageously, the cell killing effects are observed whilst the antigen binding proteins (e.g. antibodies) bind with relatively low strength to target. The antigen binding proteins (e.g. antibodies) thus achieve advantageous cytotoxicity whilst maintaining an advantageous safety profile (reduced risk of off-target effects). Indeed, the antigen binding proteins (e.g. antibodies) of the invention have also been shown to have outstanding specificity, in particular in terms of their demonstrated lack of binding to/ cross-reactivity with many HLA-A*02:01 restricted peptides with high and very high sequence identity to the target peptide PRAME425'433. The antigen binding proteins (e.g. antibodies) of the invention also redirect T-cell activity preferentially against to HLA-A*02:01/PRAME425'433 positive cells, to preferentially induce activation, differentiation and proliferation of T cells directed against HLA-A*02:01/PRAME425'433 positive cells and to induce T-cell mediated cytotoxicity preferentially against HLA- A*02:01/PRAME425'433 positive cells. The antigen binding proteins (e.g. antibodies) of the invention have also been shown to have advantageous thermal stability.
To the inventors’ knowledge, no other anti-PRAME pMHC antibodies have been disclosed to have this advantageous combination of properties, and antigen binding proteins (e.g. antibodies) with one or more, preferably all, of these properties are preferred.
Such antigen binding proteins (e.g. antibodies) of the invention comprise a HLA-A*02:01/PRAME425'433 antigen binding domain as described herein, and can conveniently and advantageously be used for the treatment of diseases associated with HLA-A*02:01/PRAME425'433 expression, in particular for the treatment of cancer.
In one aspect, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ I D NO: 111 ) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or
MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113) or a sequence substantially homologous thereto; or wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto;
and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
It is the antigen binding domain that confers the binding ability of the antigen binding protein. Thus, alternatively viewed, the present invention provides an antigen binding protein comprising at least one antigen binding domain which binds to, or specifically binds to, HLA-A*02:01/ A*02:01/PRAME425'433, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said VH and VL domains are as defined above.
By “at least one” antigen binding domain means “at least a first antigen binding domain”. As discussed elsewhere herein, further (i.e. second, third) etc. antigen binding domains may be present. However, it is the “first” antigen binding domain referred to herein that binds to, or specifically binds to, HLA-A*02:01/ A*02:01/PRAME425'433.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or
MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
A1 In another aspect, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 132, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 132, or a sequence substantially homologous thereto, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
Substantially Homologous Sequences
The invention provides the specific antigen binding proteins (e.g. antibodies) disclosed in Table A. Table A provides the specific amino acid and nucleotide sequences of these antigen binding proteins (e.g. antibodies) and the regions/domains thereof, including the CDR sequences, the framework region sequences, and the VH and VL domain sequences. These sequences of the specific antibodies of the invention disclosed in Table A, i.e. those having a given SEQ ID NO., are referred to herein as “specific”, “reference”, “given” or “disclosed” sequences.
Throughout this application, reference is also made to “substantially homologous” sequences, i.e. sequences that are “substantially homologous” to a specific sequence disclosed in Table A. The invention also encompasses antigen binding proteins (e.g. antibodies) that comprise i) one or more CDRs, and/or ii) one or more framework regions, and/or iii) a VH domain and/or iv) a VL domain that have a sequence substantially homologous to a specific sequence disclosed in Table A. Substantially homologous sequences are defined by reference to a “given”,
“reference”, “disclosed” or “specific” sequence, which is a sequence disclosed in Table A, having a specific SEQ ID NO. Alternatively viewed, the substantially homologous sequences are “based on” a specific sequence of Table A.
The discussion below relating to substantially homologous sequences applies to all aspects and embodiments of the invention described elsewhere herein, wherever the terms “substantially homologous”, “or a sequence substantially homologous thereto”, or similar terms, are used.
In all aspects and embodiments, the specific antigen binding protein (e.g. antibody), which provides the “specific”, “given”, “disclosed”, or “reference” sequence(s) on which the substantially homologous sequence(s) is/are based is an antibody disclosed in Table A, i.e. the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody. Preferably, the “specific”, “given”, “disclosed” or “reference” antigen binding protein (e.g. antibody) is the 5-A05/1-C04 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody. More preferably, the “specific”, “given”, “disclosed” or “reference” antigen binding protein (e.g. antibody) is the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
Antigen binding proteins (e.g. antibodies) of the invention comprising one or more CDR sequences that are “substantially homologous” to one or more of the specific CDRs disclosed in Table A, e.g. to SEQ ID Nos: 5 to 7 (or SEQ ID NOs: 111 to 113) and/or to SEQ ID Nos: 8 to 10, and antigen binding proteins (e.g. antibodies) of the invention comprising heavy and/or light chain variable domains that comprise an amino acid sequence that is substantially homologous to a specific heavy and/or light chain variable domain disclosed in Table A, e.g. to SEQ ID NO:3 (or SEQ ID NO: 132) and/or to SEQ ID NO: 4, respectively, are termed “substantially homologous antigen binding proteins” (e.g. substantially homologous antibodies”) herein. Thus, “substantially homologous antigen binding proteins” (e.g. substantially homologous antibodies”) are any such antigen binding protein/antibody comprising one or more CDR or variable domain sequence that is substantially homologous to the sequence of a specific CDR or variable domain sequence with a given SEQ ID NO herein.
In all aspects and embodiments, antigen binding proteins, e.g. antibodies, containing substantially homologous sequences retain the ability to bind to HLA- A*02:01/PRAME425'433. Preferably, antigen binding proteins, e.g. antibodies, containing substantially homologous sequences retain one or more (preferably all) of the other properties of a specific antigen binding protein (e.g. antibody) of the
invention, i.e. an antigen binding protein (e.g. antibody) disclosed in Table A, e.g. the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody; preferably the 5-A05/1-C04 antibody, the PAM18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody, more preferably the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
The term "substantially homologous" as used herein in connection with an amino acid or nucleic acid sequence includes sequences having at least 30%, 40%, 50%, 55%. 60%, 62%, 65%, 70%, 74% or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the specific amino acid or nucleic acid sequence disclosed. Substantially homologous sequences of the invention thus include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the specific sequences of the invention.
Other preferred examples of substantially homologous sequences are sequences containing conservative amino acid substitutions of the specific amino acid sequences disclosed.
The antigen binding proteins (e.g. antibodies) of the invention preferably comprise at least one heavy chain variable domain (region) that includes an amino acid sequence region of at least 60%, 62%, 65%, 70%, 74% or 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a heavy chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g. SEQ ID NO: 3, 132, or 142 or 144, preferably SEQ ID NO: 3 or 132, more preferably SEQ ID NO: 132; and/or (preferably “and”) at least one light chain variable domain (region) that includes an amino acid sequence region of at least 60%, 62%, 65%, 70%, 74% or 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a light chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g. SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, preferably SEQ ID NO: 4, 140 or 147, more preferably SEQ ID NO: 4 or 147, most preferably SEQ ID NO: 4.
Preferably, the antigen binding proteins (e.g. antibodies) of the invention comprise at least one heavy chain variable domain that includes an amino acid sequence region of at least 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a heavy chain variable
domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g. SEQ ID NO: 132, 142 or 144, preferably SEQ ID NO: 132; and/or (preferably “and”) at least one light chain variable domain that includes an amino acid sequence region of at least 74%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% amino acid sequence identity to the amino acid sequence of a light chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), e.g. SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, preferably SEQ ID NO: 4, 140 or 147, more preferably SEQ ID NO: 4 or 147, most preferably SEQ ID NO: 4.
Preferably, sequences that are substantially homologous to a given VH domain sequence have at least 90% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 85%, more preferably at least 95% identity) to said given sequence.
Preferably, sequences that are substantially homologous to a given VH domain sequence have at least 95% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 90%, more preferably at least 95% identity) to said given sequence.
Alternatively, or in addition, at the amino acid level, preferred substantially homologous antigen binding proteins (e.g. antibodies) contain up to 27, 26 or 25, more preferably up to 15, more preferably up to 10, e.g. only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably up to 5, for example only 1 , 2, 3, 4 or 5, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids, in the VH domain and/or the VL domain as compared to the VH and/or VL domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1- C04).
Preferred substantially homologous antigen binding proteins (e.g. antibodies) of the invention contain up to 5, for example only 1 , 2, 3, 4 or 5, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids, or no altered amino acids, in the VH domain as compared to the VH domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1- C04, i.e. SEQ ID NO: 132); and/or (preferably “and”) contain up to 27, more preferably up to 15, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids,
or no altered amino acids, in the VL domain as compared to the VL domain of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO: 4).
Preferred substantially homologous antigen binding proteins (e.g. antibodies) of the invention contain i) up to 4, e.g. only 1, 2, 3 or 4, altered amino acids in the VH domain as compared to the VH domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO: 132), and/or ( preferably “and”) contain no altered amino acids in the VL domain as compared to the VL domain of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO:4); or ii) no altered amino acids in the VH domain as compared to the VH domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO:132), and/or (preferably “and”) contain up to 27, more preferably up to 15, more preferably up to 4, e.g. only 1 , 2, 3 or 4, more preferably up to 3, e.g. only 1, 2 or 3, altered amino acids in the VL domain as compared to the VL domain of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04, i.e. SEQ ID NO:4).
Preferably, the antigen binding proteins (e.g. antibodies) of the invention comprise at least one heavy chain variable domain and/or (preferably “and”) at least one light chain variable domain as defined above, and further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of the VH CDR1, VH CDR2, VH CDR3 of a specific antibody of the invention (disclosed in Table A, e.g. 5-A05/1-C04), or sequences substantially homologous thereto; and/or (preferably “and”) said light chain variable domain comprises three CDRs comprising the amino acid sequences of the VL CDR1, VL CDR2, VL CDR3 of a (preferably said) specific antibody of the invention (disclosed in Table A, e.g. 5-A05/1-C04), or sequences substantially homologous thereto.
Sequences substantially homologous to a given CDR sequence are preferably as described below.
Other preferred examples of “substantially homologous” sequences are sequences having at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 62%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, amino acid sequence identity to the amino acid sequence of one or more of the CDR regions or one or more of the FR regions disclosed in Table A. Thus, in some embodiments, a “substantially homologous” CDR sequence may be a sequence having at least 30%, at least 40%, at least 50%,
at least 55%, at least 60%, at least 62%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a given CDR sequence described herein.
In some embodiments, in antigen binding proteins (e.g. antibodies) having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the altered amino acid residues(s) are not in a CDR region. For example, in some embodiments, in antigen binding proteins (e.g. antibodies) having a VH domain that has a certain degree of sequence identity to a given VH domain sequence of a particular antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05, 5-A05/1-C04, PAM6, PAM8, PAM 17, PAM 18, PAM22, PAM23, NGS55 antibody, or NGS17; preferably 5-A05/1-C04, PAM 18, PAM22, PAM 23 or NGS55, more preferably 5-A05/1-C04, PAM22, PAM 23 or NGS55), the altered (or variant) residue(s) are not in a CDR region. Thus, in some embodiments, in antigen binding proteins (e.g. antibodies) having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the altered amino acid residues(s) are in one or more framework regions.
As is evident from elsewhere herein, in other embodiments, in antigen binding proteins (e.g. antibodies) having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the altered amino acid residues(s) may be in one or more CDR regions.
At the amino acid level preferred substantially homologous sequences contain up to 5, e.g. only 1 , 2, 3, 4 or 5, preferably up to 4, e.g. only 1 , 2, 3, or 4, preferably up to 3, e.g. 1 , 2 or 3, more preferably up to 2, e.g. 1 or 2, more preferably only 1 altered amino acids, in one or more of the framework regions and/or one or more of the CDRs making up the sequences of the invention (disclosed in Table A). Such alterations might be amino acid substitutions, e.g. conserved or non-conserved amino acid substitutions, or a mixture thereof.
Thus, a sequence substantially homologous to a given CDR sequence preferably comprises up to 4, e.g. only 1 , 2, 3, or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions) as compared to the given CDR sequence. This is particularly the case for sequences substantially homologous to a given VH CDR3, VL CDR1 , and/or VL CDR3 sequences herein (e.g. disclosed in Table A). Sequences substantially homologous to a given VH CDR1 and/or VH CDR2 sequence herein (e.g. disclosed in Table A) preferably comprise up to 2, e.g. 1 or 2,
more preferably only 1 altered amino acids (preferably substitutions) as compared to the given CDR sequence. Sequences substantially homologous to a given VL CDR2 sequence herein (e.g. disclosed in Table A) preferably comprise only 1 altered amino acid (preferably a substitution) as compared to the given CDR sequence.
In certain embodiments, if a given starting (reference) sequence is relatively short (e.g. three amino acids in length), then fewer amino acid substitutions may be present in sequences substantially homologous thereto as compared with the number of amino acid substitutions that might optionally be made in a sequence substantially homologous to a longer starting (reference) sequence. For example, in certain preferred embodiments the VL CDR2 sequence of antigen binding proteins of the invention is three amino acids in length. A sequence substantially homologous to a starting (reference) VL CDR2 sequence in accordance with the present invention, e.g. a starting (reference) VL CDR2 sequence which is three amino acid residues in length, preferably has only 1 or 2, more preferably only 1 altered amino acid in comparison with the starting (reference) sequence. A sequence substantially homologous to a starting (reference) VH CDR3 sequence in accordance with the present invention, e.g. a starting (reference) VH CDR3 sequence which is twelve amino acid residues in length, preferably has up to 4, preferably up to 3, more preferably 1 or 2 altered amino acid in comparison with the starting (reference) sequence.
Accordingly, in some embodiments the number of altered amino acids in substantially homologous sequences (e.g. in substantially homologous CDR sequences) can be tailored to the length of a given starting CDR sequence. For example, different numbers of altered amino acids can be present depending on the length of a given starting CDR sequence such as to achieve a particular % sequence identity in the CDRs, for example a sequence identity of at least 30%, 40%, 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.
Thus, in all aspects and embodiments herein:
A sequence substantially homologous to a given VH CDR1 sequence (i.e. a VH CDR1 sequence of the invention (shown in Table A) is preferably a sequence containing up to 3 (e.g. only 1 , 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
Alternatively or in addition, a sequence substantially homologous to a given VH CDR2 sequence is preferably a sequence containing up to 3 (e.g. only 1, 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
Alternatively or in addition, a sequence substantially homologous to a given VH CDR3 sequence is preferably a sequence containing up to 4 (e.g. only 1 , 2, 3 or 4), more preferably up to 3 (e.g. only 1 , 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 , amino acid substitutions as compared to the given CDR sequence.
Alternatively or in addition, a sequence substantially homologous to a given VL CDR1 sequence is preferably a sequence containing up to 4 (e.g. only 1 , 2, 3 or 4), preferably up to 3 (e.g. only 1 , 2 or 3), more preferably up to 2 (e.g. only 1 or 2), more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
Alternatively or in addition, a sequence substantially homologous to a given VL CDR2 sequence is preferably a sequence containing only 1 amino acid substitution as compared to the given CDR sequence.
Alternatively or in addition, a sequence substantially homologous to a given VL CDR3 sequence is preferably a sequence containing up to 4 (e.g. only 1 , 2, 3 or 4), preferably up to 3 (e.g. only 1 , 2 or 3), more preferably only 1 , amino acid substitutions as compared to the given CDR sequence.
Preferably, in all aspects and embodiments, a sequence substantially homologous to a given VH CDR1 or VH CDR2 sequence is a sequence containing up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given CDR sequence; a sequence substantially homologous to a given VH CDR3 sequence is a sequence containing up to 3 (e.g. only 1 , 2 or 3) amino acid substitutions as compared to the given CDR sequence; a sequence substantially homologous to a given VL CDR1 or VL CDR3 sequence is a sequence containing up to 4 (e.g. only 1, 2, 3 or 4) amino acid substitutions, preferably up to 3 (e.g. only 1 , 2 or 3) or up to 2 (e.g. only 1 or 2) amino acid substitutions, as compared to the given CDR sequence; and a sequence substantially homologous to a given VL CDR2 sequence is a sequence containing only 1 amino acid substitution as compared to the given CDR sequence.
Preferably, in all aspects and embodiments, the antigen binding protein (e.g. antibody) of the invention comprises a VH CDR1 comprising the amino acid sequence of the given VH CDR1 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 111) or a sequence substantially homologous thereto, wherein said
substantially homologous sequence comprises up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given VH CDR1 sequence; a VH CDR2 comprising the amino acid sequence of the given VH CDR2 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 112, 114 or 115), or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given VH CDR2 sequence; a VH CDR3 comprising the amino acid sequence of the given VH CDR3 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 113) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g. only 1, 2 or 3) amino acid substitutions as compared to the given VH CDR3 sequence; a VL CDR1 comprising the specific amino acid sequence of the given VL CDR1 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 8, 116, 117, 118, 119, or 120) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g. only 1 , 2, or 3), preferably up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given VL CDR1 sequence; a VL CDR2 comprising the specific amino acid sequence of the given VL CDR2 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises only 1 amino acid substitution as compared to the given VL CDR2 sequence; and a VL CDR3 comprising the specific amino acid sequence of the given VL CDR3 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 10 or 121) or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g. only 1 , 2 or 3), amino acid substitutions as compared to the given VL CDR3 sequence.
Preferably, in all aspects and embodiments, the antigen binding protein (e.g. antibody) of the invention comprises a VH CDR1 comprising the amino acid sequence of the given VH CDR1 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 111); a VH CDR2 comprising the amino acid sequence of the given VH CDR2 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 112, 114 or 115);
a VH CDR3 comprising the amino acid sequence of the given VH CDR3 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 113); a VL CDR1 comprising the specific amino acid sequence of the given VL CDR1 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 8, 116, 117, 118, 119, or 120), or a sequence substantially homologous thereto, wherein said substantially homologous sequence comprises up to 3 (e.g. only 1 , 2, or 3), preferably up to 2 (e.g. only 1 or 2) amino acid substitutions as compared to the given VL CDR1 sequence; a VL CDR2 comprising the specific amino acid sequence of the given VL CDR2 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 9); and a VL CDR3 comprising the specific amino acid sequence of the given VL CDR3 of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 10, or 121).
In a substantially homologous antigen binding protein (e.g. antibody) of the invention, each (i.e. all) of the substantially homologous sequences are substantially homologous to a given sequence derived from the same specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. 5-A05/1-C04).
In all aspects and embodiments, preferably, each sequence substantially homologous to a given CDR sequence comprises only 1 amino acid substitution as compared to the given CDR sequence.
In all aspects and embodiments, in a substantially homologous antigen binding protein (e.g. antibody) of the invention, each (i.e. all) of the substantially homologous sequences are substantially homologous to a given sequence derived from the same specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A). Preferred specific antigen binding proteins (e.g. antibodies) of the invention (disclosed in Table A) are described elsewhere herein.
In some embodiments, in an antigen binding protein (e.g. antibody) having a “substantially homologous” sequence as compared to a given sequence, or having a certain degree of sequence identity as compared to a given sequence, the three VH CDR amino acid sequences (i.e. all three VH CDR sequences taken together) and the three VL CDR amino acid sequences (i.e. all three VL CDR sequences taken together), to make up a set of six CDRs in total, are considered together to be the whole (or entire) CDR complement of the antigen binding protein I antibody, and the amino acid sequence of said whole CDR complement of said antigen binding protein/
antibody is at least 70%, preferably at least 80%, or at least 85%, or at least 90%, or at least 95% identical to the corresponding whole (or entire) CDR complement of a given starting (or reference) antigen binding protein I antibody. The starting (or reference) antigen binding protein (e.g. antibody) may have the CDR sequences of a specific antibody of the present invention shown in Table A, e.g. the 5-A05/1-C04 antibody.
Preferred substantially homologous antigen binding proteins (e.g. antibodies) contain up to 12, e.g. only 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably up to 8, e.g. only 1 , 2, 3, 4, 5, 6, 7, or 8, preferably up to 7, e.g. only 1, 2, 3, 4, 5, 6, or 7, preferably up to 6, e.g. only 1, 2, 3, 4, 5 or 6, preferably up to 5, for example only 1, 2, 3, 4 or 5, preferably up to 4, for example only 1, 2, 3 or 4, preferably up to 3, e.g. only 1, 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions), in the whole CDR complement as compared to in the whole CDR complement of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody). Preferably, the antigen binding protein (e.g. antibody) contains up to 6, e.g. only 1, 2, 3, 4, 5 or 6 altered amino acids (preferably substitutions) as compared to the whole complement of the given CDR sequences.
At the amino acid level, preferred substantially homologous antigen binding proteins (e.g. antibodies) contain up to 12, e.g. only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably up to 8, e.g. only 1, 2, 3, 4, 5, 6, 7, or 8, preferably up to 7, e.g. only 1, 2, 3, 4, 5, 6, or 7, more preferably up to 3, e.g. only 1 , 2 or 3, altered amino acids, more preferably no altered amino acids, in the combined CDRs (i.e. the three CDR regions) of the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); and/or (preferably “and”), contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, preferably up to 4, e.g. only 1 , 2, 3 or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, altered amino acids (preferably substitutions), more preferably no altered amino acids, in the combined CDRs (i.e. the three CDR regions) of the VL domain as compared to those of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody).
At the amino acid level, preferred substantially homologous antigen binding proteins (e.g. antibodies) contain i) up to 12, e.g. only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, preferably up to 8, e.g. only 1 , 2, 3, 4, 5, 6, 7, or 8, preferably up to 7, e.g. only 1, 2, 3, 4, 5, 6, or 7
altered amino acids in the combined CDRs (i.e. the three CDR regions) of the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); and/or (preferably “and”), contain no altered amino acids in the combined CDRs (i.e. the three CDR regions) of the VL domain as compared to those of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); or i) no altered amino acids in the combined CDRs (i.e. the three CDR regions) of the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody); and/or (preferably “and”), contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, preferably up to 4, e.g. only 1 , 2, 3 or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, altered amino acids (preferably substitutions), in the combined CDRs (i.e. the three CDR regions) of the VL domain as compared to those of a (preferably said) specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. the 5-A05/1-C04 antibody).
In addition, at the amino acid level, preferred substantially homologous sequences contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, for example 1 , 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids, in the combined framework regions (e.g. the four framework regions), and/or the combined CDRs (e.g. the three CDR regions) making up the VL or VH domains of the antigen binding proteins (e.g. antibodies) of the invention. In addition, at the amino acid level, preferred substantially homologous sequences contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, for example 1 , 2, 3, 4 or 5, preferably 1 , 2, 3 or 4, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids, in the VH domain and/or the VL domain of the antigen binding proteins (e.g. antibodies) of the invention.
At the amino acid level, preferred substantially homologous antigen binding proteins (e.g. antibodies) contain up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions), more preferably no altered amino acids, in the combined framework regions (e.g. the four framework regions), and/or the combined CDRs (e.g. the three CDR regions) making up the VH domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A); and/or (preferably “and”), contain up to 6, e.g. only 1, 2, 3, 4, 5 or 6, preferably up to 5, for example only 1, 2, 3, 4 or 5, preferably up to 4, e.g. only 1, 2, 3 or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably up to 2, e.g. only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions), more preferably
no altered amino acids, in the combined framework regions (e.g. the four framework regions), and/or the combined CDRs (e.g. the three CDR regions) making up the VL domain as compared to those of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A).
Optionally, the antigen binding protein (e.g. antibody) comprises a VH CDR1 , a VH CDR2, and a VH CDR3 comprising, respectively, the amino acid sequence of the given VH CDR1 , VH CDR2, and VH CDR3 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), i.e. rather than sequences substantially homologous thereto; and/or the antigen binding protein (e.g. antibody) comprises a VL CDR1 , a VL CDR2, and a VL CDR3 comprising, respectively, the amino acid sequence of the given VL CDR1 , VL CDR2, and VL CDR3 of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A), i.e. rather than sequences substantially homologous thereto. In other words, optionally the variant amino acids lie only in either the VH CDRs, or only in the VL CDRs.
In some embodiments, the antigen binding proteins (e.g. antibodies) of the invention comprise at least one heavy chain variable domain that includes an amino acid sequence region of at least 95% and most preferably at least 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of a heavy chain variable domain of a specific antigen binding protein (e.g. antibody) of the invention (disclosed in Table A, e.g. SEQ ID NO: 132), further wherein said heavy chain variable domain comprises a VH CDR1 , a VH CDR2, and a VH CDR3 comprising the amino acid sequences of the specific VH CDR1 , VH CDR2, and VH CDR3 of said specific antigen binding protein (e.g. antibody) of the invention (shown in Table A, e.g. SEQ ID NOs: 111 , 112 and 113, respectively), or sequences substantially homologous thereto, and/or (preferably “and”) at least one light chain variable domain that includes an amino acid sequence region of at least 74%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% amino acid sequence identity to the amino acid sequence of a specific light chain variable domain of said specific antigen binding protein (e.g. antibody) of the invention (shown in Table A, e.g. SEQ ID NO: 4), further wherein said light chain variable domain comprises a VL CDR1 , a VL CDR2, and a VL CDR3, preferably comprising the amino acid sequences of the specific VL CDR1 , VL
CDR2, and VL CDR3 of said specific antigen binding protein (e.g. antibody) of the invention (shown in Table A, e.g. SEQ ID NOs: 8, 9 and 10, respectively), or sequences substantially homologous thereto, wherein said sequences substantially homologous to a given CDR sequence are as defined anywhere else herein.
In all embodiments, said alterations can be with conservative or nonconservative amino acids. Preferably said alterations are conservative amino acid substitutions.
Altered residues might be conserved or non-conserved amino acid substitutions, or a mixture thereof. In such embodiments, preferred alterations are conservative amino acid substitutions.
A "conservative amino acid substitution", as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). In other examples, families of amino acid residues can be grouped based on hydrophobic side groups or hydrophilic side groups.
Routine methods in the art such as alanine scanning mutagenesis and/or analysis of crystal structure of the antigen-antibody complex can be used in order to determine which amino acid residues of the CDRs do not contribute or do not contribute significantly to antigen binding and therefore are good candidates for alteration or substitution in the embodiments of the invention involving substantially homologous sequences.
Once identified, the addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent antigen binding protein (e.g. antibody) to form a new antigen binding protein (e.g. antibody), wherein said parent antigen binding protein (e.g. antibody) is one of the antigen binding proteins (e.g. antibodies) of the invention as defined elsewhere herein (e.g. disclosed in Table A), and testing the resulting new antigen binding protein (e.g. antibody) to identify antigen binding proteins (e.g. antibodies) that bind to HLA-A*02:01/PRAME425'433 in accordance with the invention can be carried out using techniques which are routine in the art. Such methods can be used to form multiple new antigen binding proteins
(e.g. antibodies) that can all be tested for their ability to bind HLA- A*02:01/PRAME425'433. Preferably said addition, deletion, substitution or insertion of one or more amino acids takes place in one or more of the CDR domains.
For example, said manipulations could conveniently be carried out by genetic engineering at the nucleic acid level wherein nucleic acid molecules encoding appropriate binding proteins and domains thereof are modified such that the amino acid sequence of the resulting expressed protein is in turn modified in the appropriate way. Testing the ability of one or more of the modified antigen binding proteins (e.g. antibodies) to bind to HLA-A*02:01/PRAME425'433 can be carried out by any appropriate method, which are well known and described in the art. Suitable methods are also described elsewhere herein and in the Examples section.
New antigen binding proteins (e.g. antibodies) produced, obtained or obtainable by these methods form a yet further aspect of the invention.
The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the proteins or nucleic acid molecules of the invention in substantially the same way. For example, any substantially homologous antigen binding protein (e.g. antibody) should retain the ability to bind to the antigen (pMHC antigen) as described herein. Preferably, any substantially homologous antigen binding protein (e.g. antibody) should retain one or more (or all) of the functional capabilities of the starting antigen binding protein (e.g. antibody).
Substantially homologous sequences of proteins of the invention include, without limitation, conservative amino acid substitutions, or for example alterations that do not affect the VH, VL or CDR domains of the antigen binding proteins (e.g. antibodies), e.g. antigen binding proteins (e.g. antibodies) where tag sequences, toxins or other components are added that do not contribute to the binding of antigen, or alterations to convert one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g. conversion from Fab to scFv or whole antibody or vice versa), or the conversion of an antibody molecule to a particular class or subclass of antibody molecule (e.g. the conversion of an antibody molecule to IgG or a subclass thereof, e.g. lgG2).
Homology or sequence identity may be assessed by any convenient method. However, for determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used together with BLOSLIM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1 , so that the highest
order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Bio!., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.
Generally, computer programs will be employed for such calculations. Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).
By way of providing a reference point, sequences according to the present invention having at least 30%, 40%, 50%, 55%, 60%, 62%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, Montpellier, France).
Further examples of substantially homologous amino acid sequences in accordance with the present invention are described elsewhere herein.
In all aspects and embodiments, the antigen binding proteins (e.g. antibodies) of the invention preferably comprise the specific sequences disclosed, and not sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity
determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GYTFTGYY (SEQ ID NO: 5), or GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto,
(b) a VH CDR 2 that comprises the amino acid sequence of
INPNSGGT (SEQ ID NO:6), IIPIFGTA (SEQ ID NO: 112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDRWELLGSSDDY (SEQ ID NO:7), or ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto,
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or
QQANSFPFT (SEQ ID NO:121),
or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto,
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto,
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or QQANSFPFT (SEQ ID NO:121), or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto;
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10), or or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto;
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112),
LVVGRA (SEQ ID NO: 114), or
MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), or
QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or
or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto;
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112), or a sequence substantially homologous thereto; and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto, preferably
QGVSNW (SEQ ID NO:8), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or
QQANSFPFT (SEQ ID NO:121), or a sequence substantially homologous thereto, preferably
QQYNRLPLT (SEQ ID NO: 10), or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto;
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112),
LVVGRA (SEQ ID NO: 114), or
MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, preferably
LVVGRA (SEQ ID NO: 114), or
MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or (preferably “and”) wherein
said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), or a sequence substantially homologous thereto;
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH CDR1 VH CDR2 VH CDR3 a) GYTFTGYY INPNSGGT ARDRWELLGSSDDY ; or
(SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) b) GGTFSSYA IIPIFGTA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) c) GGTFSSYA LVVGRA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 114) (SEQ ID NO: 113) d) GGTFSSYA MQVGPTRHPLWA ARDGYNYGQFDY
(SEQ ID NO: 111) (SEQ ID NO: 115) (SEQ ID NO: 113) preferably b) to d) above, and/or (preferably “and”) a light chain variable domain (VL domain) that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT ;
(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) or b) DWISGW AAS QQYNRLPLT ;
(SEQ ID NO: 116) (SEQ ID NO: 9) (SEQ ID NO: 10) or
c) AFISSW AAS QQYNRLPLT ;
(SEQ ID NO: 117) (SEQ ID NO: 9) (SEQ ID NO: 10) or d) EFISQW AAS QQYNRLPLT ;
(SEQ ID NO: 118) (SEQ ID NO: 9) (SEQ ID NO: 10) or e) EFVSSW AAS QQYNRLPLT ;
(SEQ ID NO: 119) (SEQ ID NO: 9) (SEQ ID NO: 10) or f) QGISSW AAS QQYNRLPLT
(SEQ ID NO: 120) (SEQ ID NO: 9) (SEQ ID NO: 10) or g) QGISSW AAS QQANSFPFT
(SEQ ID NO: 120) (SEQ ID NO: 9) (SEQ ID NO: 121)
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH CDR1 VH CDR2 VH CDR3 a) GGTFSSYA IIPIFGTA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) b) GGTFSSYA LVVGRA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 114) (SEQ ID NO: 113) c) GGTFSSYA MQVGPTRHPLWA ARDGYNYGQFDY
(SEQ ID NO: 111) (SEQ ID NO: 115) (SEQ ID NO: 113) and/or (preferably “and”) a light chain variable domain (VL domain) that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT ; (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) or b) EFVSSW AAS QQYNRLPLT ;
(SEQ ID NO: 119) (SEQ ID NO: 9) (SEQ ID NO: 10) or c) QGISSW AAS QQYNRLPLT
(SEQ ID NO: 120) (SEQ ID NO: 9) (SEQ ID NO: 10)
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds
to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH CDR1 VH CDR2 VH CDR3 a) GGTFSSYA IIPIFGTA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) b) GGTFSSYA LVVGRA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 114) (SEQ ID NO: 113) c) GGTFSSYA MQVGPTRHPLWA ARDGYNYGQFDY
(SEQ ID NO: 111) (SEQ ID NO: 115) (SEQ ID NO: 113) and/or (preferably “and”) a light chain variable domain (VL domain) that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT ;
(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) or b) QGISSW AAS QQYNRLPLT
(SEQ ID NO: 120) (SEQ ID NO: 9) (SEQ ID NO: 10)
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH CDR1 VH CDR2 VH CDR3 a) GGTFSSYA IIPIFGTA ARDGYNYGQFDY (SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) and/or (preferably “and”) a light chain variable domain (VL domain) that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT ; (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) or b) EFVSSW AAS QQYNRLPLT ; (SEQ ID NO: 119) (SEQ ID NO: 9) (SEQ ID NO: 10) or c) QGISSW AAS QQYNRLPLT (SEQ ID NO: 120) (SEQ ID NO: 9) (SEQ ID NO: 10) preferably a) or c), above.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises a VH CDR1 , a VH CDR2 and a VH CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH CDR1 VH CDR2 VH CDR3 a) GGTFSSYA IIPIFGTA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) b) GGTFSSYA LVVGRA ARDGYNYGQFDY ; or
(SEQ ID NO: 111) (SEQ ID NO: 114) (SEQ ID NO: 113) c) GGTFSSYA MQVGPTRHPLWA ARDGYNYGQFDY
(SEQ ID NO: 111) (SEQ ID NO: 115) (SEQ ID NO: 113) preferably b) or c) above, and/or (preferably “and”) a light chain variable domain (VL domain) that comprises a VL CDR1 , a VL CDR2 and a VL CDR3 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL CDR1 VL CDR2 VL CDR3 a) QGVSNW AAS QQYNRLPLT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity
determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 that comprise, respectively, the amino acid sequences of the VH CDR1 , VH CDR2 and VH CDR3 of a specific antibody of the invention (disclosed in Table A), or sequences substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises a variable light (VL) CDR1 , a VL CDR2 and a VL CDR3 that comprise, respectively, the amino acid sequences of the VL CDR1 , VL CDR2 and VL CDR3 of a (preferably said) specific antibody of the invention (disclosed in Table A), or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of the VH domain of a specific antibody of the invention (disclosed in Table A) or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of the VL domain of a (preferably said) specific antibody of the invention (disclosed in Table A).
In all aspects and embodiments, the specific antibody of the invention (disclosed in Table A) may be the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody; preferably the 5-A05/1-C04 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody; more preferably, the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
Alternatively viewed, in another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of a specific antibody of the invention (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said specific antibody, or sequences substantially homologous thereto; and/or (preferably “and “)
ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said specific antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the 5-A05 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the 5- A05/1-C04 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the PAM6 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the PAM8 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the PAM 17 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or
(preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the PAM 18 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the PAM22 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the PAM23 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the NGS55 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein (e.g. antibody) that binds to, or specifically binds to, HLA- A*02:01/PRAME425'433, and comprises i) the three VH CDR sequences of the NGS17 antibody (disclosed in Table A) and/or (preferably “and”) the three VL CDR sequences of said antibody, or sequences substantially homologous thereto; and/or (preferably “and “) ii) the VH domain sequence and/or (preferably “and”) the VL domain sequence) of said antibody, or sequences substantially homologous thereto.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2 or 3 amino acid substitutions compared to the given CDR sequence.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114) or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequences is separately a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3) amino acid substitutions compared to the given CDR sequence.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence,
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3, 4 or 5 amino acid substitutions compared to the given CDR sequence; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 amino acid substitution compared to the given CDR sequence, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially
homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence,
(b) a VH CDR2 that comprises: the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence; the amino acid sequence of LVVGRA (SEQ ID NO: 114) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence, or the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1, 2, or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence, and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2, 3 or 4, (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 amino acid substitution compared to the given CDR sequence, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence,
(b) a VH CDR2 that comprises: the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence; the amino acid sequence of LVVGRA (SEQ ID NO: 114) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence; or the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2 or 3, more preferably 1 or 2) amino acid substitutions compared to the given CDR sequence; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; wherein said substantially homologous sequence
is a sequence containing 1 , 2, 3 or 4 (preferably 1 , 2, or 3) amino acid substitutions compared to the given CDR sequence, and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO: 117), EFISQW (SEQ ID NO: 118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NO: 120), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 amino acid substitution compared to the given CDR sequence, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10) or QQANSFPFT (SEQ ID NO: 121), a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 (preferably 1 or 2) amino acid substitutions compared to the given CDR sequence.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, wherein said heavy chain variable domain comprises: (a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5),
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6), and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7); and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8),
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5),
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6), and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7); and wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8),
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111),
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114) or MQVGPTRHPLWA (SEQ ID NO: 115), and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113); and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8),
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises three complementarity determining regions (CDRs), and a light chain variable domain that comprises three CDRs, said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111),
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113); and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NO: 120),
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10).
A2 In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
A3 In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of EFVSSW (SEQ ID NO: 119) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
A4 In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of LVVGRA (SEQ ID NO:114) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein
said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
A5 In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO:115) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
A6 In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113) or a sequence substantially homologous thereto; and/or (preferably “and”) wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGISSW (SEQ ID NQ:120) or a sequence substantially homologous thereto,
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto, and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
It is expressly disclosed that in each of the aspects/embodiments described in paragraphs A1 , A2, A3, A4, A5, and A6 above, the substantially homologous sequences may be as described anywhere else herein, and are preferably as follows: a sequence substantially homologous to a given VH CDR1 sequence (i.e. a VH CDR1 is preferably a sequence containing up to 2 (e.g. only 1 or 2), preferably only 1 amino acid substitutions as compared to the given CDR sequence; a sequence substantially homologous to a given VH CDR2 sequence is preferably a sequence containing up to 2 (e.g. only 1 or 2), preferably only 1 amino acid substitutions as compared to the given CDR sequence;
a sequence substantially homologous to a given VH CDR3 sequence is preferably a sequence containing up to 3 (e.g. only 1 , 2, or 3), preferably up to 2 (e.g. only 1 or 2), preferably only 1 amino acid substitutions as compared to the given CDR sequence; a sequence substantially homologous to a given VL CDR1 sequence is preferably a sequence containing up to 2 (e.g. only 1 or 2), preferably only 1 amino acid substitutions as compared to the given CDR sequence; a sequence substantially homologous to a given VL CDR2 sequence is preferably a sequence containing only 1 amino acid substitution as compared to the given CDR sequence; and a sequence substantially homologous to a given VL CDR3 sequence is preferably a sequence containing up to 3 (e.g. only 1 , 2 or 3), more preferably only 1 or 2, more preferably only 1 amino acid substitutions as compared to the given CDR sequence.
Preferably, each of the sequences substantially homologous to a given CDR sequence comprises only 1 amino acid substitution as compared to the given CDR sequence.
Preferably, the antigen binding protein comprises the given VL CDR2 sequence (i.e. rather than a sequence substantially homologous thereto).
Optionally, the heavy chain variable domain comprises the given CDR sequences (i.e. rather than sequences substantially homologous thereto), and/or the light chain variable domain comprises the given CDR sequences (i.e. rather than sequences substantially homologous thereto).
Preferably, the antigen binding protein (e.g. antibody) contains up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, preferably up to 5, for example only 1 , 2, 3, 4 or 5, preferably up to 4, for example only 1 , 2, 3 or 4, preferably up to 3, e.g. only 1 , 2 or 3, more preferably only 1 or 2, more preferably only 1 altered amino acids (preferably substitutions) as compared to the whole complement of the given CDR sequences.
CDR sequences of certain antibodies of the invention are set forth herein in Table A. In some other embodiments, CDR sequences of antibodies of the invention may be CDR sequences in the VH domains and VL domains of antibodies of the invention as identified using any suitable method (or tool), for example as identified using the well-known ImMunoGeneTics information system method, i.e. the IMGT numbering scheme (e.g. Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imqt.org), e.g. as shown in Table A, or as identified according to the well-known methods of Kabat (e.g. Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD,
647-669, 1991) or Chothia (e.g. Chothia C, et al. (1989) Nature, 342:877-883, or Al- Lazikani et al., (1997) JMB 273,927-948), or by AbM numbering (e.g. Abhinandan and Martin, 2008, Mol. Immunol. 45:3832-3839).
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3, 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 140, or 147, or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, or 147, or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain and/or (preferably “and”) a VL domain comprising the following amino acid sequences or sequences substantially homologous thereto:
VH domain VL domain i) SEQ ID NO:3 SEQ ID NO:4 ; or ii) SEQ ID NO:132 SEQ ID NO:4 ; or iii) SEQ ID NO:132 SEQ ID NO:134 ; or iv) SEQ ID NO: 132 SEQ ID NO:136 ; or v) SEQ ID NO: 132 SEQ ID NO:138 ; or vi) SEQ ID NO: 132 SEQ ID NQ:140 ; or vii) SEQ ID NO:142 SEQ ID NO:4 ; or viii) SEQ ID NO:144 SEQ ID NO:4 ; or ix) SEQ ID NO: 132 SEQ ID NO:147 ; or x) SEQ ID NO: 132 SEQ ID NQ:150
Preferably the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising: i) a VH domain comprising SEQ ID NO: 132 or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150, or a sequence substantially homologous thereto; or ii) a VH domain comprising SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain and/or (preferably “and”) a VL domain comprising the following amino acid sequences or sequences substantially homologous thereto:
VH domain VL domain i) SEQ ID NO: 132 SEQ ID NO:4 ; or ii) SEQ ID NO:132 SEQ ID NQ:140 ; or iii) SEQ ID NO:142 SEQ ID NO:4 ; or iv) SEQ ID NO:144 SEQ ID NO:4 ; or
v) SEQ ID NO:132 SEQ ID NO:147
Preferably the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising: i) a VH domain comprising SEQ ID NO: 132 or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, 140, or 147, or a sequence substantially homologous thereto; or ii) a VH domain comprising SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain and/or (preferably “and”) a VL domain comprising the following amino acid sequences or sequences substantially homologous thereto:
VH domain VL domain i) SEQ ID NO: 132 SEQ ID NO: 4 ; or ii) SEQ ID NO:142 SEQ ID NO:4 ; or iii) SEQ ID NO:144 SEQ ID NO:4 ; or iv) SEQ ID NO: 132 SEQ ID NO:147
Preferably the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising: i) a VH domain comprising SEQ ID NO: 132 or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4 or 147, or a sequence substantially homologous thereto; or ii) a VH domain comprising SEQ ID NO: 132, 142 or 144, or a sequence substantially homologous thereto and/or (preferably “and”) a VL domain comprising SEQ ID NO: 4, or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds
to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 140 or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 142 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 144 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto.
In another aspect and in certain embodiments, the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132 or a sequence substantially homologous thereto, and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 147 or a sequence substantially homologous thereto.
Preferably, as described elsewhere herein, sequences that are substantially homologous to a given VH domain sequence have at least 90% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to
a given VL domain sequence have at least 74% identity (preferably at least 85%, more preferably at least 95% identity) to said given sequence.
Preferably, as described elsewhere herein, sequences that are substantially homologous to a given VH domain sequence have at least 95% identity to said given sequence; and/or (preferably “and”) sequences that are substantially homologous to a given VL domain sequence have at least 74% identity (preferably at least 90%, more preferably at least 95% identity) to said given sequence.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 78%, 80%, 85%, 90%, 95% or 98%) and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 85%, 90%, 95% or 98%).
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:132, 142 or 144 (preferably SEQ ID NO: 132), or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 80%, 85%, 90%, 95% or 98%) and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO:4, 134, 136, 138, 140, 147 or 150 (preferably SEQ ID NO: 4), or a sequence having at least 74% sequence identity thereto (e.g. at least 75%, 80%, 85%, 90%, 95% or 98%).
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:132, 142 or 144 (preferably SEQ ID NO: 132), or a sequence having at least 95% sequence identity thereto and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO:4, 140, or 147 (preferably SEQ ID NO: 4), or a sequence having at least 85% sequence identity thereto (e.g. at least 95%).
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs, preferably comprising the amino acid sequences of SEQ ID NO:5, 6 and 7, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs, preferably comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:132, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically
binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO: 119, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 142, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 114 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 144, or a sequence having at least 80% sequence
identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 115 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 132, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113, or sequences substantially homologous thereto, as defined elsewhere herein; and/or (preferably “and”) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 147, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NQ:120, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:5, 6 and 7; and/or (preferably “and”)
a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:132, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113; and/or (preferably “and”) i) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10; or ii) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ I D NO: 119, 9 and 10; or iii) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 147, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO: 120, 9 and 10.
In another aspect and in certain embodiments, the present invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising i) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:132, or a sequence having at least 80% sequence
identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 112 and 113; or ii) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 142, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 114 and 113; or iii) a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 144, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said heavy chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:111 , 115 and 113; and/or (preferably “and”) i) a light chain variable domain that comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), further wherein said light chain variable domain comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10.
In alternative embodiments of the invention, sequences indicated as having sequence identity to a sequence with a given SEQ ID NO: can have at least 60%, 65%, 70% or 75% identity to the sequence with the given SEQ ID NO.
In another aspect and in certain embodiments the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 3 and/or (preferably “and”) a VL domain that comprises the amino acid sequence of SEQ ID NO: 4.
In another aspect and in certain embodiments the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144, preferably 142 or 144, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4.
In another aspect and in certain embodiments the invention provides an antigen binding protein, for example an antibody, which binds to, or specifically binds
to, HLA-A*02:01/PRAME425'433, said antigen binding protein comprising at least one antigen binding domain, said antigen binding domain comprising a VH domain that comprises the amino acid sequence of SEQ ID NO: 132, and a VL domain that comprises the amino acid sequence of SEQ ID NO: 4, 140 or 147, preferably SEQ ID NO: 4 or 147.
A preferred antigen binding protein of the invention is or comprises an antibody defined herein (Table A) selected from the group consisting of the 5-A05 antibody, the 5-A05/1-C04 antibody, the PAM6 antibody, the PAM8 antibody, the PAM 17 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM23 antibody, the NGS55 antibody, or the NGS17 antibody.
A preferred antigen binding protein of the invention is or comprises an antibody defined herein (in Table A) selected from the group consisting of the 5- A05/1-C04 antibody, the PAM 18 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody.
A particularly preferred antigen binding protein of the invention is or comprises the 5-A05/1-C04 antibody, the PAM22 antibody, the PAM 23 antibody or the NGS55 antibody defined herein (in Table A).
An antigen binding protein according to any aspect of the present invention and disclosure may be defined as a binding protein comprising an antigen-binding domain obtained or derived from an antibody, or based on an antigen binding domain of an antibody. Thus, for example, light and heavy chain variable domains (i.e. light and heavy chain variable regions) as described herein are those obtained or derived from an antibody, or based on an antigen binding domain of an antibody.
As described above, the present invention provides antigen binding proteins, for example antibodies, or antigen binding proteins comprising antibodies or the antigen binding domain of an antibody, which bind to (or specifically recognise or specifically bind to) HLA-A*02:01/PRAME425'433. Preferred antigen binding proteins of the invention are antibodies. However, embodiments as described herein which relate to antibodies, apply equally, mutatis mutandis, to other types of antigen binding proteins, or vice versa. Thus, other antigen binding proteins can comprise the antibodies of the invention or can comprise the antigen binding domains of the antibodies of the invention, e.g. the three VL CDR regions and the three VH CDR regions of the antibodies of the invention, or a VL and VH domain of the antibodies of the invention (a domain typically comprising the three VH or the three VL CDR regions (CDR1 , CDR2 and CDR3) and the four VH or the four VL framework (“FR”) regions (FR1 , FR2, FR3 and FR4)).
Appropriate types of antigen binding protein which could be used in the invention are known in the art. For example, in some embodiments immunoglobulin based polypeptides are used, which generally comprise CDR regions (and optionally FR regions or an immunoglobulin based scaffold), such that the CDR regions (and optionally FR regions) of the antibodies of the invention can be grafted onto an appropriate scaffold or framework, e.g. an immunoglobulin scaffold. Alternatively, the antigen binding domains or the antibodies of the invention can be incorporated into any appropriate antigen binding fragment or antibody containing format, e.g. can be incorporated into a chimeric antigen receptor (CAR) format or a CAR-T cell format.
For the avoidance of doubt, in accordance with the present invention the antigen binding domain that binds to (or specifically binds to) HLA- A*02:01/PRAME425-433 is not from, or does not correspond to, a T-cell receptor (TCR). Thus, for the avoidance of doubt, antigen binding proteins in accordance with the present invention do not include TCRs as an antigen binding domain specific for HLA-A*02:01/PRAME425'433. Thus, the antigen binding proteins of the present invention are not TCRs, i.e. do not comprise a T-cell receptor a-chain, and/or preferably (“and”) do not comprise a T-cell receptor p-chain.
Preferably, the antigen binding protein (i.e. the protein having an antigen binding domain) is an antibody or an antigen binding fragment thereof (an antibody fragment). The term “antibody” is used as shorthand to refer to “antibody or an antigen binding fragment thereof” unless otherwise clear from context.
The terms "antibody" and "immunoglobulin", as used herein, refer broadly to any immunological binding agent that comprises an antigen binding domain (e.g. a human antigen binding domain), including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM and the antibodies of the invention may be in any one of these classes. Several of these are further divided into subclasses or isotypes, such as lgG1 , lgG2, lgG3, lgG4, and the like. The heavy-chain constant domains that correspond to the difference classes of immunoglobulins are termed a, 5, s, y and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types: kappa (K) and lambda ( ), based on the amino acid sequences of their constant domains and some amino acids in the framework regions of their variable domains.
The term "heavy chain complementarity determining region" ("heavy chain CDR") as used herein refers to regions of hypervariability within the heavy chain
variable region (VH domain) of an antibody molecule. The heavy chain variable region (domain) has three CDRs termed heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus. The heavy chain variable region (domain) also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.
The term "heavy chain variable region" (VH domain) as used herein refers to the variable region of a heavy chain of an antibody molecule.
The term "light chain complementarity determining region" ("light chain CDR") as used herein refers to regions of hypervariability within the light chain variable region (VL domain) of an antibody molecule. The light chain variable region (domain) has three CDRs termed light chain CDR1 , light chain CDR2 and light chain CDR3 from the amino terminus to the carboxy terminus. The light chain variable region (domain) also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.
The term "light chain variable region" ( L domain) as used herein refers to the variable region of a light chain of an antibody molecule.
As will be understood by those in the art, the immunological binding reagents encompassed by the term "antibody" includes or extends to all antibodies, antigen binding fragments thereof and antibody “formats”, including whole antibodies, dimeric, trimeric and multimeric antibodies; bispecific antibodies; trispecific antibodies; multispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof. The terms “antibody format” and “antibody construct” are used interchangeably herein. In the field, and herein, the term “antibody format” encompasses both antibody fragments, whole antibodies and multimeric antibodies.
The term "antibody fragment" as used herein refers to fragments of biological relevance, e.g. fragments that comprise the above mentioned antigen binding domain, i.e. contribute to antigen binding, e.g. form part of the antigen binding domain. Certain preferred fragments comprise a heavy chain variable region (VH domain) and a light chain variable region (VL domain) of the antibodies of the invention.
Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies,
diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art.
The term "antibody" is thus used to refer to any antibody-like molecule that has an immunoglobulin (Ig) antigen binding domain, and this term includes antibody fragments and formats that comprise an antigen binding domain including but not limited to Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, scFv/FcKIH, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively, including scFv/Fab-Fc, and scFv/Fab-FcKIH); sc-diabody; single chain bispecific diabody (scDb); kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-lg (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (destabilized diabody "Dual Affinity ReTargeting"), DART-Fc; Duabody, CrossMab, DuetMab, DNL, small antibody mimetics.
In embodiments, the antigen binding protein (e.g. antibody) of the present invention may be in a single chain format, e.g. a single chain antibody format. In other embodiments, the antigen binding protein (e.g. antibody) of the present invention may be in a multichain format.
Antibody formats, and their preparation, is within the general competencies of the person of ordinary skill in the antibody field.
In certain embodiments, the antigen binding protein (e.g. antibody) of the present invention comprises all or a portion of a heavy chain constant region, such as an lgG1, lgG2, lgG3, lgG4, lgA1 , lgA2, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region or a portion thereof. I gG 1 and lgG4 are examples of appropriate formats for the antibodies of the invention. Antibodies of the invention may comprise or contain human heavy-chain constant regions.
Furthermore, the antigen binding protein (e.g. antibody) of the invention can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof. Antigen binding proteins (e.g. antibodies) of the invention may comprise or contain human light-chain constant regions.
All or part of such constant regions may be produced naturally or may be wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art. In embodiments, full length heavy chain sequences of the antigen binding proteins (e.g. antibodies) of the invention comprise
an amino acid sequence comprising or consisting of SEQ ID NOs: 109, 153, 164 or 153, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%)).
In embodiments, full length light chain sequences of the antigen binding proteins (e.g. antibodies) of the invention comprise an amino acid sequence comprising or consisting of SEQ ID NOs: 110 or 171 , or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%)).
Preferably, the CDR, VH and/or VL sequences in said full heavy and light chain sequences are as defined anywhere else herein.
Thus, for instance in embodiments, the antigen binding proteins (e.g. antibodies) of the invention may comprise: a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 109, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), and in which said heavy chain comprises a heavy chain variable domain that comprises three CDRs comprising the amino acid sequences of SEQ ID NO:5, 6 and 7, or sequences substantially homologous thereto, as defined elsewhere herein; and/or a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 110, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%), and in which said light chain comprises a light chain variable domain that comprises three CDRs comprising the amino acid sequences of SEQ ID NO:8, 9 and 10, or sequences substantially homologous thereto, as defined elsewhere herein.
When a full complement of constant regions from the heavy and light chains are included in the antigen binding proteins (e.g. antibodies) of the invention, such antigen binding proteins (e.g. antibodies) are typically referred to herein as "full length" antibodies or "whole" antibodies. In some embodiments such full length or whole antibodies are provided.
The antigen binding proteins (e.g. antibodies) can be produced naturally or can be wholly or partially synthetically produced.
The antigen binding protein (e.g. antibodies) may be from any appropriate source, for example recombinant sources and/or produced in transgenic animals or transgenic plants, or in eggs using the IgY technology. Thus, the antigen binding protein (e.g. antibody) molecules can be produced in vitro or in vivo.
The antigen binding domains of the antigen binding proteins (e.g. antibodies) of the invention generally comprise an antibody light chain variable region (domain)
(VL) that comprises three CDR domains and an antibody heavy chain variable region (domain) (VH) that comprises three CDR domains.
However, it is well documented in the art that the presence of three CDRs from the light chain variable domain and three CDRs from the heavy chain variable domain of an antigen binding protein (e.g. antibody) is not always necessary for antigen binding. Thus, constructs smaller than the above classical antigen binding domain are known to be effective.
For example, camelid VHH antibodies and other single domain antibodies comprising VH domains alone show that these domains can bind to antigen with acceptably high affinities. Thus, three CDRs (or even a single CDR) can effectively bind antigen and form an antigen binding domain.
Thus, although preferred antigen binding domains in the antigen binding proteins (e.g. antibodies) of the invention might comprise six CDR regions (three from a light chain and three from a heavy chain), antigen binding proteins (e.g. antibodies) with antigen binding domains with fewer than six CDR regions (e.g. 3 CDR regions) are encompassed by the invention. Antigen binding proteins (e.g. antibodies) with antigen binding domains with CDRs from only the heavy chain or light chain are also contemplated.
Preferred light chain CDR regions for use in conjunction with the specified heavy chain CDR regions to form the antigen binding domain are described elsewhere herein. However, other light chain variable regions (domains) that comprise three CDRs for use in conjunction with the heavy chain variable regions (domains) of the invention are also contemplated. Appropriate light chain variable regions (domains) which can be used in combination with the heavy chain variable regions (domains) of the invention and which give rise to an antibody which binds to HLA-A*02:01/PRAME425'433 in accordance with the invention can be readily identified by a person skilled in the art.
For example, a heavy chain variable region (domain) of the invention can be combined with a single light chain variable region (domain) or a repertoire of light chain variable regions (domains) and the resulting antigen binding proteins (e.g. antibodies) tested for binding to HLA-A*02:01/PRAME425'433.
If desired, similar methods could be used to identify alternative heavy chain variable regions (domains) for use in combination with preferred light chain variable regions (domains) of the invention.
The techniques for preparing and using various antigen binding protein-based constructs (e.g. antibody-based) constructs and fragments are well known in the art.
Diabodies, in particular, are further described in EP404097 and WO 93/11161 ; whereas linear antibodies are further described in the art.
For convenience, an antigen binding protein (e.g. antibody) of the invention that binds to HLA-A*02:01/PRAME425'433, may be referred to elsewhere herein simply as a PRAME pMHC antigen binding protein, or a PRAME antigen binding protein, e.g. a PRAME pMHC antibody, or a PRAME antibody.
As described above, the present invention provides antigen binding proteins (e.g. antibodies), for example isolated antigen binding proteins (e.g. isolated antibodies), which bind to (or specifically recognise or specifically bind to) HLA- A*02:01/PRAME425'433. This is the “target” antigen of the antigen binding proteins (e.g. antibodies) of the invention. The antigen binding proteins (e.g. antibodies) of the invention are described as having a “specificity” for this target antigen. Due to their binding to (or specifically recognising or specifically binding to) a H LA-restricted peptide, the antigen binding proteins (e.g. antibodies) of the invention are termed “TCR-like” antigen binding proteins (e.g. TCR-like antibodies).
For the avoidance of doubt, the antigen binding proteins, e.g. antibodies, of the invention comprise at least one antigen binding domain that binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, i.e. it is this antigen binding domain that confers onto the antigen binding proteins of the invention the ability to bind to (or specifically recognise or specifically bind to) this antigen. The antigen binding proteins (e.g. antibodies) of the invention thus comprise a “first” antigen binding domain, with a “first” binding specificity, also termed “specificity A”, which is for HLA- A*02:01/PRAME425'433. The presence of an antigen binding domain described herein as a “first” antigen binding domain does not imply that the presence of one or more further antigen binding domains is essential; such further antigen binding domains are optional.
Within an antigen binding domain, the heavy chain variable domain and the light chain variable domain can also each be described as having a “specificity” for the antigen to which the antigen binding domain binds. Thus, the antigen binding proteins, e.g. antibodies, of the invention, comprise at least one antigen binding domain that comprises a heavy chain variable domain with specificity for HLA- A*02:01/PRAME425'433, and a light chain variable domain with specificity for HLA- A*02:01/PRAME425'433. As above, in the present disclosure, such specificity for HLA- A*02:01/PRAME425'433 is termed the “first” specificity, or “specificity A”.
HLA-A*02 (also termed “HLA-A2”, “HLA-A02”, and “HLA-A*2”) is a human class I major histocompatibility complex (MHC) allele group at the HLA-A locus. HLA-A*02 is a human leukocyte antigen serotype within the HLA-A serotype group.
The HLA-A*02 protein (encoded by the respective HLA gene) constitutes the a chain of the respective class I MHC (major histocompatibility complex) protein, which further comprises a p2 microglobulin subunit, also termed the “P chain”, which is encoded by the p2 microglobulin (P2M) locus.
A specific HLA-A2 protein is HLA-A*02:01 (also referred to as HLA-A02:01 , HLA-A0201 , or HLA-A*02.01), which is a common MHC class I allele in the HLA- A*02 group. In the present invention, the HLA-A2 protein described is HLA-A*02:01 (I PD Accession: HLA00005 and HLA00006).
The amino acid sequence of HLA-A*02:01 encoded at these loci is set forth herein in SEQ ID NO: 21. This protein comprises a N-terminal leader peptide, an extracellular domain (ECD), a transmembrane domain and a C-terminal intracellular domain. In the pHLA complexes to which the antigen binding proteins (e.g. antibodies) of the invention bind, it is the extracellular domain of the HLA that is bound, the amino acid sequence of which is set forth in SEQ ID NO: 22. Recombinant forms of the protein typically comprise only the extracellular domain.
The antigen binding proteins (e.g. antibodies) of the invention may bind to functionally equivalent pHLA-A2 complexes in which PRAME425'433 is displayed, and in which the HLA-A2 comprises an amino acid sequence substantially homologous to SEQ ID NO: 21.
The amino acid sequence of human p2 microglobulin is set forth herein in SEQ ID NO: 23.
HLA-A*02:01 can present peptides that are fragments of intracellular proteins, including PRAME-derived peptides, i.e. peptides derived from PRAME proteins.
“PRAME” stands for Preferentially Expressed Antigen in Melanoma. The PRAME protein is a Cancer Testis Antigen (CTA).
“PRAME” as used herein, refers to any native PRAME from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PRAME as well as any form of PRAME that results from processing in the cell. The term also encompasses naturally occurring variants of PRAME, e.g., splice variants or allelic variants.
Preferably, PRAME is human PRAME, which is described in UniProt (www.uniprot.org) accession no. P78395. An amino acid sequence of human PRAME is set forth in SEQ ID NO: 20 herein. Recombinant human PRAME is commercially available.
The antigen binding proteins (e.g. antibodies) of the present invention bind to (or specifically bind to) the HLA-A*02:01 restricted PRAME derived peptide
PRAME425'433. By “PRAME425'433” is meant the PRAME derived peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 19; found at positions 425-433 of the PRAME protein of SEQ ID NO: 20).
Thus, HLA-A*02:01/PRAME425'433 refers to a complex of PRAME425'433 presented I displayed on the class I MHC molecule comprising HLA-A*02:01 , i.e. HLA-A*02:01 restricted PRAME425'433. In other words, HLA-A*02:01/PRAME425'433 means a HLA-A*02:01 molecule that is presenting (or “loaded” with) the PRAME425' 433 peptide. Put another way, HLA-A*02:01/PRAME425'433 means a HLA-A*02:01 - peptide complex (pMHC) in which the PRAME425'433 epitope is presented in the antigen binding groove (or accommodated in the antigen binding groove) of the MHC.
Preferred and convenient forms of HLA-A*02:01/PRAME425'433to which the antigen binding proteins (e.g. antibodies) of the invention can bind may comprise recombinant PRAME425'433, e.g. a recombinant human PRAME425'433, or a native or natural form of PRAME425'433, for example PRAME425'433 when presented via HLA- A*02:01 on the cell surface, e.g. on the surface of a cancer cell.
The antigen binding proteins (e.g. antibodies) of the invention do not bind to (or do not cross-react with), e.g. do not significantly bind to (or do not significantly cross-react with) the HLA-A*02:01 molecule itself/ alone (i.e. the MHC molecule itself), i.e. in the absence of a presented PRAME425'433 peptide, i.e. without the PRAME425'433 peptide presented or complexed thereto, i.e. to an unloaded HLA- A*02:01 molecule.
The antigen binding proteins (e.g. antibodies) of the invention do not bind to (or do not cross-react with), e.g. do not significantly bind to (or do not significantly cross-react with) the soluble form of PRAME425'433, i.e. they do not bind (or do not significantly bind) to PRAME425'433 unless it is presented by a HLA-A*02:01 complex, i.e. unless it is HLA-A*02:01 restricted.
Thus, the antigen binding proteins (e.g. antibodies) of the invention typically bind to, or specifically bind to, the PRAME425'433 epitope (or peptide), solely (or strictly) in the context of the MHC, i.e. HLA-A*02:01.
A convenient and appropriate method for assessing binding would include in vitro binding assays such as ELISA assays to assess binding of antigen binding proteins (e.g. antibodies) to immobilised antigen, such as immobilised forms of HLA- A*02:01/PRAME425'433 as described elsewhere herein.
Thus, in certain embodiments, antigen binding proteins (e.g. antibodies) of the present invention can bind to HLA-A*02:01/PRAME425'433 in an ELISA assay.
The skilled person will be familiar with ELISA assays and readily able to establish suitable conditions to assess the ability of an antibody to bind to HLA- A*02:01/PRAME425'433 in such an assay. Suitable assays are discussed elsewhere herein. Particularly preferred ELISA assays are described in the Examples section herein
In certain embodiments, antigen binding proteins (e.g. antibodies) of the present invention bind to HLA-A*02:01/PRAME425'433 in (as determined in) a Surface Plasmon Resonance (SPR) assay (e.g. a BIACore assay, e.g. using a BIAcore S200 instrument). Suitable SPR assays are known in the art and are discussed elsewhere herein. Particularly preferred SPR assays are described in the Examples section herein.
Thus, in some embodiments, antibodies (or binding proteins) of the invention are able to bind to HLA-A*02:01/PRAME425'433 in an SPR assay, or in an ELISA assay, preferably both.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding.
In preferred embodiments, an antigen binding protein (e.g. antibody) of the invention binds to, or specifically binds to, the HLA-A*02:01/PRAME425'433 complex when said complex is present on cells, e.g. cancer cells.
In other words, the antigen binding proteins (e.g. antibodies) of the invention are capable of binding to HLA-A*02:01/PRAME425'433 positive cells, i.e. HLA-A*02:01 positive, PRAME425'433 positive cells. Such cells are preferred “target cells” herein; target cells being cells displaying an antigen to which the antibodies bind, or specifically bind.
Preferred target cells are cancer cells, which includes tumour cells and blood cancer cells. Cancer cells may be any HLA-A*02:01/PRAME425'433 positive cancer cell, i.e. any cancer cell on the surface of which is displayed HLA- A*02:01/PRAME425'433. In certain embodiments, the cancer cells are selected from the group consisting of lung cancer cells (e.g. NCI-H1703 cells), melanoma cells (e.g. A-375 cells), and monocytes (e.g. THP-1 cells). Preferably the cancer cells are cancer cells in or from a subject suffering from cancer. In certain embodiments, the cancer is lung cancer, skin cancer or leukaemia. Cancer cells may be cancer cell line cells, which may be used in the assays described herein.
Alternatively, the cells may be T2 cells, which are well-known and widely used in the field. T2 cells can be induced (“pulsed”) to display MHC class I molecules complexed with exogenously administered peptides. T2 cells are deficient in a
peptide transporter involved in endogenous antigen processing (TAP) and therefore have markedly reduced ability to display MHC class I molecules complexed with endogenous peptides. Thus the antigen binding proteins (e.g. antibodies) of the invention are capable of binding to T2 cells induced to display exogenously administered PRAME425'433 in a HLA-A*02:01 restricted manner.
Methods of assessing binding to HLA-A*02:01/PRAME425'433 on or in cells (e.g. cancer cells or peptide pulsed T2 cells) would be well-known to a person skilled in the art and any appropriate method can be used.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding.
As described above, the antigen binding proteins (e.g. antibodies) of the invention comprise a first antigen binding domain which binds to (or specifically binds to) HLA-A*02:01/PRAME425'433.
The antigen binding proteins (e.g. antibodies) of the invention may comprise further antigen binding domains. The number of antigen binding domains possessed by an antigen binding protein (e.g. antibody) determines its valency. Thus, in certain embodiments, the antigen binding proteins (e.g. antibodies) of the invention comprise a second antigen binding domain (i.e. are bivalent), and optionally a third antigen binding domain (i.e. are trivalent), and optionally further antigen binding domains (i.e. are multivalent).
In certain embodiments some or all of the further antigen binding domains may also bind to (or specifically bind to) HLA-A*02:01/PRAME425'433. In these embodiments, any further antigen binding domain(s) may incorporate, singly or in combination, any of the features described herein in relation to the first antigen binding domain.
However, in preferred embodiments, some or all of the further antigen binding domains bind to (or specifically bind to) an antigen other than HLA- A*02:01/PRAME425'433. Thus, the antigen binding proteins (e.g. antibodies) of the invention may be bispecific, trispecific or multispecific. The term “-specific” or "- specificity" in the context of bispecific, trispecific, multispecific, etc. as used herein refers to the ability of a domain (or antigen binding domain) to recognize a particular target antigen and refers to the capability of binding that target antigen. Bispecific antibodies are therefore capable of recognizing and binding to (or specifically binding to) two different target antigens.
Thus, in addition to a first antigen binding domain that binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, bispecific antigen binding proteins (e.g.
antibodies) of the invention comprise a “second antigen binding domain” having a “second antigen binding specificity” (or simply a “second specificity” or “specificity B”) which is for an antigen other than HLA-A*02:01/PRAME425'433. Trispecific antigen binding proteins (e.g. antibodies) of the invention comprise a “third antigen binding domain” having a “third antigen binding specificity” (or simply a “third specificity”, or “specificity C”), which is for an antigen other than HLA-A*02:01/PRAME425'433 and which is different from the specificity of the second antigen binding domain (i.e. the second antigen binding specificity). Multispecific antigen binding proteins (e.g. antibodies) comprise four or more antigen binding domains each of which have a different antigen binding specificity.
The bivalent, trivalent, multivalent, bispecific, trispecific and multispecific antigen binding proteins (e.g. antibodies) of the invention may comprise antigen binding domains fused to each other, optionally via peptide linkers. The production of bivalent, trivalent, multivalent, bispecific, trispecific and multispecific antigen binding proteins (e.g. antibodies), the selection of (e.g. length and sequence of) peptide linkers, and the orientation of domains and peptide linkers, are well-known in the field, and would be within the competencies of the person of ordinary skill in the art.
For instance, in a bispecific antigen binding protein (e.g. antibody) of the invention, the first and second antigen binding domains may be fused to each other, optionally via a peptide linker. The first and the second antigen binding domain may be fused in any orientation, for instance (i) the C-terminus of the first antigen binding domain may be fused, optionally via a peptide linker, to the N-terminus of the second antigen binding domain, or (ii) the C-terminus of the second antigen binding domain may be fused, optionally via a peptide linker, to the N-terminus of the first antigen binding domain.
In certain antibody formats, particularly single chain bispecific antibody formats (e.g. BiTE and scDb formats), the variable light (VL) and variable heavy (VH) domains from the first (A) and second (B) antigen binding domains may be fused (optionally via peptide linkers) in any sequence/ orientation (i.e. order), provided that upon proper folding of the resulting polypeptide, two functional antigen binding domains are formed. Again, the production of such antigen binding proteins (e.g. antibodies), the selection of (e.g. length and sequence of) peptide linkers, and the orientation of domains and peptide linkers, are well-known in the field, and would be within the competencies of the person of ordinary skill in the art.
Preferred orientations are, in the N to C direction, i) VLA-VHB-VLB-VHA, ii) VLB-VHA-VLA-VHB; iii) VHA-VLB-VHB-VLA and iv) VHB-VLA-VHA-VLB, most
preferred are i) VLA-VHB-VLB-VHA, and ii) VLB-VHA-VLA-VHB, particularly i) VLA- VHB-VLB-VHA.
In a preferred embodiment, the antigen binding protein (e.g. antibody) is at least bispecific, e.g. is a bispecific antibody. In other words, the antigen binding protein (e.g. antibody) of the invention preferably comprises a second antigen binding domain with a second specificity, i.e. which binds to, or specifically binds to, a second antigen, i.e. to an antigen other than HLA-A*02:01/PRAME425'433.
The (at least) bispecific antigen binding protein (e.g. antibody) of the present invention may be of any antibody fragment, format or construct described herein, including single chain formats and multichain formats.
Preferred antigen binding proteins (e.g. antibodies) of the invention are in a T- cell engager format, i.e. are T-cell engagers. This is a well-understood term in the art, which refers to antigen binding proteins (e.g. antibodies) that bind to (or specifically bind to) HLA-A*02:01/PRAME425'433 and to an antigen present on T cells (T lymphocytes) via different antigen binding domains. Such T-cell engagers (TCE) are also termed T-cell engaging antibodies (or antigen binding proteins). TCEs physically recruit T cells to target cells (e.g. cancer cells) by binding simultaneously via separate antigen binding domains to both the target cell antigen HLA- A*02:01/PRAME425'433 and to a T-cell surface antigen (referred to herein as simply a T-cell antigen). A T-cell surface antigen is an antigen present on the surface of T cells. A T-cell specific antigen is an antigen present in or on T-cells specifically. This dual binding and recruitment of T cells to target cells leads to T-cell activation, proliferation and T-cell mediated target cell killing. T-cell engagers may be trispecific or multispecific, but they are at least bispecific, as described above.
Thus, in a preferred embodiment the antigen binding protein (e,g. antibody) of the invention further comprises a second antigen binding domain that binds to (or specifically binds to) a T-cell surface antigen.
The T-cell surface antigen is preferably a T-cell specific antigen, particularly CD3, most particularly CD3E. Alternatively the second antigen binding domain may be specific for another T-cell surface antigen, e.g. the T-cell receptor (i.e. a polypeptide comprised in the T-cell receptor). Preferably the T-cell surface antigen, e.g. CD3, is a human T-cell antigen, e.g. human CD3.
Such (at least) bispecific antibodies trigger T-cell activation in a target specific manner, i.e. they redirect T cell activity (e.g. T-cell mediated lysis) against target cells (e.g. cancer cells and tumours) displaying the HLA-A*02:01/PRAME425'433 antigen.
Antibodies against suitable T-cell surface antigens are numerous and widely available, for instance OKT3 and LICHT1. The second antigen binding domain which
is specific for a T-cell surface antigen may be the antigen binding domain of any such suitable antibody against a T-cell surface antigen, preferably CD3. The skilled person will readily be able to identify suitable antibodies and binding domains for their purposes.
A preferred anti-CD3 antibody in this regard is LICHT1, the heavy chain and light chain variable domain amino acid sequences of which are well-known (SEQ ID NOs: 24 and 25 herein, respectively). These VH and VL domain sequences represent preferred VH and VL domains of the second antigen binding domain of the (at least) bispecific antigen binding proteins (e.g. antibodies) of the present invention.
Thus, in embodiments, the antigen binding proteins (e.g. antibodies) of the invention preferably comprise a second antigen binding domain that binds to (or specifically binds to) CD3, said antigen binding domain comprising a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:24, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto), and/or (preferably “and”) a light chain variable region that comprises the amino acid sequence of SEQ ID NO:25, or a sequence substantially homologous thereto (e.g. having at least 80% sequence identity thereto). The meaning of “substantially homologous” is as defined elsewhere herein.
Thus, antigen binding proteins (e.g. antibodies) of the invention that are in the T-cell engager format (i.e. that comprise a second antigen binding domain that is specific for a T-cell surface antigen) redirect T cells displaying said T-cell surface antigen to the site of the target antigen HLA-A*02:01/PRAME425'433, e.g. to cancer cells or tumours, and induce activation, differentiation, proliferation of T-cells directed to said target antigen, and thus T-cell mediated cytotoxicity against said target cells.
The T cells redirected in this manner are termed “effector T cells” herein, and may be CD8+ T cells, CD4+ T cells, CD4+CD8+ T cells, regulatory T cells, gammadelta T cells, memory T cells, natural killer T cells (NKTs) or any combination thereof.
The T cells may be comprised within an effector cell population, which may also comprise other effector cells. “Effector cell” is a term well-known in the art to mean any immune cell that can effect or enhance an immune response. Preferred effector cells are peripheral blood mononuclear cells (PBMCs), and pan-T cells derived therefrom. PBMCs are a mixed composition of various effector cell types including lymphocytes (T cells, B cells, and NK cells) and monocytes. These cells can be extracted from whole blood or buffy coat samples, e.g. by using a hydrophilic colloid and density gradient centrifugation, which separates the blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes. Pan T-cells are a population of CD3+ cells that be isolated from PBMCs. Obtaining PBMCs, pan-T
cells and other effector cell populations is within the competencies of the person of ordinary skill in the art, and suitable methods are provided in the Examples herein.
CD3 is a preferred T cell surface antigen, and is a T cell specific antigen, i.e. is displayed specifically on T cells, including on CD8+ T cells, CD4+ T cells, CD4+CD8+ T cells, regulatory T cells, gamma-delta T cells, memory T Cells, and natural killer T cells (NKTs).
It is possible to recruit and activate specific subsets of T cells to the site of the target antigen HLA-A*02:01/PRAME425'433, e.g. to cancer cells or tumours. In embodiments the second antigen binding domain may bind to (or specifically bind to) a T-cell surface antigen specific to the desired subset of T cells.
For instance, in certain embodiments the second antigen binding domain may bind to (or specifically bind to) a “public” T-cell receptor (i.e. polypeptides thereof), i.e. TCRs of a sequence known to be shared by a large number of individuals within a population. Antigen binding proteins (e.g. antibodies) comprising such a second antigen binding domain would have likely therapeutic utility in a wide range of patients, given that the mechanism of action would be to redirect T cells known to be present in a large number of individuals against the target antigen HLA- A*02:01/PRAME425'433. Such public T-cell receptors, and the sequences of their TCRs, are well documented in the field. Such public TCRs typically arise in response to a common infectious agent, e.g. a virus. Thus, preferred public TCR to which the second antigen binding domain of the antigen binding proteins (e.g. antibodies) binds (or specifically binds), are virus specific public TCRs, preferably human virus specific public TCRs.
Alternatively, gamma-delta T cells may be redirected to the site of the target antigen HLA-A*02:01/PRAME425'433, e.g. to cancer cells or tumours, and activated, in embodiments wherein the second antigen binding domain of the antigen binding proteins (e.g. antibodies) of the invention binds to (or specifically binds to) an antigen present on the surface of the gamma-delta T cells. For instance, the second antigen binding domain may be bind to (or specifically bind to) the public Vy9V52 TCR, preferably to human TCRs.
Alternatively, NKT cells may be redirected to the site of the target antigen HLA-A*02:01/PRAME425'433, e.g. to cancer cells or tumours, and activated, in embodiments wherein the second antigen binding domain of the antigen binding proteins (e.g. antibodies) of the invention binds to (or specifically binds to) an antigen present on the surface of the NKT cells. For instance, the second antigen binding domain may bind to (or specifically bind to) the public Va24 TCR (Va14 in mice), preferably to human TCRs.
Furthermore, other effector cells (non-T cells) may be redirected to the site of the target antigen HLA-A*02:01/PRAME425'433 antigen, e.g. to cancer cells or tumours, and activated, in embodiments wherein the second antigen binding domain of the antigen binding proteins (e.g. antibodies) of the invention binds to (or specifically binds to) an antigen present on the surface of the effector cells. For instance, the second antigen binding domain may bind to (or specifically bind to) CD16, NKG2D, NKp30, or NKp46 receptors, in which case natural killer (NK) cells may be redirected and activated. Alternatively, the second antigen binding domain may be bind to (e.g. specifically bind to) SIRPa, in which case macrophages may be recruited and activated.
In an embodiment, the antigen binding protein (e.g. antibody) of the invention is bispecific, e.g. is a bispecific antibody, which comprises a third, and optionally further, antigen binding domain(s). In this embodiment, the second antigen binding domain binds to, or specifically binds to, a second antigen, i.e. to an antigen other than HLA-A*02:01/PRAME425'433, preferably as described elsewhere herein, and the third antigen binding domain binds to, or specifically binds to HLA- A*02:01/PRAME425'433. The third antigen binding domain may incorporate, singly or in combination, any of the features described herein in relation to the first antigen binding domain. In one embodiment, the third antigen domain is identical to the first antigen binding domain. Further antigen binding domains may bind to, i.e. specifically bind to HLA-A*02:01/PRAME425'433, or to the same antigen as the second binding domain.
In an embodiment, the antigen binding protein (e.g. antibody) of the invention is trispecific, e.g. is a trispecific antibody. In other words, the antigen binding protein (e.g. antibody) of the invention preferably comprises a second antigen binding domain which binds to, or specifically binds to, a second antigen, preferably as described elsewhere herein, and a third antigen binding domain which binds to, or specifically binds to, a third antigen, wherein said first, second and third antigens are different from each other. The trispecific antigen binding proteins (e.g. antibodies) of the invention are preferably also T-cell engagers (as defined and described above).
The bispecific, trispecific or multispecific antigen binding protein (e.g. antibody) may be of any known format, including one selected from the group consisting of single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a bispecific T-cell engager (BiTE; tandem di-scFv), a tandem tri-scFv, a tribody (Fab-(scFv)2) or bibody (Fab- (scFv)1), triabody, scDb-scFv, bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc- scFv fusion, di-diabody, DVD-lg, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-
scFv fusions, such as bsAb (scFv linked to C-terminus of light chain), Bs1Ab (scFv linked to N-terminus of light chain), Bs2Ab (scFv linked to N-terminus of heavy chain), Bs3Ab (scFv linked to C-terminus of heavy chain), Ts1Ab (scFv linked to N- terminus of both heavy chain and light chain), Ts2Ab (dsscFv linked to C-terminus of heavy chain), Knob-into-Hole antibodies (KiHs), a MATCH and DuoBodies.
As mentioned above, the antigen binding proteins (e.g. antibodies) of the present invention are preferably T-cell engaging antibodies (or antigen binding proteins), also termed “T-cell engagers” (TCEs). These are (at least) bispecific molecules, and in a preferred embodiment are T-cell engaging bispecific antigen binding proteins (e.g. antibodies).
In a preferred embodiment, the antigen binding protein (e.g. antibody) of the invention is a single-chain T-cell engaging bispecific antigen binding protein (e.g. antibody).
As mentioned above, in such antigen binding proteins (e.g. antibodies), the second antigen binding preferably binds to (or specifically binds to) CD3. The antigen binding domains in such bispecific molecules are as defined elsewhere herein.
In a preferred embodiment, the antigen binding protein of the invention is a single-chain bispecific diabody, i.e. a bispecific antibody in the single-chain bispecific diabody (scDb) format.
Preferably, the scDb has the capability of binding to (or specifically binding to) both HLA-A*02:01/PRAME425'433 via a first antigen binding domain, as described above, and to a T-cell surface antigen, preferably CD3, via a second antigen binding domain as described above. The antigen binding domains in such bispecific molecules are as defined elsewhere herein.
The scDb format has a more compact form in comparison to other bispecific antibody formats and allows the formation of immunocytolytic synapses.
Thus, in a further aspect and in certain embodiments, the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME425'433, wherein said VHA comprises a variable heavy (VH) CDR1 , a VH CDR2 and a VH CDR3 that comprise, respectively, the amino acid sequences of the VH CDR1 , VH CDR2 and VH CDR3 of a specific antibody of the invention (disclosed in Table A), or sequences substantially homologous thereto;
ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises a variable light (VL) CDR1, a VL CDR2 and a VL CDR3 that comprise, respectively, the amino acid sequences of the VL CDR1 , VL CDR2 and VL CDR3 of a (preferably said) specific antibody of the invention (disclosed in Table A), or sequences substantially homologous thereto; iii) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and iv) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker. Preferred specific antibodies of the invention and the CDR sequences thereof that are disclosed in Table A are as described above.
Thus, in a further aspect and in certain embodiments, the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME425'433, wherein said VHA comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5) or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6) or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7) or a sequence substantially homologous thereto; ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto; iii) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and iv) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker.
In a further aspect and in certain embodiments, the present invention provides a single-chain bispecific diabody comprising: i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME425'433, wherein said VHA comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO: 111) or a sequence substantially homologous thereto,
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115) or a sequence substantially homologous thereto, and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO:117), EFISQW (SEQ ID NO:118), EFVSSW (SEQ ID NO:119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO:10), or QQANSFPFT (SEQ ID NO: 121), or a sequence substantially homologous thereto; iii) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and iv) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker. Preferred CDRs and substantially homologous sequences are as described anywhere else herein.
Thus, in a further aspect and in certain embodiments, the present invention provides a single-chain bispecific diabody comprising: iii) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME425'433, wherein said VHA comprises the amino acid sequence of the VH domain of a specific antibody of the invention (disclosed in Table A) or a sequence substantially homologous thereto; iv) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises the amino acid sequence of the VL domain of a (preferably said) specific antibody of the invention (disclosed in Table A); v) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and vi) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker. Preferred specific antibodies of the invention and the VH and VL sequences thereof that are disclosed in Table A are as described above.
In a further aspect and in certain embodiments, the present invention provides a single-chain bispecific diabody comprising:
i) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME425'433, wherein said VHA comprises the amino acid sequence of SEQ ID NO: 3 or a sequence substantially homologous thereto; ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially homologous thereto; iii) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and iv) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker.
In a further aspect and in certain embodiments, the present invention provides a single-chain bispecific diabody comprising: ii) a first heavy chain variable domain of an immunoglobulin with a first specificity (VHA), wherein said first specificity is for HLA- A*02:01/PRAME425'433, wherein said VHA comprises the amino acid sequence of SEQ ID NO: 132, 142 or 144 or a sequence substantially homologous thereto; ii) a first light chain variable domain of an immunoglobulin with said first specificity (VLA), wherein said VLA comprises the amino acid sequence of SEQ ID NO: 4, 134, 136, 138, 140, 147 or 150 or a sequence substantially homologous thereto; v) a second heavy chain variable domain of an immunoglobulin with a second specificity (VHB), wherein said second specificity is for a T cell CD3; and vi) a second light chain variable domain of an immunoglobulin with said second specificity (VLB); wherein the VLA, VHB, VLB and VHA domains are connected within a single polypeptide chain, and wherein each domain is connected to the adjacent domain(s) via a peptide linker. Preferred VH and VL domains and substantially homologous sequences are as described anywhere else herein.
The VLA, VHB, VLB and VHA domains are connected within (i.e. in) a single polypeptide chain, i.e. are fused, i.e. connected linearly, via peptide linkers. There are thus three peptide linkers connecting the four domains. The domains may be connected in any order/ orientation, provided that upon proper folding of the resulting polypeptide, two functional antigen binding domains are formed.
Preferred orders/ orientations are, in the N to C direction: i) VLA-VHB-VLB- VHA, ii) VLB-VHA-VLA-VHB; iii) VHA-VLB-VHB-VLA and iv) VHB-VLA-VHA-VLB, most preferred are i) VLA-VHB-VLB-VHA, and ii) VLB-VHA-VLA-VHB, particularly i) VLA-VHB-VLB-VHA.
As stated above, the antigen binding proteins of the invention comprise at least one antigen binding domain, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs. The scDbs of the invention comprise two such antigen binding domains: one comprising VHA and VLA, and one comprising VHB and VLB.
In the scDbs of the invention, optional and preferred features and definitions of the first heavy and light chain variable domains, including the CDR and domain sequences, and the substantially homologous sequences, are as defined elsewhere herein. Optional and preferred features and definitions of the second heavy and light chain variable domains, including the CDR and domain sequences, and the substantially homologous sequences, are as defined elsewhere herein.
As mentioned above, the selection of (e.g. length and sequence of) peptide linkers, and the orientation of domains and peptide linkers, are well-known in the field, and would be within the competencies of the person of ordinary skill in the art. Linkers suitable for use have been studied in detail and are well known in the field, e.g. as described in Vdlkel et al., (2001) Protein Engineering, Design and Selection, Volume 14, Issue 10, Pages 815-823.
The scDbs comprise a peptide linker between each of: i) the VLA and the VHB domains (or vice versa, depending on which orientation of domains is present); and ii) the VLB and the VHA domains (or vice versa, depending on which orientation of domains is present); and iii) the VHB and the VLB domains (or vice versa, depending on which orientation of domains is present); or iv) the VHA and VLA domains (or vice versa) depending on which orientation of domains is present.
Thus, the sDbs comprise both linker i) and linker ii) and either linker iii) or linker iv), depending on the orientation of domains therein.
Typically, the peptide linker between the VLA and the VHB domains (or vice versa) (linker (i)) and the peptide linker between the VLB and the VHA domains (or vice versa) (linker (ii)), is relatively short, e.g. 3 to 7 amino acids, e.g. 4 to 7 amino acids or 3 to 5 amino acids, i.e. 3, 4 or preferably 5 amino acids. These are termed the “scDb short linkers” herein. In contrast, the peptide linker between the VHB and the VLB domains (or vice versa) (linker (iii)) or in other arrangements between the VHA and VLA domains (or vice versa) (linker (iv)), is relatively longer, e.g. 12 to 21, preferably 13-19, more preferably 14 to 17 amino acids, e.g. 14, 15, 16 or 17 amino acids. This is termed the “scDb short linker” herein.
Typically, the peptide linker between the VHB and the VLB domains (or vice versa) (linker (iii)) or in other arrangements between the VHA and VLA domains (or vice versa) (linker (iv)), is 2.5 to 3.5 times longer, e.g. about 3 times longer, than the peptide linker between the VLA and the VHB domains (or vice versa) (linker (i)), and the peptide linker between the VLB and the VHA domains (or vice versa) (linker (ii)). The presence of a longer peptide linker between the two central domains of the single polypeptide chain, and the presence of shorter peptide linkers between each of the N and C terminal domains and their respective adjacent domains, achieves correct folding and formation of the two antigen binding domains.
The short linker between the VLA and the VHB domains (or vice versa) (linker
(i)) and the short linker between the VLB and the VHA domains (or vice versa) (linker
(ii)), may be of identical length and/or (preferably “and”) amino acid sequence.
The amino acid sequences of the various peptide linkers are not particularly limited; suitable linker sequences could be identified and prepared by the person of ordinary skill in the art, e.g. with reference to Vdlkel et al., (2001) Protein Engineering, Design and Selection, Volume 14, Issue 10, Pages 815-823, and any suitable linker sequence may be used.
In some embodiments, the peptide linker sequences comprise only hydrophilic amino acids (e.g. arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine) and aliphatic amino acids (e.g. alanine, glycine, isoleucine, leucine, proline, and valine).
In some embodiments, the peptide linker sequences comprise only amino acids selected from the group consisting of threonine, asparagine, serine, aspartic acid, glycine and alanine.
In certain embodiments relating to single-chain bispecific diabodies, said peptide linker between the VLA and the VHB domains (or vice versa) (linker (i)) and between the VLB and the VHA domains (or vice versa) (linker (ii)) consists of four
Glycine amino acid residues and one Serine amino acid residue (GGGGS, SEQ ID NO: 26), and the peptide linker between the VHB and the VLB domains (or vice versa) (linker (iii)), or between the VHA and VLA domains (or vice versa) (linker (iv)) consists of three contiguous sequences consisting each of four Glycine amino acid residues and one Serine amino acid residues (GGGGS, SEQ ID NO: 26), i.e. GGGGSGGGGSGGGGS (SEQ ID NO: 27).
In one embodiment, the antigen binding proteins are chimeric antigen receptors (CARs). As used herein, the term “chimeric antigen receptor” or “CAR” refers to a receptor that is capable of activating an immune cell in response to antigen binding. CARs are recombinant membrane spanning molecules and are advantageously expressed on immune cells. Their structure typically comprises (i) an extracellular domain (“ectodomain” or “antibody domain”), (ii) a transmembrane domain and (iii) a cytoplasmic domain (endodomain or intracellular signalling domain).
The ectodomain (i.e., antibody domain) comprises an antigen binding domain that binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, as defined elsewhere herein. Typically, the ectodomain comprises an antigen binding protein in the scFv format, but other antibody fragments/formats may also be used. A spacer sequence generally connects the ectodomain and the transmembrane domain, which in turn is connected to an endodomain. Upon binding of the ectodomain to the antigen, the receptors cluster and an activation signal is transmitted to the immune cell which results in initiation of an immune response against the cell on which the target antigen (HLA-A*02:01/PRAME425'433) is displayed.
First generation CARs have a simply structured endodomain comprising CD3- zeta. To increase the activation signal, a co- stimulatory domain was added in the second-generation CARs; and third generation CARs include two or more costimulatory domain. Said co-stimulatory domains may be selected from the group consisting of CD28, 0X40 and/or 4-1 BB. Apart from CD3-zeta, other ITAM- containing domains have been explored including the Fc receptor for IgE-y domain.
Thus, in one embodiment, the invention provides a chimeric antigen receptor (CAR) that specifically recognizes HLA-A*02:01/PRAME425'433, comprising: i) an ectodomain that comprises an antigen binding domain which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433, as defined anywhere elsewhere herein; ii) a transmembrane domain; and iii) an intracellular signalling domain.
In certain embodiments, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domains of a type I transmembrane protein, an alpha, beta or zeta chain of a T-cell receptor,
CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
In certain embodiments, the intracellular signalling domain is selected from the group consisting of cytoplasmic signalling domains of a human CD3 zeta chain, FcyRIII, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
Other suitable components, e.g. domains, of CARs, and means for their inclusion are well-known in the field, and any suitable component or domain may be included in the CARs of the present invention.
In a further aspect, the invention provides immune cells engineered to express CARs comprising the antigen binding proteins described herein.
Suitable immune cells include, without being limited to, T cells, Natural Killer T (NKT) cells, natural killer (NK) cells, human embryonic stem cells, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS). Such T cells may be a cytotoxic T lymphocyte (CTL), a regulatory T lymphocyte, an inflammatory T- lymphocytes, or a helper T-lymphocyte or a gamma-delta T cell. The T cell may be a CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells. T cells expressing a CAR are termed CAR-T cells and in embodiments, immune cells engineered to express CARs comprising the antigen binding proteins described herein are CAR-T cells.
Footprint/ Binding Specificity
Example 1 herein describes the examination of the fine-specificity of an antibody of the invention, 5-A05, towards the HLA-A*02:01 presented PRAME425'433 peptide. Positional alanine (Ala) scanning was performed. An array of PRAME425'433 variant peptides were produced, in each of which one residue of the PRAME425'433 sequence was mutated to alanine. The variant peptides sequences are shown in Table C. The binding of 5-A05 to the Ala-mutated peptides was assessed using pHLA phage capture ELISA, as described in Example 1 , relative to the binding to HLA-A*02:01 presented PRAME425'433.
As shown in Figure 2, 5-A05 exhibited peptide-dependent binding. Specific single Ala mutations in the PRAME425'433 peptide affected the extent of binding. Specifically, the antibody-pHLA binding was abrogated with peptide P2, P3, P5, P6, P7 and P9, indicating that amino acids at positions 2, 3, 5, 6, 7 and 9 of WT PRAME425'433 (SEQ ID NO: 19) were important for antibody binding.
Positions 4, 5, 7, and 8 of the PRAME425'433 peptide sequence (SEQ ID NO: 19) are solvent-exposed when presented in the HLA-A*02:01 complex (see Figure 1). Thus, the results showed that clone 5-A05 exhibited restricted positional binding dependency towards 2 of the 4 solvent exposed residues - namely the amino acids at positions 5 and 7 of PRAME425'433 (SEQ ID NO: 19). This indicates a central binding mode and high degree of specificity for 5-A05 since a binding footprint covering the N- or C-terminal ends of the presented peptide is more likely to result in increased potential for binding promiscuity, i.e. confer a binding specificity less dependent on the particular sequence of the presented peptide. The central binding mode of 5-A05 indicates a high specificity for the particular peptide sequence of PRAME425'433 in the HLA-A*02:01/PRAME425'433 complex.
The same analysis was performed using the other antibodies disclosed in Table A, i.e. the 5-A05/1-C04, PAM6, PAM8, PAM17, PAM18, PAM22, PAM23, NGS55 and NGS17 antibodies, with similar peptide-dependent binding observed, and some of the antibodies also exhibiting restricted positional binding dependency towards the amino acid at position 8 of PRAME425'433 (SEQ ID NO: 19) (data not shown).
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention bind to (i.e. are capable of binding to) the amino acid at position 5 or/and (preferably “and”) to the amino acid at position 7 of the HLA-A*02:01 restricted peptide PRAME425'433 (SEQ ID NO: 19). In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention bind to (i.e. are capable of binding to) the amino acids at positions 5, 7 and 8 of the HLA-A*02:01 restricted peptide PRAME425' 433 (SEQ ID NO: 19).
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention preferentially bind to (or selectively bind to) HLA-A*02:01/PRAME425'433 as compared to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 35 and/or (preferably “and”) as compared to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37 (P5 and P7 of Table C).
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention exhibit binding to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 35 and/or (preferably “and”) to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37 (P5 and P7 of Table C), which is at most 25%, e.g. at most 20%, of the exhibited binding of the same antigen binding protein (e.g. antibody) to HLA-A*02:01/PRAME425'433
Alternatively viewed, in an embodiment the antigen binding proteins (e.g. antibodies) of the invention exhibit a decrease of at least 75%, e.g. at least 80%, in binding to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 35 and/or (preferably “and”) to a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 37 (P5 and P7 of Table C), as compared to binding to HLA-A*02:01/PRAME425- 433
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding.
Preferably such binding is exhibited when the antigen binding protein is an antibody in the scFv format, preferably wherein the scFV is present on the surface of a phage particle.
ASSAY 1 : Assay for binding specificity (ELISA)
Binding of an antigen binding protein (e.g. antibody) to a H LA-restricted peptide (pH LA) can be assessed by any appropriate means and the skilled person is familiar with suitable methods (e.g. an ELISA assay such as an ELISA assay in which the pHLA is coated on ELISA plates/wells).
In some embodiments, binding of an antigen binding protein (e.g. antibody) of the invention to H LA-restricted peptide (pHLA) may be as determined by (or as assessed by) an ELISA assay, e.g. an ELISA assay that comprises:
(a) Coating an ELISA plate (i.e. wells of the plate) with NeutrAvidin (e.g. 5 pg/mL) in PBS (phosphate-buffered saline) and incubating for about 16 hours at e.g. 4°C;
(b) Washing the coated plate, for example with PBS containing 0.05% Tween (from hereon “PBST” means “PBS containing 0.05% Tween”), at least once, e.g. three times;
(c) Incubating the avidin coated plate (e.g. for about 1 hour), e.g. at room (or ambient) temperature (e.g. 25°C) with a blocking buffer (e.g. 5% skimmed milk powder in PBST (from hereon “PBSTM”);
(d) Washing the blocked plate, for example with PBST, at least once, e.g. three times;
(e) Incubating the ELISA plate with biotinylated pHLA (e.g. in blocking buffer, e.g. PBSTM) and incubating for about 1h at room (or ambient) temperature (e.g. 25°C);
(f) Washing the coated plate (wells of the coated plate), for example with PBST, at least once, e.g. three times
(g) Incubating the antigen binding protein (e.g. antibody) to be tested (e.g. in scDb, IgG, or scFv format wherein scFv is present on the surface of phage particles) diluted in blocking buffer (e.g. PBSTM), e.g. over a dilution series, e.g. starting at 385 nM for scDb, 250 nM for IgG, or 109 phage/ml for scFv-phage, in wells of the ELISA plate, e.g. for 1h at for example room (or ambient) temperature (e.g. 25°C) for scDb or IgG, or 37 °C for scFv-phage;
(h) Washing the plate (wells of the plate), for example with PBST, at least once, e.g. three times;
(i) Incubating in the wells of the plate, e.g. for 1 h, for example at room (or ambient) temperature (e.g. 25°C), a secondary antibody having (e.g. conjugated to) a detectable label (e.g. horse radish peroxidase, “HRP”), e.g. anti-M13-HRP (e.g. stock solution diluted 1 :5000 in 100 pl blocking buffer (e.g. PBSTM)/well) for scFv-phage, or protL (e.g. diluted 1 :10,000 in 100 pl blocking buffer (e.g. PBSTM)/well) for scDb or IgG;
(j) Washing the plate (wells of the plate), for example with PBST, at least once, e.g. three times;
(k) Detecting (and quantifying) the detectable label. For example, if HRP (horseradish peroxidase) is used as the detectable label, then a HRP substrate (e.g. TMB substrate (3,3',5,5'-Tetramethylbenzidine)) may be added to the wells and incubated (e.g. for 10 min at room (or ambient) temperature (e.g. 25°C), and the reaction may then be stopped (e.g. with 100pl/well of 1M HCI) and absorbance measured, e.g. at 450nm, e.g. using a microplate reader.
A preferred ELISA assay for assessing the ability of an antigen binding protein (e.g. antibody) to bind to a H LA-restricted peptide (pH LA) is described in the Examples section herein.
In such an ELISA assay used to determine binding, the antigen binding protein may be an antibody in any format. In a preferred assay, the antibody is in the scDb, IgG or scFv format. If the scFv format is used, then the scFv is present on the surface of a phage particle. If phage particles are used, then preferably 109 phages/well are used in step (g) of the above method.
Specificity of T-cell re-direction
Example 3 herein describes the assessment of the specificity with which an antibody of the invention, 5-A05 in the scDb format, redirects T-cell activity against
cells displaying the target antigen HLA-A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 restricted peptides comprising (or consisting of) sequences of human genomic origin identified as having high levels of sequence similarity to PRAME425'433 (termed “risk peptides”). The identified human peptides are shown in Table F. Such HLA-A2 restricted peptides represent a potential crossreactivity risk, should antigen binding proteins (e.g. antibodies) directed to HLA- A*02:01/PRAME425'433 also bind to them and induce T-cell activation and redirection against the cells displaying them.
A panel of genomic risk peptides was prepared (Table F), and exogenously added to a culture of T2 cells which were pulsed to induce HLA-A*02:01 presentation of said peptides. A Jurkat-NFAT activation assay was performed as described in Example 3. This is a well-known and widely used assay in the field, which comprises the use of Jurkat T cell line (TCR/CD3 “Effector Cells”) that expresses a luciferase reporter driven by a Nuclear factor of activated T-cells-response element (NFAT- RE). When the Jurkat T cells are engaged with an anti-CD3 antibody, their T-cell receptor (TCR) transduces intracellular signals resulting in NFAT-RE-mediated luminescence. Thus, the assay indicates the ability of a T-cell engaging antigen binding protein (e.g. antibody) with a first antigen binding domain specific for a target antigen, and a second antigen binding domain specific to a T-cell surface antigen (e.g. CD3), to redirect T cell activity in a specific manner against cells displaying the target antigen.
Figures 7A and 7B demonstrate that the assay is a valid proxy for T-cell mediated killing, i.e. the level of T-cell activation seen via NFAT-RE-mediated luminescence, correlates with the level of target cell killing. Figure 8A shows the extent to which 5-A05 induced Jurkat T-cell activation/ redirection against pHLA restricted risk peptides relative to HLA-A*02:01/PRAME425'433 and relative to nonpulsed T2 cells (i.e. cells lacking any pHLA restricted exogenous peptide).
As shown in Figure 8A, 5-A05 induced T-cell activation (redirected T-cell activity) with remarkable specificity against cells displaying HLA-A*02:01/PRAME425' 433 over cells displaying all of the HLA-A*02:01 restricted risk peptides. Jurkat activation was only observed when T2 cells were pulsed with PRAME425'433 peptide and SACS and TRGV10-2 peptides; the level of activation observed with all (each) of the other risk peptides was comparable to non-pulsed T2 cells, despite these risk peptides sharing high sequence similarity with PRAME425'433. The three peptides activating Jurkat cells in Figure 8A (PRAME425'433, SACS and TRGV10-2 peptides) were analyzed further by 3-fold peptide titration, and T2 cells pulsed with SACS or
TRGV10-2 peptides only activated Jurkat NFAT cells at 10pM peptide, but not lower peptide concentrations (Figure 8B). 10pM is a non-physiological concentration.
In silico assessment of these peptides in terms of HLA binding and processing ability strongly indicated lack of both key features to represent true epitopes (data not shown). Both peptides received a negative total score for TAP and MHC binding using an MHC-p processing prediction program, i.e. it is predicted that in vivo, neither peptide is processed into 9-mers, transported and presented on HLA- A2. NetMHCpan 4.1 predicts both SACS and TRGV10-2 as weak binders to HLA- A2, and neither peptide is predicted as a T-cell epitope using NetCTLpan. These data, combined with the very low T cell activation potential of these two alternative peptides, excludes them as potential cross-reactivity risks of further relevance.
Example 3 herein also describes the assessment of the specificity with which the antibodies of the invention, 5-A05/1-C04, PAM22, PAM23 and NGS55, in the scDb format, redirect T-cell activity against cells displaying the target antigen HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 restricted peptides comprising (or consisting of) sequences of human genomic origin identified as having high levels of sequence similarity to PRAME425'433 (termed “PRAME425'433 related degenerate risk peptides”, or simply “risk peptides”). The identified human peptides are shown in Table K. Such HLA-A2 restricted peptides represent a potential cross-reactivity risk, should antigen binding proteins (e.g. antibodies) directed to HLA-A*02:01/PRAME425'433 also bind to them and induce T- cell activation and redirection against the cells displaying them.
A panel of PRAME425'433 related degenerate risk peptides was prepared (Table K), and exogenously added to a culture of T2 cells which were pulsed to induce HLA-A*02:01 presentation of said peptides. A Jurkat-NFAT activation assay was performed as described in Example 3. The assay indicates the ability of a T- cell engaging antigen binding protein (e.g. antibody) with a first antigen binding domain specific for a target antigen, and a second antigen binding domain specific to a T-cell surface antigen (e.g. CD3), to redirect T cell activity in a specific manner against cells displaying the target antigen.
Figures 11A to 11 D show the extent to which 5-A05/1-C04, PAM22, PAM23 and NGS55 induced Jurkat T-cell activation/ redirection against pHLA restricted PRAME425'433 related degenerate risk peptides relative to HLA-A*02:01/ PRAME425' 433 and relative to non-pulsed T2 cells (i.e. cells lacking any pH LA restricted exogenous peptide). Jurkat activation was only observed when T2 cells were pulsed with PRAME425'433 peptide; the level of activation observed with all (each) of the risk
peptides was comparable to non-pulsed T2 cells, despite these risk peptides sharing high sequence similarity with PRAME425'433.
This further demonstrates the remarkable specificity of the antigen binding proteins (e.g. antibodies) of the invention, e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 and NGS55 antibodies, in terms of redirecting potent and relevant T-cell activity specifically against HLA-A*02:01/PRAME425'433 positive cells.
The antigen binding proteins (e.g. antibodies) of the invention, when in a T- cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect (i.e. are capable of preferentially (or selectively) redirecting) T-cell activity against cells displaying HLA-A*02:01/PRAME425'433 (i.e. HLA-A*02:01/PRAME425'433 positive cells).
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against cells displaying HLA-A*02:01/PRAME425'433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against cells displaying HLA-A*02:01/PRAME425'433 as compared to against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of i) SEQ ID NOs: 40 to 105 (genomic risk peptides), and/or ii) SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME425-433 related degenerate risk peptides).
In this context, the term “redirect T-cell activity” against cells displaying a given (target) antigen is meant that the T-cell engaging antigen binding protein (e.g. antibody) forms an immunological synapse by binding to the T-cell antigen (e.g. CD3) and to the given target antigen displayed on target cells, leading to recruitment of T cells to the target cells and activation of T-cell function against said target cells. In other words, the term means induce (i.e., promote, enhance or increase) T-cell activity against the cells displaying the target antigen. Induce need not mean that the starting activity level is zero, i.e. it may mean “induce further”.
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), redirect T-cell activity against pulsed T2 cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of i) SEQ ID NOs: 40 to 105 (genomic risk peptides), and/or ii) SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME425'433 related degenerate risk peptides), to an extent that is
not different, e.g. is not significantly different, from the extent of T-cell activity redirected by the same antigen binding protein (e.g. antibody) against negative control cells. Suitable negative control cells may be non-pulsed T2 cells, i.e. T2 cells lacking any pH LA-restricted exogenous peptide.
Alternatively viewed, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format (e.g. in the scDb format), redirect T-cell activity against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of i) SEQ ID NOs: 40 to 105 (genomic risk peptides), and/or ii) SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME425-433 related degenerate risk peptides), to an extent that is not different, e.g. is not significantly different, from the extent of T-cell activity redirected by the same antigen binding protein (e.g. antibody) against negative control cells. Suitable negative control cell cells may be cells displaying neither said HLA-A*02:01 risk peptides, nor HLA-A*02:01/PRAME425-433, i.e. cells against which merely background levels of redirected T-cell activity are observed.
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), i) redirect T-cell activity against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of SEQ ID NOs: 40 to 105 (genomic risk peptides) to an extent that is at most 25% of the T-cell activity redirected by the same antigen binding protein (e.g. antibody) against cells displaying HLA-A*02:01/PRAME425-433; and/or ii) redirect T-cell activity against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of SEQ ID NOs: 57, 77, 80, 82 and 172- 199 (PRAME425-433 related degenerate risk peptides) to an extent that is at most 10% of the T-cell activity redirected by the same antigen binding protein (e.g. antibody) against cells displaying HLA-A*02:01/PRAME425-433.
Alternatively viewed, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format, i) exhibit a decrease of at least 75% in T-cell activity redirected against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of SEQ ID NOs: 40 to 105 (genomic risk peptides) as compared to the T-cell activity redirected by the same antigen binding protein (e.g. antibody) against cells displaying HLA-A*02:01/PRAME425-433 and/or ii) exhibit a decrease of at least 90% in T-cell activity redirected against cells displaying a HLA-A*02:01 restricted peptide comprising (or consisting of) the amino acid sequence of any one, preferably each, of SEQ ID NOs: 57, 77, 80, 82 and 172-199 (PRAME425-433 related degenerate risk peptides) as compared to the T-cell activity redirected by the same
antigen binding protein (e.g. antibody) against cells displaying HLA- A*02:01/PRAME425'433.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such redirection of T-cell activity.
Preferably such redirection of T-cell activity is exhibited when the antigen binding protein is any T-cell engager format, preferably in a bispecific format. Preferably, the antibody is in the scDb format.
Preferably such redirection of T cell activity is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME425' 433 (“target cells”) is about 1 :1. Thus, preferably such redirection of T cell activity is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME425'433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of T cell activity. Preferably, such detection and quantification is as described below.
Example 3 herein also describes, again using a Jurkat-NFAT activation assay, that 5-A05 effectively redirects T-cell activity against cancer cells displaying HLA-A*02:01/PRAME425'433 but not against cancer cells displaying HLA-A*02:01 lacking the PRAME425'433 peptide (Figure 9B).
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect (i.e. are capable of preferentially (or selectively) redirecting) T- cell activity against cells displaying HLA-A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against cells displaying HLA-A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA- A*02:01 restricted peptide that is not PRAME425'433.
Alternatively viewed, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) redirect T-cell activity against HLA-
A*02:01/PRAME425'433 positive cells as compared to against HLA-A*02:01 positive, PRAME425'433 negative cells.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such redirection of T-cell activity.
Preferably such redirection of T-cell activity is exhibited when the antigen binding protein is any T-cell engager format, preferably a bispecific format. Preferably, the antibody is in the scDb format.
Preferably such redirection of T cell activity is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME425' 433 (“target cells”) is about 1 :1. Thus, preferably such redirection of T cell activity is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME425'433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of T cell activity. Preferably, such detection and quantification is as described below.
ASSAY 2 : Assay for redirection of T-cell activity (Jurkat)
The ability of an antigen binding protein (e.g. antibody) in a T-cell engager format to redirect T-cell activity against cells displaying a H LA-restricted peptide (pHLA) can be assessed by any appropriate means and the skilled person is familiar with suitable methods, e.g. a Jurkat activation assay in which the pH LA is displayed on the surface of a target cell (e.g. a T2 cell or a cancer cell).
Jurkat assays may comprise the co-culturing of Jurkat NFAT reporter cells representing effector cells (“E”), target cells (“T”) (e.g. peptide pulsed T2 cells (described elsewhere herein) or cancer cells e.g. cancer cell line cells), preferably at an E:T ratio of about 1 :1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently applying the cultured cells to wells comprising a luciferase substrate and detecting luminescence.
In some embodiments, the ability of an antigen binding protein (e.g. antibody) in a T-cell engager format to redirect T-cell activity against cells displaying a HLA- restricted peptide (pHLA) may be as determined by (or as assessed by) a Jurkat activation, e.g. a Jurkat activation assay that comprises:
(a) Culturing target cells (e.g. T2 cells, or cancer cells e.g. cancer cell line cells) in cell culture medium (e.g. RPMI1640 supplemented with 10% FBS (Fetal Bovine Serum) and 5U/ml PS (Penicillin-Streptomycin);
(b) Harvesting said target cells, e.g. by transferring suspension cells to a Falcon tube (e.g. of 50ml volume), or by removing cell culture supernatant from adherent cells (cancer cells may be adherent cells), washing the adherent cells once with sterile 1xPBS, incubating with an appropriate amount of Trypsin (e.g. 1-2ml) at about 37°C in a humidified incubator until the cells detach from the cell culture vessel, adding an appropriate amount of cell culture medium (e.g. 10ml) and transferring the suspension of adherent cells to a new vessel e.g. to a 50ml Falcon tube;
(c) Washing said harvested target cells, e.g. by pelleting the cells (e.g. at 300g for 5 minutes), resuspending in cell culture medium (e.g. 10ml), pelleting a second time (e.g. at 300g for 5 minutes), then resuspending pelleted cells to an appropriate volume (e.g. 5-10ml);
(d) Counting said target cells and diluting said target cells to an appropriate density (e.g. 500000/ml);
(e) Seeding said target cells in the wells of cell culture plates (e.g. white 96 well plates*) in cell culture medium (e.g. at 50000 cancer cells/well the day before coculturing (step (q)) to allow for monolayer formation of adherent cells, or 25000 T2 cells/well on the day of co-culture);
(f) If cancer cells are seeded in step (e), then incubating said seeded adherent cancer cells e.g. for 18h at 37°C in a humidified incubator to allow for monolayer formation;
(g) Culturing Jurkat NFAT reporter cells e.g. in cell culture medium (e.g. RPMI1640 supplemented with 10% FBS and 5U/ml PS);
(h) Harvesting said Jurkat NFAT reporter cells e.g. by transferring cells to a Falcon tube (e.g. of 50ml volume);
(i) Washing said Jurkat NFAT reporter cells, e.g. by pelleting the cells (e.g. at 300g for 5 minutes), resuspending in cell culture medium (e.g. 10ml), pelleting a second time (e.g. at 300g for 5 minutes), then resuspending pelleted cells to an appropriate volume (e.g. 5-10ml);
(j) Counting said Jurkat NFAT reporter cells and diluting to an appropriate density (e.g. 500000/ml);
(k) Adding said re-suspended Jurkat NFAT reporter cells to the target cells at a ratio of about 1 :1 Jurkat NFAT reporter cells to target cells;
(l) Diluting the antigen binding protein (e.g. antibody) to be tested in assay medium (e.g. RPMI1640 supplemented with 10% FBS and 5U/ml PS), for example at a concentration range of 0.2nM to 200nM;
(m) Adding the antigen binding protein (e.g. antibody) to be tested to the coculture of cells of step (k) (e.g. at a final concentration range from 0.02nM to 20nM);
(n) Diluting H LA-restricted peptides (e.g. PRAME425'433 or other peptides) in assay medium (e.g. RPMI1640 supplemented with 10% FBS and 5U/ml PS), for example at a concentration range of 2.4nM to 200pM (if cancer cells are used as target cells, this step is not performed);
(o) If T2 cells are used as target cells, transfer (e.g. about 50 pl) of peptide dilutions (e.g. in duplicates or triplicates) to a 96-well cell culture plate (i.e. wells of the plate) e.g. at final concentration in the range of 600pM to 50pM (if cancer cells are used as target cells, this step is not performed)
(p) Adding culture medium (e.g. to a final volume of 200 pl per well);
(q) Co-culturing by incubating the co-culture e.g. for about 16h at about 37°C in a humidified incubator;
(r) Diluting luciferase substrate (e.g. D-Luciferin, Monopotassium Salt (Promega)) e.g. at 1 :10 in Milli-Q water;
(s) Adding diluted luciferase substrate (e.g. D-Luciferin Monopotassium Salt (Promega)) from step (r), e.g. at a ratio of 1 :3 luciferase substrate to cell co-culture (e.g. by adding 50pl of luciferase substrate to the 150 pl of co-culture from step (q)); and
(t) Recording luminescence, e.g. using Varioscan LUX (Thermo Fisher).
* Step (t) typically requires the co-culture and D-Luciferin to be present in the wells of a white cell culture plate in order for the luminescence to be recorded, so it is preferable that cells are seeded in white cell culture plates in step (e). However, this is not essential; step (e) may alternatively comprise seeding said target cells in the wells of cell culture plates that are not white. If so, then step (q) further comprises pelleting the co-culture plates (e.g. at 300g for 5min), discarding excess cell culture supernatant, e.g. 100pl; and re-suspending pelleted cells in remaining cell culture supernatant (e.g 100pl). Subsequently, in step (s), an aliquot of the diluted luciferase substrate from step (r) and an aliquot of the resuspended cells would be added at a ratio of 1 :3 luciferase substrate to cell co-culture in the wells of a white cell culture plate (e.g. a white 96-well plate), e.g. 25pl per well of luciferase substrate and 75pl per well of cell co-culture, prior to the performance of the luminescence recordal step (t).
A preferred Jurkat activation assay is described in the Examples section herein.
In such a Jurkat activation assay, the antigen binding protein may be an antibody in any T-cell engager format, preferably a bispecific format. In a preferred assay, the antibody is in the scDb format.
T-cell activation, differentiation, proliferation
The antigen binding proteins (e.g. antibodies) of the invention, when in a T- cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA-A*02:01/PRAME425'433.
Alternatively viewed, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), are capable of preferentially (or selectively) inducing activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA-A*02:01/PRAME425'433.
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
Alternatively viewed, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce activation and/or (preferably “and”) differentiation and/or (preferably “and”) proliferation of T cells directed against HLA-
A*02:01/PRAME425'433 positive cells as compared to against HLA-A*02:01 positive, PRAME425'433 negative cells.
The HLA-A*02:01/PRAME425'433 displaying, or positive or negative cells referred to herein, may be any cell type including the preferred target and cancer cell types disclosed elsewhere herein.
The T-cells may be as defined anywhere else herein, and are preferably CD4+ T cells or CD8+ T cells.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such inducement of T-cell activation, differentiation, or proliferation.
The term "induce” as used herein means promote, i.e. enhance or increase the stated property or activity. Induce need not mean that the starting level of the property or activity is zero, i.e. it may mean “induce further”.
Preferably such induction of activation, differentiation or proliferation of T cells is exhibited when the antigen binding protein is any T-cell engager format, preferably a bispecific format. Preferably, the antibody is in the scDb format.
Preferably such induction of activation, differentiation or proliferation of T cells is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME425'433 (“target cells”) is about 1 :1. Thus, preferably such induction of activation, differentiation or proliferation of T cells is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME425'433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of T cell activation, differentiation or proliferation. Preferably, such detection and quantification is as described below.
ASSAY 3 : Assay for induced T-cell activation/ differentiation
The ability of an antigen binding protein (e.g. antibody) in a T-cell engager format to induce activation or differentiation of T cells directed against cells displaying a H LA-restricted peptide (pH LA) (“target cells”) can be assessed by any appropriate means, and the skilled person is familiar with suitable methods, for example coculturing the target cells (“T”), and effector cells (“E”) comprising T cells, such as PBMCs, preferably at an E:T ratio of about 1 :1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting and quantifying markers of T-cell activation or differentiation.
Suitable T-cell activation/ differentiation markers are well-known, and include CD25 and CD71 (both markers of T-cell activation), and Granzyme B (a marker of T- cell differentiation). Markers can be detected by any suitable means, including using fluorescently-labelled antibodies against said markers, with subsequent quantification thereof, e.g. by flow cytometry techniques, which are well-known. If desired, signal specifically from T cells, or specifically from CD8+ or specifically from CD4+ T cells in particular can be assessed by also performing detection using fluorescently labelled antibodies against CD3, CD8+ and CD4+, respectively.
Target cells may be T2 cells pulsed with exogenously applied PRAME425'433 peptide. In these assays, control cells are non-pulsed T2 cells, and T2 cells pulsed with an unrelated peptide. Alternatively, target cells may be HLA- A*02:01/PRAME425'433 positive cancer cells. In these assays control cells are HLA- A*02:01/PRAME425'433 negative cancer cells.
In assays of the ability of a T-cell engager to induce T-cell activity, proliferation, differentiation and/or cytotoxicity, it is possible to use T cells, or a subset of T cells as the effector cells (e.g. PBMCs or pan T-cells isolated from PBMCs).
In such assays, the apparent efficacy of an antibody can be artificially inflated through use of a high E:T ratio in the co-culturing steps. The E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity against the target cells. E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested. Therefore, it is preferred that an E:T ratio of 1 :1 is used.
Thus, a suitable assay may comprise
(a) Co-culturing in wells of an assay plate target cells (“T”) (e.g. cancer cells (e.g. cancer cell line cells), or peptide pulsed T2 cells), e.g. as discussed above, and effector cells (“E”) comprising T cells (e.g. peripheral blood mononuclear cells (PBMCs)), e.g. as discussed above, preferably at an E:T ratio of about 1 :1 , and the antigen binding protein (e.g. antibody) to be tested (e.g. 100 ng/ml), e.g. for about 72h at about 37°C; and
(b) Performing (multi-colour) flow cytometry staining and analysis, wherein
(i) staining may be performed with anti-human fluorescently-labelled antibodies against one or more biomarkers of T-cell activity, e.g. CD25 (e.g. APC-CD25 (clone BC96)) or CD71 (e.g. PercpCy5.5-CD71 (clone CY1G4)); and/or
(ii) staining may be performed with anti-human fluorescently-labelled antibodies against one or more biomarkers of T-cell differentiation, e.g.
Granzyme B (e.g. PE-Granzyme B (clone QA18A28)); and
(c) Quantifying the signal of the fluorescent label, e.g. using Zombie Yellow fixable viability kit, using appropriate fluorescence minus one (FMO) controls to exclude background staining.
If peptide pulsed T2 cells are used as target cells, then T2 cells are pulsed with the relevant peptide prior to step (a), e.g. by resuspension in assay buffer containing the peptide (e.g. about 50pM of peptide) and incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide.
In some embodiments, such an assay comprises the use of peptide pulsed T2 cells or cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs as effector cells, and the assay comprises:
(a) If peptide pulsed T2 cells are used as target cells, then T2 cells are resuspended in assay buffer (e.g. RPMI/10% FBS/1% P/S) containing peptide (e.g. about 50pM of peptide), which may be PRAME425'433 or a comparator peptide, incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide (if cancer cells are used as target cells, this step is not performed);
(b) Diluting the antigen binding protein (e.g. antibody) to be tested e.g. to 10x the final concentrations required in assay buffer;
(c) Preparing PBMCs, e.g. by thawing frozen PBMCs obtained from liquid nitrogen storage (e.g. in a 37°C water bath), and transferring said PBMCs drop wise into pre-warmed assay buffer, centrifuging and resuspending the PBMCs in fresh assay buffer, counting and resuspending the PBMCs at a desired concentration;
(d) Preparing a co-culture of PMBCs (effector cells “E”) and target cells (“T”) in wells of a culture plate at an E:T ratio of about 1:1 (e.g. 10,000 PBMCs and 10,000 target cells) e.g. in a total volume of about 180pl;
(e) Adding e.g. about 20pl of the antigen binding protein (e.g. antibody) to be tested to the wells (e.g. about 100ng/ml) e.g. to make a final volume of about 200pl;
(f) Incubating the co-culture at about 37°C e.g. for about 72hrs; and
(g) Combining contents of a number (e.g. 4) wells of identical setup, and centrifuging and washing cells therein (e.g. by discarding supernatant and resuspending cells in PBS and centrifuging cells);
(h) Discarding supernatant, and resuspending cells in flow cytometry staining buffer (e.g. PBS/2%FBS) containing Fc block, to prevent any non-specific binding, and incubating e.g. for about 15 minutes, at about 4°C in the dark;
(i) Centrifuging, and washing cells, and discarding supernatant, then resuspending cells in PBS containing a viability marker e.g. for about 15 minutes in the dark at about 25°C;
(j) Centrifuging and washing cells and discarding the supernatant; and
For assaying T cell activation:
(k) Resuspending cells in staining buffer e.g. for about 30 minutes in the dark at about 4°C, said staining buffer containing fluorescently labelled antibodies against T cell antigens (e.g. CD3, CD4, CD8), and against markers of T-cell activation (e.g. CD25, CD71);
(l) Centrifuging, washing and resuspending cells in fixation buffer, e.g. BD cytofix, for e.g. about 20 mins, at about 4°C in the dark (the fixation buffer preserves the light-scattering characteristics and fluorescence intensities of the anti-bound T cells);
(m) Centrifuging cells, twice washing cells and resuspending cells in staining buffer (and optionally storing cells at about 4°C); and
(n) Analysing fluorescence (of cells) using a flow cytometer (e.g. a BD Canto II); or
For T cell differentiation after steps (a) to (j) above
(k) Resuspending cells in staining buffer e.g. for about 30 minutes, in the dark at about 4°C, said staining buffer containing fluorescently labelled antibodies against T cell antigens (e.g. CD3, CD4, CD8);
(l) Centrifuging, washing and resuspending cells in fixation buffer, e.g. BD cytofix, for e.g. about 20 mins, at about 4°C in the dark;
(m) Centrifuging cells, twice washing cells and resuspending cells in staining buffer;
(n) Permeabilizing cells by i) Centrifuging cells and discarding supernatant, and resuspending cells in a permeabilization buffer (e.g. eBiosciences Perm/Wash (1x)) e.g. for about 10 minutes at about 4°C in the dark; and ii) centrifuging cells, washing and discarding supernatant, and resuspending cells in permeabilization buffer (e.g. eBiosciences
Fixation/permeabilization buffer) e.g. for about 30 minutes, at about 4°C in the dark;
(o) Centrifuging cells, washing cells with a permeabilization/wash buffer (e.g. eBioscience Perm/Wash (1x)) and discarding the supernatant;
(p) Resuspending cells in a permeabilization/wash buffer (e.g. eBioscience Perm/Wash (1x)) containing fluorescently labelled antibodies against markers of T- cell differentiation, e.g. anti-human Granzyme B antibody, e.g. for about 45 minutes at about 4°C in the dark;
(q) Centrifuging cells, twice washing cells with a permeabilization/wash buffer (e.g. eBioscience Perm/Wash (1x)) and resuspending cells in staining buffer; and
(r) Analysing fluorescence (of cells) using a flow cytometer (e.g. a BD Canto II).
In such assays, fluorescence (of cells) is from differently labelled antibodies against different ? cell antigens and activation/differentiation markers. The different fluorescence signals can be distinguished using standard flow cytometry techniques, thereby permitting detection of particular activation/differentiation markers of interest, either from all T cells and/or from particular T cell types of interest.
In such an assay, the antigen binding protein may be an antibody in any bispecific antibody format. In a preferred assay, the antibody is in the scDb format.
ASSAY 4 : Assay for T-cell proliferation
The ability of an antigen binding protein (e.g. antibody) in a T-cell engager format to induce proliferation of T cells directed against cells displaying a HLA- restricted peptide (pH LA) (“target cells”) can be assessed by any appropriate means and the skilled person is familiar with suitable methods, for example co-culturing the target cells (“T”) (e.g. cancer cells (e.g. cancer cell line cells), or peptide pulsed T2 cells), e.g. as discussed above, and fluorescently-labelled effector cells (“E”) comprising T cells, such as CFSE-labelled PBMCs, preferably at an E:T ratio of about 1:1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting the fluorescent-label via flow cytometry. If desired, signal from specifically T cells, or CD8+ or CD4+ T cells in particular can be assessed by also performing detection using fluorescently labelled antibodies against CD3, CD8+ and CD4+, respectively.
Thus, a suitable assay may comprise:
(a) Co-culturing in wells of an assay plate target cells (“T”) (e.g. cancer cells (e.g. cancer cell line cells), or peptide pulsed T2 cells), e.g. as discussed above, and effector cells comprising T cells (“E”) (e.g. PBMCs), e.g. as discussed above, labelled
with a fluorescent label (e.g. Carboxyfluorescein succinimidyl ester (CFSE)), at an E:T ratio of about 1:1 , and the antigen binding protein (e.g. antibody) to be tested (e.g. about 100 ng/ml), e.g. for about 72h at about 37°C;
(b) Detecting the fluorescent-label (e.g. CFSE) e.g. using (multi-colour) flow cytometry; and
(c) Quantifying the signal of the fluorescent label, e.g. using Zombie Yellow fixable viability kit, using appropriate fluorescence minus one (FMO) controls to exclude background staining.
If peptide pulsed T2 cells are used as target cells, then T2 cells are pulsed with the relevant peptide prior to step (a), e.g. by resuspension in assay buffer containing the peptide (e.g. about 50pM of peptide) and incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide.
In some embodiments, such an assay comprises the use of peptide pulsed T2 cells or cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs as effector cells, and the assay comprises
(a) If peptide pulsed T2 cells are used as target cells, then T2 cells are resuspended in assay buffer (e.g. RPMI/10% FBS/1% P/S) containing peptide (e.g. about 50pM of peptide), which may be PRAME425'433 or a comparator peptide, incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide (if cancer cells are used as target cells, then this step is not performed);
(b) Diluting the antigen binding protein (e.g. antibody) to be tested, e.g. to 10x the final concentrations required in assay buffer;
(c) Preparing PBMCs, e.g. by thawing frozen PBMCs obtained from liquid nitrogen storage (e.g. in a 37°C water bath), and transferring said PBMCs drop wise into pre-warmed assay buffer, centrifuging and resuspending the PBMCs in fresh assay buffer;
(d) Pelleting and resuspending the PBMCs in PBS (e.g. 1ml Dulbecco's phosphate-buffered saline (DPBS)) containing CFSE (e.g. about 0.5pM CFSE), incubating for e.g. about 8 minutes at room (or ambient) temperature (e.g. about 25°C), and adding assay buffer (e.g. about 9ml of assay buffer);
(e) Pelleting the PBMCs, washing the PBMCs once in assay buffer, counting and resuspending the PBMCs at a desired concentration;
(f) Preparing a co-culture of PMBCs (effector cells “E”) and target cells (“T”) in wells of a culture plate at an E:T ratio of about 1:1 (e.g. 10,000 PBMCs and 10,000 target cells) e.g. in a total volume of about 180pl;
(g) Adding e.g. about 20pl of the antigen binding protein (e.g. antibody) to be tested to the wells e.g. to make a final volume of about 200pl;
(h) Incubating the co-culture at about 37°C e.g. for about 72hrs;
(i) Combining the contents of a number (e.g. 4) wells of identical setup, and centrifuging cells and washing cells (e.g. by discarding supernatant and resuspending cells in PBS), discarding supernatant;
(j) Resuspending cells in flow cytometry staining buffer (e.g. PBS/2%FBS) containing Fc block, to prevent any non-specific binding, and incubating e.g. for about 15 minutes, at about 4°C in the dark;
(k) Centrifuging cells, washing cells, and discarding supernatant, then resuspending cells in PBS containing a viability marker, e.g. for about 15 minutes in the dark, at about 25°C;
(l) Centrifuging cells, washing cells and discarding the supernatant;
(m) Resuspending cells in staining buffer, e.g. for about 30 minutes in the dark at about 4°C, said staining buffer containing fluorescently labelled antibodies against T cell antigens (e.g. CD3, CD4, CD8);
(n) Centrifuging cells, washing cells and resuspending cells in fixation buffer, e.g. BD cytofix, e.g. for about 20 mins at about 4°C in the dark (the fixation buffer preserves the light-scattering characteristics and fluorescence intensities of the antibound T cells);
(o) Centrifuging cells, twice washing cells and resuspending cells in staining buffer (and optionally storing cells at about 4°C); and
(p) Analysing fluorescence (of cells) using a flow cytometer (e.g. a BD Canto II).
In such assays, fluorescence (of cells) is from differently labelled antibodies against different T cell antigens, and from CFSE. The different fluorescence can be distinguished using standard flow cytometry techniques, thereby permitting detection of proliferation signal either from all T cells and/or from particular T cell types of interest.
In such assays, fluorescence (from cells) is from differently labelled antibodies against different T cell antigens and activation/differentiation markers. The different fluorescence signals can be distinguished using standard flow cytometry techniques, thereby permitting detection of proliferation either from all T cells and/or from particular T cell types of interest.
In such an assay, the antigen binding protein may be an antibody in any T- cell engager format. In a preferred assay, the antibody is in the scDb format.
T-cell mediated cytotoxicity
Example 4 herein also describes that a 7-Aminoactinomycin D (7-AAD) viability staining assay was used to assess the ability of antibodies of the invention to specifically induce T-cell mediated cytotoxicity of cells displaying the HLA- A*02:01/PRAME425'433 antigen, as compared to cells displaying HLA-A*02:01 absent the PRAM E425'433 peptide complexed thereto.
The present inventors performed assays in which target (“T”) cells (positive or negative for HLA-A*02:01/PRAME425-433), CFSE-labelled effector cells (“E”) comprising T cells (e.g. pan-T cells isolated from PBMCs), and 5-A05, were coincubated, as described above, with subsequent staining with the non-viable cell stain 7-AAD, detection thereof via flow cytometry and quantification of the signal.
Specifically, the assay was used to assess the capacity of 5-A05 to induce T- cell mediated cytotoxicity of HLA-A*02:01/PRAME425'433 positive and negative cancer cells (Figure 10A and 10B).
A 1 :1 ratio of effector cells to target cells was again used. As shown in Figures 10A and 10B, 5-A05 induced T-cell mediated target cell killing only when co-cultured with HLA-A*02:01/PRAME425'433 positive cells cancer cell lines. No T-cell mediated cytotoxicity resulted against cells lacking HLA-A*02:01/PRAME425'433, i.e. HLA-A*02:01/PRAME425'433 negative cancer cells. Cytotoxicity against HLA- A*02:01/PRAME425'433 positive tumor cell lines representing both solid tumors (10A) and liquid tumors (10B) was demonstrated.
The same analysis was performed using the other antibodies disclosed in Table A, i.e. 5-A05/1-C04, PAM6, PAM8, PAM17, PAM18, PAM22, PAM23, NGS55 and NGS17, with similarly specific cytotoxicity effects being observed as for 5-A05 (data not shown).
The antigen binding proteins (e.g. antibodies) of the invention, when in a T- cell engager format (e.g. in the scDb format), are capable of preferentially (or selectively) inducing T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME425'433.
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
Alternatively viewed, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce T-cell mediated cytotoxicity against HLA- A*02:01/PRAME425'433 positive cells as compared to against HLA-A*02:01 positive, PRAME425'433 negative cells.
The HLA-A*02:01/PRAME425'433 displaying, or positive or negative cells referred to herein, may be any cell type including the preferred target and cancer cell types disclosed elsewhere herein.
The T-cells may be as defined anywhere else herein, and are preferably CD4+ T cells or CD8+ T cells.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such inducement of T-cell mediated cytotoxicity.
The term "induce” as used herein means promote, i.e. enhance or increase the stated property or activity. Induce need not mean that the starting level of the property or activity is zero, i.e. it may mean “induce further”.
Preferably such T-cell mediated cytotoxicity is exhibited when the antigen binding protein is an antibody in any T-cell engager format, preferably a bispecific format. Preferably, the antibody is in the scDb format.
Preferably such T-cell mediated cytotoxicity is exhibited when the ratio of T- cells (or effector cells comprising T cells) to cells displaying HLA-A*02:01/PRAME425' 433 (“target cells”) is about 1 :1. Thus, preferably such T-cell mediated cytotoxicity is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME425'433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and
quantification of a marker of cell death. Preferably, such detection and quantification is as described below.
ASSAY 5 : Assay for T-cell mediated cytotoxicity
The ability of an antigen binding protein (e.g. antibody) in a T-cell engager format to induce T-cell mediated cytotoxicity against cells displaying a H LA-restricted peptide (pH LA) (“target cells”) can be assessed by any appropriate means and the skilled person is familiar with suitable methods, e.g. co-culturing CFSE-labelled target cells (“T”), and effector cells (“E”) comprising T cells, such as PBMCs or preferably pan-T-cells derived therefrom, preferably at an E:T ratio of about 1 :1, together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting and quantifying a marker of cytotoxicity. Such detection and quantification may be performed by staining for non-viable cells (e.g. with 7-amino actinomycin D (7-AAD)) and detecting said stain, e.g. via flow cytometry.
Thus, a suitable assay may comprise:
(a) Co-culturing in wells of an assay plate CFSE-labelled target cells (“T”), e.g. cancer cells (e.g. cancer cell line cells), or peptide pulsed T2 cells, e.g. as described above, and effector cells comprising T cells (“E”), e.g. PBMCs or preferably pan-T-cells derived therefrom, e.g. as described above, at an E:T ratio of about 1 :1 , and the antigen binding protein (e.g. antibody) to be tested, e.g. at a single concentration (e.g. about 100 ng/ml), or over a concentration range (e.g. of about 0.01-1000ng/ml), and co-culturing e.g. for about 72h at about 37°C;
(b) Harvesting assay supernatants and cells from the assay plates;
(c) Adding a fluorescent stain for non-viable cells (e.g. 7-AAD);
(d) Detecting the fluorescent-stain (e.g. 7-AAD) using multi-colour flow cytometry; and
(e) Quantifying the signal of the stain, e.g. using Zombie Yellow fixable viability kit, using appropriate fluorescence minus one (FMO) controls to exclude background staining.
If peptide pulsed T2 cells are used as target cells, then T2 cells are pulsed with the relevant peptide prior to step (a), e.g. by resuspension in assay buffer containing the peptide (e.g. 50pM of peptide) and incubating e.g. for 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide.
In some embodiments, such an assay comprises the use of peptide pulsed T2 cells or cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs or pan- T cells as effector cells, and the assay comprises:
(a) If peptide pulsed T2 cells are used as target cells, then T2 cells are resuspended in assay buffer (e.g. RPMI/10% FBS/1% P/S) containing peptide (e.g. about 50pM of peptide), which may be PRAME425'433 or a comparator peptide, incubating e.g. for about 4.5hrs at about 37°C, and subsequently centrifuging the resulting peptide pulsed T2 cells and resuspending them in fresh assay buffer to remove excess peptide (if cancer cells are used as target cells then this step is not performed);
(b) Pelleting and resuspending target cells (peptide pulsed T2 cells or cancer cells) in PBS (e.g. 1ml Dulbecco's phosphate-buffered saline (DPBS)) containing CFSE (e.g. about 0.5pM CFSE), incubating for e.g. about 8 minutes at room (or ambient) temperature (e.g. about 25°C), and adding assay buffer (e.g. about 9ml of assay buffer);
(c) Pelleting the target cells, washing the target cells once in assay buffer, counting and resuspending the target cells at a desired concentration;
(d) Diluting the antigen binding protein (e.g. antibody) to be tested e.g. to 10x the final concentrations required in assay buffer;
(e) If PBMCs are used as effector cells, preparing PBMCs, e.g. by thawing frozen PBMCs obtained from liquid nitrogen storage (e.g. in a 37°C water bath), and transferring said PBMCs drop wise into pre-warmed assay buffer, centrifuging and resuspending the PBMCs in fresh assay buffer, counting and resuspending the PBMCs at a desired concentration; or
If pan-T cells are used as effector cells, as is preferable, preparing pan-T cells from PBMCs (e.g. prepared as in step (e) wherein counting and resuspending the PBMCs at a desired concentration in not necessary), using any known protocol, e.g. using the EasySep™ Human T cell isolation kit /protocol, e.g. said kit /protocol described in the Examples herein;
(f) Preparing a co-culture of PMBCs or preferably pan-T cells (effector cells “E”) and target cells (“T”) (i.e. peptide pulsed T2 or cancer cells (e.g. cancer cell line cells)) in wells of a culture plate at an E:T ratio of about 1 :1 (e.g. 10,000 PBMCs and 10,000 target cells) e.g. in a total volume of about 180pl;
(g) Adding e.g. about 20pl of the antigen binding protein (e.g. antibody) to be tested to the wells e.g. to make a final volume of about 200pl;
(h) Incubating the co-culture at about 37°C e.g. for about 72hrs;
(i) If adherent cancer cells were used as target cells, then detaching said adherent cells by incubation with Trypsin/EDTA, and adding cell culture supernatant back to detached cells (this step is not performed if the target cells are T2 cells or non-adherent cancer cells);
(j) Transferring target cells to a fresh culture plate (e.g. a 96-well plate);
(k) Staining dead cells using 7-AAD (e.g. about 1 l 7-AAD/well); and
(l) Analysing fluorescence (of cells) using a flow cytometer (e.g. a BD Canto II).
In such assays, fluorescence (from cells) is from CFSE and 7-AAD labelled cells. The different fluorescence signals can be distinguished using standard flow cytometry techniques, thereby permitting detection of proliferation either from all T cells and/or from particular T cell types of interest detection of dead cell signal (via 7- AAD fluorescence) specifically from target cells (via CFSE fluorescence).
In such an assay, the antigen binding protein may be an antibody in any T- cell engager antibody format, preferably a bispecific format. In a preferred assay, the antibody is in the scDb format.
A preferred assay for assessing the ability of an antigen binding protein (e.g. antibody) to induce T-cell mediated cytotoxicity against a target cell is described in the Examples section herein.
EC50
The T-cell mediated cytotoxicity assay described above (Assay 5) was performed in Example 4 (results shown in Figure 10), which allowed for the determination of the EC50 value for many antibodies of the invention disclosed in Table A, 5-A05, which was assayed over a concentration range of 0.01-1000ng/ml.
The parameter “ECso“ is the concentration of the antigen binding protein (e.g. antibody) of the invention that is necessary to achieve half of the maximum possible effect, which in the present case is half of the observed T-cell mediated cytotoxicity of target cells.
For example, the exemplified 5-A05 antibody of the invention in the scDb format shows (Figure 10A & 10B, and Example 4): an EC50 (for cytotoxicity) of 14.2 pM as measured in the cancer cell line NCI- H1703; an EC50 (for cytotoxicity) of 22.2 pM as measured in the cancer cell line A375; an EC50 (for cytotoxicity) of 13.2 pM as measured in the cancer cell line
THP1 ; and an EC50 (for cytotoxicity) of 6.9 pM as measured in the cancer cell line SKM1.
Example 4 (Table H) further demonstrates EC50 values for many of the antibodies of the invention disclosed in Table A in the scDb format, as measured in the cancer cell line NCI-H1703, with EC50 values ranging from 3.8 pM to 32.8 pM,
and most below 25 pM. Such cytotoxicity values in the low (single digit or low double digit) picomolar range are markedly advantageous.
Example 4 (Table I) further demonstrates EC50 values for many of the antibodies of the invention disclosed in Table A in the scDb format, as measured in the cancer cell line THP-1 , with EC50 values ranging from 6.1 pM to 17.7 pM. Such cytotoxicity values in the low (single digit or low double digit) picomolar range are markedly advantageous.
As mentioned above, in assays of the capability of an antigen binding protein (e.g. antibody) in a T-cell engager format to induce T-cell activity, including cytotoxicity, against target cells, the apparent activity/efficacy of the antigen binding protein (e.g. antibody) can be artificially inflated through use of a high E:T ratio in the step(s) of co-culturing the effector cells comprising T cells (“E”) and the target cells (“T”). The E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity against the target cells. E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested.
The presently described EC50 values exhibited by the antigen binding proteins (e.g. antibodies) of the invention are exhibited in assays in which an E:T ratio of 1 :1 is used. Lower (i.e. better) EC50 values could be achieved if a higher E:T ratio was used, e.g. 10:1.
Using the same assay protocol in each case, i.e. in which an E:T ratio of 1 :1 was used, the present inventors have demonstrated that the antigen binding proteins (e.g. antibodies) of the invention have marked cytotoxicity, exhibiting EC50 values in the picomolar range as measured in disparate cancer cell types. Preferably the antigen binding proteins (e.g. antibodies) of the invention exhibit EC50 values in the picomolar or femtomolar range as measured in cancer cells.
Thus, preferably, antigen binding proteins (e.g. antibodies) of the present invention, for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 of less than 1O'10 M as measured in cancer cells, e.g. less than 5 x 10'11 M, 2.5 x 10'11 M, 10'11 M, 10'12 M, 10'13 M, or 10'14 M as measured in cancer cells.
Preferably, antigen binding proteins (e.g. antibodies) of the present invention, for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 of less than 5 x 10'11 M, 4 x 10'11 M, 3 x 10'11 M, 2.5 x 10’11 M, 2 x 10-11 M, 1 .5 x 10'11 or 1 x 10'11 M, preferably less than 2.5 x 10’11 M, 2 x
10-11 M, 1.5 X 10-11 or 1 X 10’11 M, more preferably less than 1 x 10’11 M, 9 x 10'12, 8 x 10'12 M, 7 x 10'12 M, 6 x 10'12 M, or 5 x 10'12 M, e.g. less than 4 x 10'12 M, e.g. less than 3 x 10'12 M, less than 2 x 10'12 M or less than 1 x 10'12 M, as measured in cancer cells.
Preferably the EC50 value is as measured in HLA-A*02:01/PRAME425'433 positive cancer cells, e.g. cancer cell line cells, preferably NCI-H1703 cells, A375 cells, THP1 cells or SKMI cells, preferably NCI-H1703 cells, A375 cells, or THPI cells, more preferably NCI-H1703 cells or THPI cells, most preferably NCI-H1703 cells.
Preferably, such EC50 values are as determined in an assay comprising the co-culturing of cancer cells (target cells “T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of a marker of cell death. Preferably, such detection and quantification is performed by staining for non-viable cells (e.g. with 7-amino actinomycin D (7-AAD)) and detecting said stain, e.g. via flow cytometry.
In such assays, the effector cells may be as defined elsewhere herein. Preferred effector cells are PBMCs, or pan-T cells derived therefrom.
In such assays, preferably the step of co-culturing the target and effector cells is performed for about 72 hours, at about 37°C.
In such an assay, the antigen binding protein may be an antibody in any T- cell engager format, preferably a bispecific format. In a preferred assay, the antibody is in the scDb format.
A preferred assay for determining EC50 values is set out in Assay 5 above, and in the Examples.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such EC50 values.
The antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against HLA-A*02:01/PRAME425'433 cancer cells, preferably with the above-mentioned EC50 values. Preferably, the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against one or more of NCI-H1703 cells, A375 cells, and THP1 cells, preferably with the above-mentioned EC50 values.
Preferably, the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against NCI-H1703 cells, preferably with the above-mentioned EC50 values. Preferably, the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against NCI-H1703 cells, and one or more of A375 cells,
and THP1 cells, preferably with the above-mentioned EC50 values. Preferably, the antigen binding proteins (e.g. antibodies) of the invention exhibit cytotoxicity against NCI-H1703 cells, and THP1 cells, preferably with the above-mentioned EC50 values.
Preferably, such EC50 values are as determined in an assay comprising the co-culturing of cancer cells (target cells “T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of a marker of cell death. Preferably, such detection and quantification is performed by staining for non-viable cells (e.g. with 7-amino actinomycin D (7-AAD)) and detecting said stain, e.g. via flow cytometry.
In such assays, the effector cells may be as defined elsewhere herein. Preferred effector cells are PBMCs, or pan-T cells derived therefrom.
In such assays, preferably the step of co-culturing the target and effector cells is performed for about 72 hours, at about 37°C.
In such an assay, the antigen binding protein may be an antibody in any T- cell engager format, preferably a bispecific format. In a preferred assay, the antibody is in the scDb format.
A preferred assay for determining EC50 values is set out in Assay 5 above, and in the Examples.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such EC50 values.
Binding affinity
In embodiments, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, may have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of about 1200 nM, 1000 nM, 800 nM, 600 nM, 500 nM, 400 nM, 300nM, 250nM, 200nM, 150nM, 100nM, 50nM, 10, 5, 4, 3, 2, or 1nM, 500pM, 400pM, 300pM, 200pM, 100pM, 50pM, 20pM, 10pM, or 5pM.
Preferably, antigen binding proteins (e.g. antibodies) of the present invention, for example when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433, e.g. have a KD (equilibrium dissociation constant) in the range of 2000 nM or less, 1800 nM or less, 1600 nM or less, 1400 nM or less, preferably 1200 nM or less, 1000 nM or less, 800 nM or less, 600 nM or less, or 500 nM or less, or 300 nM or less for example when determined in an SPR assay.
Thus, antigen binding proteins (e.g. antibodies) of the invention, for example when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of less than 2000 nM, less than 1800 nM, less than 1600 nM, less than 1400 nM, preferably less than 1200 nM, less than 1000 nM, less than 800 nM, less than 600 nM, less than 500nM, less than 400nM, less than 375nM, less than 350nM, or less than 300nM.
Preferably, the antigen binding proteins (e.g. antibodies) of the invention, for example when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of less than 300nM, less than 250nM, 200nM, 150nM, 100nM, 50nM, 10, 5, 4, 3, 2, or 1 nM, or less than 500pM, less than 400pM, less than 300pM, 200pM, 100pM, 50pM, 20pM, 10pM, or 5pM.
However, the present antibodies demonstrate advantageous cytotoxicity against target-positive cancer cells, despite having relatively weak target binding affinity (in the nanomolar or high picomolar range, rather than in the mid- or low picomolar range). Far from being a disadvantage of the present antibodies, this relatively weak target binding affinity is advantageous. When a plurality of different antibodies that achieve the same or similar levels of efficacy/activity/cytotoxicity, antibodies that display a weaker binding affinity are actually advantageous in terms of their safety profile, since an antibody with a weaker affinity for the target is less likely to bind strongly to off-target molecules of a similar structure. Highly cytotoxic antibodies often bind to their targets with high affinity, but antibodies that display similar cytotoxicity with a lower affinity would be desirable given the balance of efficacy and safety
Thus, preferably, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of 1 to 1200 nM, 1 to 1000 nM, 1 to 500nM, 1 to 400nM, 1 to 375nM, or 1 to 350nM, more preferably 1 to 300nM.
Preferably, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of 1 to 300 nM, 1 to 250 nM, or 1 to 200 nM.
Preferably, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of 3 to 1200 nM, 3 to
1000 nM, 3 to 500nM, 3 to 400nM, 3 to 375nM, or 3 to 350nM, more preferably 3 to 300nM.
Preferably, the antigen binding proteins (e.g. antibodies) of the invention when in a T-cell engager format, preferably in the scDb format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of 3 to 300 nM, 3 to 250 nM, or 3 to 200 nM.
In embodiments, the antigen binding proteins (e.g. antibodies) of the invention when in the Fab format, have a binding affinity for HLA-A*02:01/PRAME425'433 that is or corresponds to a KD of 1 to 5000 nM, e.g. 50 to 4700 nM, or 100 to 4700 nM, preferably 50 to 300 nM, or 100 to 250 nM.
For example, the exemplified 5-A05 antibody of the invention shows a binding affinity of 254 nM in the scDb format (Example 2), and the 5-A05/1-C04 antibody shows a binding affinity of 155nM in the scDb format (Example 2).
For example, exemplified antibodies of the invention in the Fab format show the following binding affinities: 558 nM (5-A05), 4656 nM (5-A05/1-C04), 113 nM (PAM22) and 231 nM (NGS17) (Example 2).
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such binding affinity.
ASSAY 6 : Binding Affinity
Any suitable method of determining the binding affinity (KD) may be used and the skilled person is familiar with suitable methods. Preferably the KD is determined in a Surface Plasmon Resonance assay (e.g. a BIAcore assay), preferably in which kinetic parameters are determined.
Suitable SPR assays are known in the art and for example may involve immobilising HLA-A*02:01/PRAME425'433 on a solid support and applying the antigen binding protein (e.g. antibody) to be tested. Suitable assays are discussed elsewhere herein. Particularly preferred SPR assays are described in the Examples section herein.
In certain preferred SPR assays, HLA-A*02:01/PRAME425'433 is captured (or immobilised) on a solid support (e.g. a sensor chip) and various concentrations (e.g. a dilution series, e.g. a doubling dilution series) of the antigen binding protein (e.g. antibody) to be tested is then injected. Antigen binding protein (e.g. antibody) concentrations and response units (Rll) are generally selected at a range and level, respectively, such that the chip is not saturated and which allow robust fitting, e.g. robust 1 :1 fitting, by the SPR/Biacore software. Preferred concentrations and flow-
rates for injection, together with appropriate RU Units are described in the Examples section.
Suitable association periods and dissociation periods to be used in an SPR assay are known to a skilled person, for example, a preferred association period in the SPR assay is 2 minutes and a preferred dissociation period in the SPR assay is 2 minutes (in a single cycle analysis). In certain embodiments, all measurements may be performed at 25°C in 20mM PBS, pH7.4, 2.7mM KCI, 137 mM NaCI. Kinetic parameters may be determined or calculated by any suitable model or software, for example by fitting the sensorgram experimental data assuming a 1 :1 interaction, in other words using a 1 :1 binding model, for example using Single Cycle Kinetics software. Particularly preferred SPR assays are described in the Examples section herein. Preferably a single cycle analysis is used.
In some embodiments, binding affinity (KD) of an antigen binding protein (e.g. antibody) of the invention to H LA-restricted peptide (pHLA), e.g. HLA- A*02:01/PRAME425'433 may be as determined by (or as assessed by) an SPR assay, e.g. an SPR assay that comprises:
(a) Coupling NeutrAvidin (e.g. 10 pg/mL in 10 mM sodium acetate, pH 5.0), to a solid support (e.g. a sensor chip, such as a CM3 series S sensor chip) via amine coupling, e.g. to 500 response units (RU);
(b) Adding (capturing), pHLA (e.g. soluble, recombinant, biotinylated pHLA) (e.g. 1 pg/mL) on said solid support e.g. to 20-100 RU,
(c) Applying the antigen binding protein (e.g. antibody) to be tested to the solid support;
(d) Using an association period of 2 min for injection of samples, in a concentration series of single cycle kinetic experiments;
(e) Using dissociation periods of 2 min between injection of samples in a concentration series of single cycle kinetics experiments, and a final dissociation time of 20 min; and
(f) Obtaining sensorgram data and determining binding affinity (KD) using a suitable model or software, for example by using S200 Evaluation Software v1.1 ., e.g. by fitting the sensorgram data from Single Cycle Kinetics method to a 1 :1 Langmuir binding model after buffer subtraction and NeutrAvidin-reference-cell subtraction, wherein all measurements are performed at 25°C in 20mM PBS, pH7.4, 2.7mM KCI, 137 mM NaCI.
Thermostability
Example 2 herein describes the assessment of the thermal stability of an antibody of the invention, 5-A05, in the scDb format. As shown in Figure 6, the melting temperature of 5-A05, measured by thermal unfolding and defined as the temperature at which 50% of the molecules are unfolded, was 66.7°C. This advantageous stability was surprising. The antigen binding Fab unit of the wellperforming therapeutic antibody Trastuzumab was included as control, in a format (Fab) which normally has higher thermostability due to the CH1-CL pair, yet 5-A05 (in scDb) format had a similar melting temperature.
The same analysis was performed using the antibody 5-A05/1-C04 disclosed in Table A, and a similar thermal stability was observed as for 5-A05 (67.9°C, data not shown).
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in an scDb format, have a melting temperature of at least 60°C, preferably at least 62°C, more preferably at least 64°C, more preferably at least 66°C, e.g. 60°C to 80°C, 62°C to 80°C, 64°C to 80°C, or 66°C to 80°C, or 60°C to 75°C, 62°C to 75°C, 64°C to 75°C, or 66°C to 75°C, more preferably 63°C to 71 °C, 64°C to 70°C, 65°C to 69°C, or 66°C to 68°C.
For example, the exemplified 5-A05 antibody of the invention shows a melting temperature of 66.7°C in the scDb format (Example 2, Figure 6) and the 5-A05 antibody shows a melting temperature of 67.9°C in the scDb format (Example 2).
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such a melting temperature.
The melting temperature is the temperature at which 50% of the molecules are unfolded.
ASSAY 7 : Thermostability
Any suitable method of determining the melting temperature may be used and the skilled person is familiar with suitable methods. Preferably the melting temperature is determined in a nano differential scanning fluorimetry (nanoDSF) assay, which is a well-known assay in the field, in which tryptophan or tyrosine fluorescence is used to monitor protein unfolding, and the ratio of the fluorescence intensities at 350 nm and 330 nm is suitable to detect any changes in protein structure due to protein unfolding. Particularly preferred melting temperature assays are described in the Examples section herein.
In some embodiments, the melting temperature of an antigen binding protein (e.g. antibody) of the invention to H LA-restricted peptide (pHLA), e.g. HLA-
A*02:01/PRAME425'433 may be as determined by (or as assessed by) a nanoDSF method, e.g. a nanoDSF method that comprises:
(a) Preparing a solution of antigen binding protein (e.g. antibody) to be tested, e.g. in PBS, e.g. at a concentration of 0.04 - 0.46 mg/ml;
(b) Transferring about 10 pL of said solution to glass capillaries (e.g. NanoTemper capillaries), e.g. in triplicates;
(c) Performing a discovery scan to set a suitable instrument laser intensity (e.g. set to 40% of maximal intensity)
(d) Subjecting samples to a temperature scan e.g. from about 20 °C to about 95 °C, with 1 °C/min increments, e.g. using a Prometheus nanoDSF (NanoTemper);
(e) Applying an excitation wavelength of 295 nm, and measuring emission at 330 nm and 350 nm;
(f) Analysing collected data, e.g. using AB-Protein PR.ThermControl V2.12, to determine melting temperatures.
A preferred assay is described in the Examples section herein.
Induction of IFNy release from T-cells
Cytotoxic T cells release cytokines such as IFN-y, which induce the increased expression of MHC class I and other molecules involved in peptide loading in cancer cells, which in turn increases the chance that cancer cells will be recognized as target cells for cytotoxic attack. IFN-y also activates macrophages, recruiting them to target sites both as effector cells and as antigen-presenting cells. The ability of an antigen binding protein, particularly at low concentrations, to induce the release of IFN-y from T-cells when co-cultured with HLA-A*02:01/PRAME425'433 positive target cells would therefore be advantageous, and demonstrative of the cytotoxic and therapeutic activity of the molecules.
The antigen binding proteins (e.g. antibodies) of the invention, when in a T- cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of interferon gamma (IFNy) from T cells directed against cells displaying H LA-A*02: 01 /PRAM E425’433.
Alternatively viewed, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), are capable of preferentially (or selectively) inducing release of IFNy from T cells directed against cells displaying HLA-A*02:01/PRAME425-433.
Thus, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or
selectively) induce release of IFNy from T cells directed against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy of T cells directed against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
In an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy from T cells directed against cells displaying HLA- A*02:01/PRAME425'433 as compared to against cells displaying HLA-A*02:01 without the PRAME425'433 peptide complexed thereto and/or (preferably “and”) against cells displaying a HLA-A*02:01 restricted peptide that is not PRAME425'433.
Alternatively viewed, in an embodiment, the antigen binding proteins (e.g. antibodies) of the invention, when in a T-cell engager format (e.g. in the scDb format), preferentially (or selectively) induce release of IFNy from T cells directed against HLA-A*02:01/PRAME425'433 positive cells as compared to against HLA- A*02:01 positive, PRAME425'433 negative cells.
The HLA-A*02:01/PRAME425'433 displaying, or positive or negative cells referred to herein, may be any cell type including the preferred target and cancer cell types disclosed elsewhere herein.
The T-cells may be as defined anywhere else herein, and are preferably CD4+ T cells or CD8+ T cells.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such inducement of T-cell activation, differentiation, or proliferation.
The term "induce” as used herein means promote, i.e. enhance or increase the stated property or activity. Induce need not mean that the starting level of the property or activity is zero, i.e. it may mean “induce further”.
Preferably such induction of release of IFNy from T cells is exhibited when the antigen binding protein is any T-cell engager format, preferably a bispecific format. Preferably, the antibody is in the scDb format.
Preferably such induction of release of IFNy from T cells is exhibited when the ratio of T cells (or effector cells comprising T cells) to cells displaying HLA- A*02:01/PRAME425'433 (“target cells”) is about 1 :1. Thus, preferably such induction
of release of IFNy from T cells is exhibited in an assay comprising the co-culturing of HLA-A*02:01/PRAME425'433 positive target cells (“T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably tested over a concentration range, with subsequent detection and quantification of IFNy. Preferably, such detection and quantification is as described below.
ASSAY 8 : Assay for induced T-cell release of IFNy
The ability of an antigen binding protein (e.g. antibody) in a T-cell engager format to induce IFNy release from PBMCs, more specifically T cells when cocultured with cells displaying a H LA-restricted peptide (pH LA) (“target cells”) can be assessed by any appropriate means and the skilled person is familiar with suitable methods, e.g. co-culturing target cells (“T”), and effector cells (“E”) comprising T cells, such as PBMCs, preferably at an E:T ratio of about 1 :1 , together with the antigen binding protein (e.g. antibody) to be tested, and subsequently detecting and quantifying IFNy release from PBMCs. Such detection and quantification may be performed by measuring the concentration of IFNy in the cell culture medium following co-culture.
Target cells may be T2 cells pulsed with exogenously applied PRAME425'433 peptide. In these assays, control cells are non-pulsed T2 cells, and T2 cells pulsed with an unrelated peptide. Alternatively, target cells may be HLA- A*02:01/PRAME425'433 positive cancer cells. In these assays control cells are HLA- A*02:01/PRAME425'433 negative cancer cells.
In assays of the ability of a T-cell engager to induce T-cell release of IFNy, it is possible to use T cells, or a subset of T cells as the effector cells (e.g. PBMCs or pan T-cells isolated from PBMCs).
In such assays, the apparent efficacy of an antibody can be artificially inflated through use of a high E:T ratio in the co-culturing steps. The E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity (e.g. IFNy release) against the target cells. E:T ratios of 10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested. Therefore, it is preferred that an E:T ratio of 1 :1 is used.
Assay kits for the detection and quantification of IFN- y are well-known and widely available, for instance the ELISA Max Set from BioLegend ®, and any suitable assay or kit may be used.
Thus, a suitable assay may comprise
(a) Co-culturing in wells of an assay plate HLA-A*02:01/PRAME425'433 positive target cells (“T”), e.g. cancer cells (e.g. cancer cell line cells), as described above, and effector cells comprising T cells (“E”), e.g. PBMCs as described above, at an E:T ratio of about 1 :1 , and the antigen binding protein (e.g. antibody) to be tested, e.g. at a single concentration (e.g. about 1 nM), or over a concentration range (e.g. of about 0.1-10,000pM), and co-culturing e.g. for about 24h at about 37°C;
(b) Harvesting assay supernatants from the assay plates;
(c) Assessing the concentration of IFNy and the EC50 of the response, e.g. via ELISA, e.g. using a commercially available human IFNy ELISA kit.
It is within the competencies of the person of ordinary skill in the art to prepare and make use of suitable controls and standard curves to facilitate correct running of assays and the determination of accurate concentrations.
In some embodiments, such an assay comprises the use of HLA- A*02:01/PRAME425'433 positive cancer cells (e.g. cancer cell line cells) as target cells, and PBMCs as effector cells, and the assay comprises:
(a) Co-culturing (e.g. in a total volume of about 200pl) the HLA- A*02:01/PRAME425'433 positive target cells (“T”) and the PBMC effector cells at an E:T ratio of about 1 :1 (e.g. 50,000 PBMCs and 50,000 target cells), and the antigen binding protein (e.g. antibody) to be tested, e.g. at a single concentration (e.g. about 1 nM), or over a concentration range (e.g. of about 0.1-10,000pM), and co-culturing e.g. for about 24h at about 37°C, and harvesting supernatant;
(b) Coating an ELISA plate (i.e. wells of the plate) with IFNy capture antibody, as per manufacturer’s instructions, e.g. for about 16hrs at e.g. at 4°C;
(c) Washing the plate, for example with wash buffer (e.g. PBS with 0.05% Tween 20) at least once (e.g. 4 times);
(d) Incubating the plate (e.g. for about 1 hr), e.g. at room (or ambient) temperature (e.g. 25°C) with assay diluent);
(e) Washing the blocked plates, for example with wash buffer (e.g. PBS with 0.05% Tween 20), at least once e.g. 4 times;
(f) Adding 10OpI of supernatant obtained in step (a) (e.g. for about 2 hrs) at room (or ambient) temperature (e.g. 25°C);
(g) Washing the ELISA plate, for example with wash buffer (e.g. PBS with 0.05% Tween 20) at least once e.g. 4 times;
(h) Incubating in the wells of the plate, e.g. for about 1 hour, for example at room (or ambient) temperature (e.g. 25°C), with about 1 OOpI of a secondary antibody, e.g. a biotin labelled “detection” antibody;
(i) Washing plates, for example with wash buffer (e.g. PBS with 0.05% Tween 20) at least once (e.g. 4 times);
(j) Incubating in the wells of the plate, e.g. for about 30 minutes, for example at room (or ambient) temperature (e.g. 25°C) with about 10OpI of 1 :1000 diluted avidin-HRP;
(k) Washing plates, for example with wash buffer (e.g. PBS with 0.05% Tween 20) at least once (e.g. 4 times);
(l) Incubating in the wells of the plate, e.g. for up to 5 minutes, in the dark, about 100pl of TMB solution (3,3',5,5'-Tetramethylbenzidine);
(m) Immediately after incubation step (I), adding about 100 pl of 1M HCI (i.e. stop solution);
(n) Measuring absorbance e.g. at 450nM, e.g. using a plate reader;
(o) Determining the concentration of I FNy, e.g. by utilising a standard curve.
The T-cell I FNy release assay described above (Assay 8) was performed in Example 5 (results shown in Table J), which allowed for the determination of an EC50 value for the antibodies of the invention disclosed in Table A.
The parameter “ECso“ is the concentration of the antigen binding protein (e.g. antibody) of the invention that is necessary to achieve half of the maximum possible effect, which in the present case is half of the observed maximal amount of IFNy released from T-cells when co-cultured with HLA-A*02:01/PRAME425'433 positive target cells.
Example 5 (Table J) demonstrates EC50 values (for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME425'433 positive cancer cells) for each of the antibodies of the invention disclosed in Table A, with EC50 values ranging from 35 pM to 202 pM, and most below 150 pM. Such values are markedly advantageous.
The present inventors have demonstrated that the antibodies of the invention, advantageously, have marked ability to induce release of I FNy from T-cells when cocultured with HLA-A*02:01/PRAME425'433 positive target (cancer) cells.
As mentioned above, in assays of the capability of an antigen binding protein (e.g. antibody) in a T-cell engager format to induce T-cell activity, including IFNy release, the apparent activity/efficacy of the antigen binding protein (e.g. antibody) can be artificially inflated through use of a high E:T ratio in the step(s) of co-culturing the effector cells comprising T cells (“E”) and the target cells (“T”). The E:T ratio is simply the ratio of the number of effector cells to the number of target cells. The use of a greater number of effector cells relative to target cells is more likely to lead to a higher level of observed effector cell activity against the target cells. E:T ratios of
10:1 are seen in the art, but this is considered a high E:T ratio that can mask poor performance of the antibodies being tested.
The presently described EC50 values for IFNy release from T-cells exhibited by the antigen binding proteins (e.g. antibodies) of the invention are exhibited in assays in which an E:T ratio of 1 :1 is used. Lower (i.e. better) EC50 values could be achieved if a higher E:T ratio was used, e.g. 10:1.
Using the same assay protocol in each case, i.e. in which an E:T ratio of 1 :1 was used, the present inventors have demonstrated that the antigen binding proteins (e.g. antibodies) of the invention achieve marked IFNy release, exhibiting EC50 values in the picomolar range. Preferably the antigen binding proteins (e.g. antibodies) of the invention exhibit EC50 values for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME425'433 positive target (cancer) cells in the picomolar range.
Thus, preferably, antigen binding proteins (e.g. antibodies) of the present invention, for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME425'433 positive target (cancer) cells of less than 5 x 1O'10 M, e.g. less than 1 x 10 10, 5 x 10'11 M, 2.5 x 10’11 M, 10’11 M, 10’12 M, 10’13 M, or 10’14 M.
Preferably, antigen binding proteins (e.g. antibodies) of the present invention, for example when in a T-cell engager format, preferably a bispecific format, e.g. the scDb format, exhibit an EC50 for IFNy release from T-cells in the presence of HLA- A*02:01/PRAME425'433 positive target (cancer) cells of less than 5 x 10'1° M, less than 3 x 10'1° M, less than 2.5 x 10'1° M, less than 2 x 10'1° M, less than 1.5 x 10'1° M or less than 1 x 10'1° M.
Preferably the EC50 value is as measured in HLA-A*02:01/PRAME425'433 positive cancer cells, e.g. cancer cell line cells, preferably THP-1 cells or NCI-H1703 cells, preferably THP-1 cells.
Preferably, such EC50 values are as determined in an assay comprising the co-culturing of cancer cells (target cells “T”) and effector cells comprising T cells (“E”) at an E:T ratio of about 1 :1 (e.g. 1 :1), together with the antigen binding protein (e.g. antibody), preferably over a concentration range, with subsequent detection and quantification of IFNy. Preferably, such detection and quantification is performed by ELISA.
In such assays, the effector cells may be as defined elsewhere herein. Preferred effector cells are PBMCs, or pan-T cells derived therefrom.
In such assays, preferably the step of co-culturing the target and effector cells is performed for about 72 hours, at about 37°C.
In such an assay, the antigen binding protein may be an antibody in any T- cell engager format, preferably a bispecific format. In a preferred assay, the antibody is in the scDb format.
A preferred assay for determining said EC50 values is set out in Assay 8 above, and in the Examples.
Any substantially homologous antigen binding protein (e.g. antibody) of the invention preferably exhibits such EC50 values for IFNy release from T-cells in the presence of HLA-A*02:01/PRAME425'433 positive target (cancer) cells.
As discussed above, several of the above assays comprise the use of effector cells (“E”) and target cells (“T”). Preferably, the E:T ratio used in such assays is no greater than about 1:1, i.e. no more than about 1 :1. Preferably the number of effector cells is not greater than, or is not significantly greater than, the number of target cells. E:T ratios lower than 1 : 1 may be used, however ratios of about 1 :1, e.g. 1 :1 are preferred. It is within the competencies of the person of ordinary skill in the art to count cell densities in cell cultures, dilute cells to a desired density and provide a suitable volume of cells to provide a given number of cells for an assay. In this way, desired E:T ratios can be straightforwardly provided.
In the above discussions of antigen binding protein (e.g. antibody) properties and assays for their assessment, there are disclosed parameters/ conditions with the term “about” applied to a specific value, e.g. “about 72 hours”, “about 20°C”, “about 100 ng/ml”, “about 1:1”, etc. The person of ordinary skill in the art will appreciate that minor modifications of parameters/conditions can be tolerated, and particular parameters/conditions can be readily selected by the skilled person for their particular purposes. For the avoidance of doubt, such disclosures are also disclosures of said specific value in particular, i.e. without the term “about” applied thereto.
Preferably, any substantially homologous antigen binding protein (e.g. antibody) should retain the ability to bind to, or specifically bind to, the same epitope of the antigen as recognized by the antigen binding protein (e.g. antibody) in question, for example, the same epitope recognized by the CDR domains of the invention, or the antigen binding domains of the invention, or the VH and VL domains of the invention, as described herein, e.g. bind to the same epitope as an antibody of the invention shown in Table A, e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody.
Thus, preferably any substantially homologous antigen binding protein (e.g. antibody) should retain the ability to bind to any one or more of, preferably all of,
amino acid positions 5 and 7 of the HLA-A*02:01 restricted peptide PRAME425'433 (SEQ ID NO: 19), which binding and assessment thereof is described elsewhere herein and in the Examples.
Thus, preferably, any substantially homologous antigen binding protein (e.g. antibody) should retain the ability to compete with one or more of the antigen binding proteins of the invention (shown in Table A) for binding to the relevant antigen. Binding to the same epitope/antigen can be readily tested by methods well known and described in the art, e.g. using binding assays, e.g. a competition assay. Suitable binding assays are discussed elsewhere herein.
Binding to the same epitope can, for example be tested, e.g. by using epitope mapping assays, e.g. by analysis of the crystal structure of the antigen-antibody complex, or by mutational studies of individual residues (e.g. using alanine scanning and/or deep mutational scanning, DMS, for example yeast display in combination with DMS, see for example as described in Sierocki et al., 2021 , PLoS Negl Trop Dis.,15(3):e0009231 ; see also Van Blarcom et al., 2015, JMB, 427:6(B):1513-1534 and Medina-Cucurella and Whitehead, 2018, Methods Mol. Biol., 1764:101-121) and any of the above such analyses to determine the epitope can be used.
Thus, antibodies which bind to the same epitope as one or more of the various antibodies of the invention, for example as assessed or determined by one or more of the methods outlines above, form yet further aspects of the invention.
Retention of other functional properties, in particular binding affinity, can also readily be tested by methods well known and described in the art or herein.
Thus, a person skilled in the art will appreciate that binding assays can be used to test whether "substantially homologous" antigen binding proteins (e.g. antibodies) have the same binding specificities as the antigen binding proteins of the invention, for example, binding assays such as competition assays or ELISA assays, e.g. as described elsewhere herein. Surface Plasmon Resonance (e.g. BIAcore) assays could also readily be used to establish whether "substantially homologous" antigen binding proteins can bind to the relevant antigen. The skilled person will be aware of other suitable methods and variations.
As outlined below, a competition binding assay can be used to test whether "substantially homologous" antigen binding proteins (e.g. antibodies) retain the ability to bind to, or specifically bind to, substantially the same epitope of the relevant antigen as recognized by the antigen binding proteins (e.g. antibodies) of the invention (e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody), or have the ability to compete with one or more of the various antigen binding proteins (e.g. antibodies) of the invention. The method described below is only one example
of a suitable competition assay. The skilled person will be aware of other suitable methods and variations.
An exemplary competition assay involves assessing the binding of various effective concentrations of an antigen binding protein (e.g. antibody) of the invention to the relevant antigen in the presence of varying concentrations of a test antigen binding protein (e.g. a substantially homologous antigen binding protein e.g. antibody). The amount of inhibition of binding induced by the test antigen binding protein can then be assessed. A test antigen binding protein that shows increased competition with an antigen binding protein of the invention at increasing concentrations (i.e. increasing concentrations of the test antigen binding protein result in a corresponding reduction in the amount of antigen binding protein of the invention binding to the relevant antigen) is evidence of binding to the same or substantially the same epitope. Preferably, the test antigen binding protein significantly reduces the amount of antigen binding protein of the invention that binds to the relevant antigen. Preferably, the test antigen binding protein reduces the amount of antigen binding protein of the invention that binds to the relevant antigen by at least about 95%. ELISA assays may be used for assessing inhibition of binding in such a competition assay but other suitable techniques would be well known to a person skilled in the art.
In some embodiments, “substantially homologous” antigen binding proteins (e.g. antibodies) which retain the ability to bind to, or specifically bind to, substantially the same (or the same) epitope of the relevant antigen as recognized by antigen binding proteins (e.g. antibodies) of the invention (e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody) or which have the ability to compete with one or more of the various antigen binding proteins (e.g. antibodies) of the invention (e.g. the 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 antibody) are preferred.
Such antigen binding proteins (e.g. antibodies) which have the ability to bind (or specifically bind) to the same (or substantially the same) epitope of HLA- A*02:01/PRAME425-433 as recognized by the antibody of the invention (e.g. 5-A05, 5- A05/1-C04, PAM22, PAM23 or NGS55 of Table A) are further embodiments of the present invention. Such antigen binding proteins (e.g. antibodies) which have the ability to compete with one or more of the antibodies of the invention (e.g. with 5-A05, 5-A05/1-C04, PAM22, PAM23 or NGS55 shown in Table A) for binding to HLA- A*02:01/PRAME425'433 are further embodiments of the present invention.
Thus, a yet further aspect of the invention provides an antigen binding protein (e.g. antibody), which binds to (or specifically binds to) HLA-A*02:01/PRAME425'433 and which has the ability to bind to the same (or substantially the same) epitope for binding to HLA-A*02:01/PRAME425-433 as:
the 5-A05 antibody (Table A), i.e. an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO:3, as described herein, or the ability to bind to the same (or substantially the same) epitope as an antibody comprising the same CDRs as the 5-A05 antibody (Table A), i.e. an antibody comprising VL CDR sequences of SEQ ID NOs: 8, 9 and 10 and VH CDR sequences of SEQ ID NOs: 5, 6 and 7; the 5-A05/1-C04 antibody (Table A), i.e. an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO:132, as described herein, or the ability to bind to the same (or substantially the same) epitope as an antibody comprising the same CDRs as the 5-A05/1-C04 antibody (Table A), i.e. an antibody comprising VL CDR sequences of SEQ ID NOs: 8, 9 and 10 and VH CDR sequences of SEQ ID NOs: 111, 112 and 113; the PAM22 antibody (Table A), i.e. an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO: 142, as described herein, or the ability to bind to the same (or substantially the same) epitope as an antibody comprising the same CDRs as the PAM22 antibody (Table A), i.e. an antibody comprising VL CDR sequences of SEQ ID NOs: 8, 9 and 10 and VH CDR sequences of SEQ ID NOs: 111 , 114 and 113; the PAM23 antibody (Table A), i.e. an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO: 144, as described herein, or the ability to bind to the same (or substantially the same) epitope as an antibody comprising the same CDRs as the PAM23 antibody (Table A), i.e. an antibody comprising VL CDR sequences of SEQ ID NOs: 8, 9 and 10 and VH CDR sequences of SEQ ID NOs: 111 , 115 and 113; or the NGS55 antibody (Table A), i.e. an antibody comprising the VL of SEQ ID NO:147 and the VH of SEQ ID NO:132, as described herein, or the ability to bind to the same (or substantially the same) epitope as an antibody comprising the same CDRs as the NGS55 antibody (Table A), i.e. an antibody comprising VL CDR sequences of SEQ ID NOs: 120, 9 and 10 and VH CDR sequences of SEQ ID NOs: 111 , 112 and 113.
In a further aspect, such antigen binding proteins (e.g. antibodies) have the ability to compete with one or more of the antibodies of the invention (e.g. 5-A05, 5- A05/1-C04, PAM22, PAM23 or NGS55 (Table A)) for binding to HLA- A*02:01/PRAME425'433. Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.
The term "competing antigen binding protein", as used herein, refers to antigen binding proteins (e.g. antibodies) that bind to about, substantially or essentially the same, or even the same, epitope as a "reference antigen binding protein" (e.g. antibody). Competing antigen binding proteins are thus able to
effectively compete with a reference antigen binding protein for binding to the relevant antigen. Preferably, the competing antigen binding protein (e.g. antibody) can bind to the same epitope as the reference antigen binding protein (e.g. antibody). Alternatively viewed, the competing antigen binding protein (e.g. antibody) preferably has the same epitope specificity as the reference antigen binding protein (e.g antibody).
"Reference antigen binding proteins" as used herein are antigen binding proteins which can bind to the relevant antigen in accordance with the invention. Preferably, reference antigen binding proteins (e.g. antibodies) have a VH and a VL domain as defined herein. For example, a reference antigen binding protein which binds to, or specifically binds to, HLA-A*02:01/PRAME425'433 may comprise a VL domain and a VH domain of the 5-A05 antibody (i.e. comprise a VL domain of SEQ ID NO:4 and a VH domain of SEQ ID NO:3), or of any other antibody disclosed in Table A. Reference antigen binding proteins are preferably antibodies. Thus a preferred reference antigen binding protein may be the 5-A05 antibody as defined herein (Table A).
The identification of one or more competing antigen binding proteins (e.g. antibodies) is a straightforward technical matter now that reference antibodies such as the 5-A05 antibody and the other antibodies of the invention disclosed in Table A have been provided. As the identification of competing antigen binding proteins (e.g. antibodies) is determined in comparison to a reference antigen binding protein (e.g. antibody), it will be understood that actually determining the epitope to which either or both antigen binding proteins bind is not in any way required in order to identify a competing antigen binding protein. However, epitope mapping can be performed using standard techniques, if desired.
The CDRs of the antigen binding proteins (e.g. antibodies) of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and/or effective engineered antibodies. Thus, the VH, VL and individual CDR sequences of the invention are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding, herein HLA-A*02:01/PRAME425'433 binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1 , FR2, FR3 and/or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions.
Appropriate sequences that can be used for framework regions are well known and documented in the art and any of these may be used. Exemplary
sequences for framework regions are one or more of the framework regions making up the VH, and/or VL domains of the antibodies of the invention, e.g. one or more of the framework regions of antibodies as disclosed in Table A (e.g., 5-A05, 5-A05/1- C04, PAM6, PAM8, PAM 17, PAM 18, PAM22, PAM23, NGS55, or NGS17), or framework regions substantially homologous thereto, and in particular framework regions that allow the maintenance of antigen specificity, for example framework regions that result in substantially the same or the same 3D structure of the antibody.
Preferably, the antigen binding protein (e.g. antibody) comprises a VH domain that comprises a VH FR1 , a VH FR2, a VH FR3 and a VH FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH FR1 VH FR2 VH FR3 VH FR4 a) SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 ; or
. x SEQ ID NO: SEQ ID NO: SEQ ID NO: b) SEQ ID NO: 14 122 123 124 and/or (preferably “and”) a VL domain that comprises a VL FR1, a VL FR2, a VL FR3 and a VL FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL FR1 VL FR2 VL FR3 VL FR4 a) SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 ; or
SEQ ID NO: SEQ ID NO: SEQ ID NO:
SEQ ID NO: 18 ; or
125 127 128
SEQ ID NO: SEQ ID NO: SEQ ID NO:
SEQ ID NO: 130 .
126 127 129
Preferably, the antigen binding protein (e.g. antibody) comprises a VH domain that comprises a VH FR1 , a VH FR2, a VH FR3 and a VH FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
VH FR1 VH FR2 VH FR3 VH FR4
SEQ ID NO: SEQ ID NO: SEQ ID NO:
SEQ ID NO: 14
122 123 124 and/or (preferably “and”) a VL domain that comprises a VL FR1, a VL FR2, a VL FR3 and a VL FR4 comprising the following amino acid sequences or sequences substantially homologous thereto:
VL FR1 VL FR2 VL FR3 VL FR4 a) SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18 ; or
. . SEQ ID NO: SEQ ID NO: SEQ |D NO: 18, } 125 127
more preferably a) above.
Preferably, the antigen binding protein (e.g. antibody) comprises one or more, preferably all, of the framework regions making up the VH, and/or (preferably “and”) VL domains of a specific antibody of the invention disclosed in Table A, or framework regions substantially homologous thereto. In certain embodiments, all four of the variable heavy chain (e.g. a) SEQ ID NOs:11 , 12, 13 and 14; or b) SEQ ID NOs: 122, 123, 124 and 14) and/or variable light chain (e.g. a) SEQ ID NOs:15, 16, 17 and 18; or b) SEQ ID Nos: 125,127, 128 and 18; or c) SEQ ID Nos: 126, 127, 129 and 130) framework regions (FR), as appropriate, or FR regions substantially homologous thereto, are found in the antibodies (or binding proteins) of the invention.
The antigen binding protein (e.g. antibody) and nucleic acid molecules of the invention are generally "isolated" or "purified" molecules insofar as they are distinguished from any such components that may be present in situ within a human or animal body or a tissue sample derived from a human or animal body. The sequences may, however, correspond to or be substantially homologous to sequences as found in a human or animal body. Thus, the term "isolated" or "purified" as used herein in reference to nucleic acid molecules or sequences and proteins or polypeptides, e.g. antigen binding proteins (e.g. antibodies), refers to such molecules when isolated from, purified from, or substantially free of their natural environment, e.g. isolated from or purified from the human or animal body (if indeed they occur naturally), or refers to such molecules when produced by a technical process, i.e. includes recombinant and synthetically produced molecules.
Thus, when used in connection with a protein or polypeptide molecule such as light chain CDRs 1 , 2 and 3, heavy chain CDRs 1 , 2 and 3, light chain variable regions (domains), heavy chain variable regions (domains), and antigen binding proteins (e.g. antibodies) of the invention, including full length antibodies, the term "isolated" or "purified" typically refers to a protein substantially free of cellular material or other proteins from the source from which it is derived. In some embodiments, particularly where the protein is to be administered to humans or animals, such isolated or purified proteins are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
It can be noted that in embodiments the antigen binding proteins (e.g. antibodies) etc., of the invention do not occur in nature and are, in that respect, manmade constructs in that they do not correspond to molecules that occur naturally. For example, preferred antigen binding proteins (e.g. antibodies) can be engineered or recombinantly produced. In other words in embodiments the antigen binding proteins (e.g. antibodies) etc, of the invention are non-native or not naturally occurring.
A person skilled in the art will appreciate that the proteins and polypeptides of the invention, such as the heavy and light chain CDRs, the heavy and light chain variable regions (domains), antigen binding proteins, antibodies and antibody fragments, may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods.
In preferred embodiments the antibodies of the invention are human antibodies, more preferably fully human antibodies. In this regard, human antibodies generally have at least two potential advantages for use in human therapy. First, the human immune system should not recognize the antibody as foreign. Second, the half-life in the human circulation will be similar to naturally occurring human antibodies, allowing smaller and less frequent doses to be given.
The term "human" as used herein in connection with antibody molecules and binding proteins first refers to antibodies and binding proteins having variable regions e.g., VH, VL, CDR or FR regions) and, optionally, constant antibody regions, isolated or derived from a human repertoire or derived from or corresponding to sequences found in humans or a human repertoire, e.g., in the human germline or somatic cells.
The "human" antibodies and binding proteins of the invention further include amino acid residues not encoded by human sequences, e.g., mutations introduced by random or site directed mutations in vitro, for example mutations introduced by in vitro cloning or PCR, for example in an affinity maturation method. Particular examples of such mutations are mutations that involve conservative substitutions or other mutations in a small number of residues of the antibody or binding protein, e.g., in up to 6, 5, 4, 3, 2 or 1 of the residues of the antibody or binding protein, preferably e.g., in up to 4, 3, 2 or 1 of the residues making up one or more of the CDRs of the antibody or binding protein. Certain examples of such "human" antibodies include antibodies and variable regions that have been subjected to standard modification techniques to reduce the amount of potentially immunogenic sites.
Thus, the "human" antibodies of the invention include sequences derived from and related to sequences found in humans, but which may not naturally exist within the human antibody germline repertoire in vivo. In addition, the human antibodies
and binding proteins of the present invention include proteins comprising human consensus sequences identified from human sequences, or sequences substantially homologous to human sequences.
In addition, the human antibodies and binding proteins of the present invention are not limited to combinations of VH, VL. CDR or FR regions that are themselves found in combination in human antibody molecules. Thus, the human antibodies and binding proteins of the invention can include or correspond to combinations of such regions that do not necessarily exist naturally in humans (e.g. are not naturally occurring antibodies).
In preferred embodiments, the human antibodies will be fully human antibodies. "Fully human" antibodies, as used herein, are antibodies comprising "human" variable region domains and/or CDRs, as defined above, without substantial non-human antibody sequences or without any non-human antibody sequences. For example, antibodies comprising human variable region domains "without substantial non-human antibody sequences" are antibodies and/or domains in which only up to 6, 5, 4, 3, 2 or 1 amino acids are amino acids that are not encoded by human antibody sequences. Antibodies comprising human CDRs "without substantial non- human antibody sequences" are antibodies, and/or CDRs in which only up to 4, 3, 2 or 1 amino acids are amino acids that are not encoded by human antibody sequences. Thus, "fully human" antibodies are distinguished from "humanized" antibodies, which are based on substantially non-human variable region domains, e.g., mouse variable region domains, in which certain amino acids have been changed to better correspond with the amino acids typically present in human antibodies.
The "fully human" antibodies of the invention may be human variable region domains and/or CDRs without any other substantial antibody sequences, such as being single chain antibodies. Alternatively, the "fully human" antibodies of the invention may be human variable region domains and/or CDRs integral with or operatively attached to one or more human antibody constant regions. Certain preferred fully human antibodies are IgG antibodies with the full complement of IgG constant regions.
In other embodiments, "human" antibodies of the invention will be part-human chimeric antibodies. "Part-human chimeric" antibodies, as used herein, are antibodies comprising "human" variable region domains and/or CDRs operatively attached to, or grafted onto, a constant region of a non-human species, such as rat or mouse. Such part-human chimeric antibodies may be used, for example, in pre- clinical studies, wherein the constant region will preferably be of the same species of animal used in the pre-clinical testing. These part-human chimeric antibodies may
also be used, for example, in ex vivo diagnostics, wherein the constant region of the non-human species may provide additional options for antibody detection.
Antibodies of the present invention may also be CDR grafted antibodies. Such antibodies are antibodies comprising the CDR sequences (e.g. 3 VH CDRs and/or 3 VL CDRs) of an antibody of the invention grafted into a framework region that is different from the framework region with which the CDRs are associated in the VL and/or VH domains described herein.
Preferred heavy and light chain variable region framework sequences are set forth in the sequence tables herein.
Nucleic acid molecules (e.g. one or more nucleic acid molecules) comprising nucleotide sequences that encode the antigen binding proteins (e.g. antibodies) or immunoconjugates of the present invention as defined herein or parts or fragments thereof, or nucleic acid molecules substantially homologous thereto, form yet further aspects of the invention.
Preferred nucleic acid molecules are those encoding an antigen binding protein (e.g. antibody) of the present invention as described elsewhere herein which can bind to HLA-A*02:01/PRAME425'433, e.g. an antigen binding protein (e.g. antibody) of the invention with CDR and optionally FR and other regions as defined in Table A, or antibodies with sequences substantially homologous thereto.
Preferred nucleic acid molecules are those encoding a VH region of an antigen binding protein (e.g. antibody) of the present invention. Other preferred nucleic acid molecules are those encoding a VL region of an antigen binding protein (e.g. antibody) of the present invention. Other preferred nucleic acid molecules are those encoding a VH region of an antigen binding protein (e.g. antibody) of the present invention and a VL region of an antigen binding protein (e.g. antibody) of the present invention.
In some embodiments, preferred nucleic acid molecules are those encoding a VL region of SEQ ID NO:4 (such as SEQ ID NO:2), of SEQ ID NO: 134 (such as SEQ ID NO:133), of SEQ ID NO:136 (such as SEQ ID NO:135), of SEQ ID NO:138 (such as SEQ ID NO:137), of SEQ ID NQ:140 (such as SEQ ID NO:139), of SEQ ID NO:147 (such as SEQ ID NO:146), or of SEQ ID NQ:150 (such as SEQ ID NO:149); and/or a VH region of SEQ ID NO:3 (such as SEQ ID NO:1), of SEQ ID NO:132 (such as SEQ ID NO:131 , 145 or 148), of SEQ ID NO:142 (such as SEQ ID NO:141), or of SEQ ID NO:144 (such as SEQ ID NO:143).
Preferred nucleic acid molecules are those encoding an antigen binding protein (e.g. antibody) of the present invention (e.g. as defined in Table A) which can bind to HLA-A*02:01/PRAME425'433.
In some embodiments nucleic acid molecules are those having a nucleic acid sequence that is substantially homologous to the specific nucleic acid sequences defined herein, for example having at least 80% sequence identity to specific nucleic acid sequences defined herein.
The term "nucleic acid sequence" or "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid molecules may be double stranded or single stranded. The nucleic acid molecules may be wholly or partially synthetic or recombinant.
A person skilled in the art will appreciate that the antigen binding proteins (e.g. antibodies) and polypeptides of the invention, such as the light and heavy CDRs, the light and heavy chain variable regions (domains), antibodies, antibody fragments, and immunoconjugates, may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods.
Nucleic acid fragments encoding the light and heavy chain variable regions (domains) of the antigen binding proteins (e.g. antibodies) of the invention can be derived or produced by any appropriate method, e.g. by cloning or synthesis.
Once nucleic acid fragments encoding the light and heavy chain variable regions (domains) of the antigen binding proteins (e.g. antibodies) of the invention have been obtained, these fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region fragments into full length antigen binding protein (e.g. antibody) molecules with appropriate constant region domains, or into particular formats of antibody fragment discussed elsewhere herein, e.g. Fab fragments, scFv fragments, scDb formats, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antigen binding protein (e.g. antibody) molecules of the invention are generally
incorporated into one or more appropriate expression vectors in order to facilitate production of the antigen binding proteins (e.g. antibodies) of the invention.
Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.
The invention therefore contemplates a recombinant expression vector containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.
Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes and are well known in the art. Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
The recombinant expression vectors of the invention may also contain a selectable marker gene that facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
The recombinant expression vectors may also contain genes that encode a fusion moiety that provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification (for example appropriate C-terminal or N-terminal "tags" to enable purification and/or identification may be present, e.g., His tags or myc tags). In some embodiments, however, the antigen binding proteins (e.g. antibodies) of the invention do not comprise a C- terminal or N-terminal tag, e.g. a His-tag.
Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to encompass introduction of nucleic acid {e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sam brook et a/., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.
Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells, as will be well known to a person skilled in the art. For example, the proteins of the invention may be expressed in yeast cells or mammalian cells, for example HEK or CHO cells. Cell-free expression systems might also be used.
Given the teachings provided herein, promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.
Alternatively, the proteins of the invention may also be expressed in nonhuman transgenic animals such as, rats, rabbits, sheep and pigs.
The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.
N-terminal or C-terminal fusion proteins comprising the antigen binding proteins (e.g. antibodies) of the invention conjugated to other molecules, such as proteins, may be prepared by fusing through recombinant techniques. The resultant fusion proteins contain an antigen binding protein (e.g. antibody) of the invention fused to the selected protein or marker protein, or tag protein as described herein. The antigen binding proteins (e.g. antibodies) of the invention may also be conjugated to other proteins by known techniques. For example, the proteins may be coupled using heterobifunctional thiol-containing linkers as described in WO 90/10457, N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5 thioacetate.
A yet further aspect provides an expression construct or expression vector or expression system (e.g. a viral or bacterial or other expression construct, vector or system), e.g. one or more expression constructs or expression vectors, comprising one or more of the nucleic acid fragments or segments or molecules of the invention. Preferably the expression constructs or vectors or systems are recombinant. Preferably said constructs or vectors or systems further comprise the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.
A yet further aspect provides a host cell (e.g. a mammalian or bacterial or yeast host cell) or virus, e.g. one or more host cells or viruses, comprising one or more expression constructs or expression vectors of the invention. Also provided are host cells (e.g. a mammalian or bacterial or yeast host cell) or viruses, e.g. one or more host cells or viruses, comprising one or more of the nucleic acid molecules of the invention. A host cell (e.g. a mammalian host cell or bacterial host cell, or yeast host cell) or virus expressing an antigen binding protein (e.g. antibody) of the invention forms a yet further aspect.
A yet further aspect of the invention provides a method of producing (or manufacturing) a protein (e.g. antibody) of the present invention comprising a step of culturing the host cells of the invention. Preferred methods comprise the steps of (i) culturing a host cell comprising one or more of the recombinant expression vectors or one or more of the nucleic acid sequences of the invention under conditions suitable for the expression of the encoded antigen binding protein (e.g. antibody); and optionally (ii) isolating or obtaining the antigen binding protein (e.g. antibody) from the host cell or from the growth medium/supernatant. Such methods of production (or manufacture) may also comprise a step of purification of the antigen binding protein (e.g. antibody) product and/or formulating the antigen binding protein (e.g. antibody) into a composition including at least one additional component, such as a pharmaceutically acceptable carrier or excipient.
In embodiments when the antigen binding protein (e.g. antibody) of the invention is made up of more than one polypeptide chain (e.g. whole antibodies), then all the polypeptides are preferably expressed in the host cell, either from the same or a different expression vector, so that the complete proteins, e.g. antibody proteins of the invention, can assemble in the host cell and be isolated or purified therefrom.
One therapeutic (or diagnostic) approach is the use of antibodies that can target specific antigens expressed on cancer cells, that are not expressed or are expressed at a lower level on normal cells. These target antigens, of which HLA- A*02:01/PRAME425'433 is an example, can be exploited using antibodies to specifically kill antigen-bearing cancer cells by a variety of mechanisms including by delivering immuno- or radio labelled conjugates that, when delivered to the antigenbearing cell, specifically kill the target cell. Such targeting can also be used for diagnosis.
The invention thus also provides a range of conjugated antigen binding proteins (e.g. antibodies), i.e. “immunoconjugates”, in which the antigen binding protein (e.g. antibody) of the invention is operatively attached to at least one other
therapeutic or diagnostic agent. The term "immunoconjugate" is broadly used to define the operative association of the antibody (or binding protein) with another effective agent (e.g. therapeutic or diagnostic agent) and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical "conjugation". Fusion proteins, e.g. recombinant fusion proteins, are particularly contemplated. So long as the delivery or targeting agent (the anti- HLA- A*02:01/PRAME425'433 component) is able to bind to the target and the therapeutic or diagnostic agent is sufficiently functional upon delivery, the mode of attachment will be suitable.
For example, in some preferred embodiments, antibodies (or binding proteins) of the invention are part of an immunotoxin or are used (e.g. used therapeutically) as part of immunotoxins wherein the antibody is itself operatively associated or combined with a toxic agent (e.g. a chemotherapeutic agent or toxin or a radioactive material such as a radiotracer, e.g. for use in radioimmunotherapy). The operative attachment includes all forms of direct and indirect attachment as described herein and known in the art.
Suitable chemotherapeutic agents or toxins are well known and described in the art, for example cytotoxic proteins derived from bacteria or plants can be used. The toxin should be capable of killing target cells once it has been taken up into said cells. Thus, preferred immunoconjugates of the invention are immunotoxins comprising an antibody of the invention linked or otherwise conjugated to a toxin (also sometimes referred to as antibody drug conjugates, ADCs). In preferred immunoconjugates, active ingredients such as radionuclides, toxins (for example, the diphtheria toxin), or other cytostatic agents can be bonded (conjugated) or otherwise linked to the corresponding antibodies.
In some embodiments, antibodies of the invention are used (e.g. used therapeutically) in their "naked" unconjugated form.
An immunoconjugate comprising a therapeutic agent, or a carrier comprising a therapeutic agent, may be used in therapy. An immunoconjugate comprising a diagnostic agent, or a carrier comprising a diagnostic agent, may be used in in vivo and/or in vitro diagnostic methods.
In some embodiments, antigen binding proteins (e.g. antibodies) of the invention are used (e.g. used therapeutically) in their "naked" unconjugated form.
Compositions comprising at least a first antigen binding protein (e.g. antibody) or immunoconjugate of the invention, or at least a first nucleic acid molecule or expression vector of the invention, or at least a first host cell of the invention, constitute further aspects of the present invention. Formulations (compositions)
comprising one or more antigen binding proteins (e.g. antibodies), etc., of the invention, optionally in admixture with a suitable diluent, carrier or excipient constitute a preferred embodiment of the present invention. Such formulations may be for pharmaceutical use, and thus compositions of the invention are preferably pharmaceutically acceptable or otherwise acceptable for administration to human or non-human animals, but in particular humans. Suitable diluents, excipients and carriers are known to the skilled man.
The pharmaceutical compositions may additionally comprise further active ingredients (e.g. as described elsewhere herein) in the context of co-administration regimens or combined regimens.
Any appropriate mode of administration can be used for administering/ delivering the compositions according to the invention. The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral (e.g. intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular), topical or rectal administration, or for mucosal delivery, and any of these modes of administration, or indeed any other appropriate mode of administration, can be used. In a preferred embodiment, compositions according to the invention are presented in a form suitable for intravenous administration. In some embodiments, compositions according to the invention are presented in a form suitable for intraperitoneal (i.p.) administration. In some embodiments, compositions according to the invention are presented in a form suitable for injection directly into a tumour (intratumoural).
The active compounds (e.g. the antigen binding proteins (e.g. antibodies) of the invention) as defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, exosomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms.
Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions may then be filled into injection vials or ampoules.
Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers, either with an aerosol propellant or provided with means for manual compression.
The pharmaceutical compositions (formulations) of the present invention are preferably administered parenterally. Intravenous administration is preferred. In
some embodiments, administration is intraperitoneal (i.p.) administration. In some embodiments, administration is by injection into a tumour. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the antibody in the form of a nasal or pulmonal spray. As a still further option, the antigen binding proteins (e.g. antibodies) of the invention can also be administered transdermally, e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally.
Suitable dosage units can be determined by a person skilled in the art.
In another aspect, the invention provides a method of binding HLA- A*02:01/PRAME425'433, comprising contacting a composition comprising HLA- A*02:01/PRAME425'433 with an antigen binding protein (e.g. antibody) of the invention, or an immunoconjugate thereof.
In yet another aspect, the invention provides a method of detecting HLA- A*02:01/PRAME425'433, comprising contacting a composition suspected of containing HLA-A*02:01/PRAME425'433 with an antigen binding protein (e.g. antibody) of the invention, or an immunoconjugate thereof, under conditions effective to allow the formation of HLA-A*02:01/PRAME425'433/antigen binding protein complexes (e.g. HLA-A*02:01/PRAME425-433/antibody complexes) and detecting the complexes so formed.
In embodiments, the composition suspected of containing HLA- A*02:01/PRAME425'433 may have been obtained from a subject, e.g. may be a subject sample e.g. blood sample or biopsy.
Yet further aspects are methods of diagnosis or imaging of a subject comprising the administration of an appropriate amount of an antigen binding protein (e.g. antibody) of the invention as defined herein to the subject and detecting the presence and/or amount and/or the location of the antigen binding protein (e.g. antibody) of the invention in the subject.
Appropriate diseases to be imaged or diagnosed in accordance with the present invention are cancers, e.g. as described elsewhere herein in connection with disease treatments.
In vitro methods of diagnosis are provided. Thus, in a further aspect, the invention provides a method of diagnosing cancer in a subject comprising the step of:
(a) contacting a test sample taken from said subject with one or more of the antigen binding proteins (e.g. antibodies) of the invention.
In an embodiment, the invention provides a method of diagnosing cancer in a subject comprising the steps of:
(a) contacting a test sample taken from said subject with one or more of the antigen binding proteins (e.g. antibodies) of the invention;
(b) measuring the presence and/or amount and/or location of antigen binding protein-antigen complex (e.g. antibody-antigen complex) in the test sample; and, optionally
(c) comparing the presence and/or amount and/or location of antigen binding protein-antigen complex (e.g. antibody-antigen complex) in the test sample to a control.
In the above methods, said contacting step is carried out under conditions that permit the formation of an antigen binding protein-antigen complex (e.g. antibody-antigen complex). Appropriate conditions can readily be determined by a person skilled in the art.
In the above methods any appropriate test sample may be used, for example biopsy cells, tissues or organs suspected of being affected by disease, or histological sections.
The subject is as defined elsewhere herein, and is preferably a human subject. The subject may be a subject at risk of developing cancer or at risk of the occurrence of cancer, e.g. a healthy subject or a subject not displaying any symptoms of cancer or any other appropriate “at risk” subject. In another embodiment the subject is a subject having, or suspected of having (or developing), or potentially having (or developing) cancer.
In certain of the above methods, the presence of any amount of antigen binding protein-antigen complex (e.g. antibody-antigen complex) in the test sample would be indicative of the presence of disease. Preferably, for a positive diagnosis to be made, the amount of antigen binding protein-antigen complex (e.g. antibodyantigen complex) in the test sample is greater than, preferably significantly greater than, the amount found in an appropriate control e.g. control sample. More preferably, the significantly greater levels are statistically significant, preferably with a probability value of < 0.1 , preferably <0.05. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used.
Appropriate control controls or samples could be readily chosen by a person skilled in the art, for example, in the case of diagnosis of a particular disease, an
appropriate control would be a sample from a subject that did not have that disease. Appropriate control "values" could also be readily determined without running a control "sample" in every test, e.g. by reference to the range for normal subjects known in the art.
For use in the diagnostic or imaging applications, the antigen binding proteins (e.g. antibodies) of the invention may be labeled with a detectable marker such as a radioisotope, such as 3H, 14C, 32P, 35S, 123l, 125l, 1311; a radioactive emitter (e.g. a, p or y emitters); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, luciferin or Europium; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion; or a chemical moiety such as biotin which may be detected by binding to a specific cognate detectable moiety, e.g. labelled avidin/streptavidin. Methods of attaching a label to an antigen binding protein (e.g. an antibody) are known in the art. Such detectable markers allow the presence, amount or location of antigen binding protein-antigen complexes (e.g. antibody-antigen complexes) in the test sample to be examined.
Preferred detectable markers for in vivo use include an X-ray detectable compound, such as bismuth (III), gold (III), lanthanum (III) or lead (II); a radioactive ion, such as copper67, gallium67, gallium68, indium111, indium113, iodine123, iodine125, iodine131, mercury197, mercury203, rhenium186, rhenium188, rubidium97, rubidium103, technetium99m or yttrium90; a nuclear magnetic spin-resonance isotope, such as cobalt (II), copper (II), chromium (III), dysprosium (III), erbium (III), gadolinium (III), holmium (III), iron (II), iron (III), manganese (II), neodymium (III), nickel (II), samarium (III), terbium (III), vanadium (II) or ytterbium (III); or rhodamine or fluorescein.
The invention also includes diagnostic or imaging agents comprising the antigen binding proteins (e.g. antibodies) of the invention attached to a label or detectable marker that produces a detectable signal, directly or indirectly. Appropriate labels or detectable markers are described elsewhere herein.
In one embodiment the method of diagnosing cancer is an in vitro method.
In one embodiment the method of diagnosing cancer is an in vivo method.
Alternatively viewed, the present invention provides a method for screening for cancer in a subject.
In some embodiments, antigen binding proteins (e.g. antibodies) of the present invention can be used as companion diagnostics.
In some aspects, diagnostic methods of the invention are provided which further comprise a step of treating cancer by therapy, e.g. using an antigen binding protein (e.g. antibody) of the present invention. For example, if the result of a
method of the invention is indicative of cancer in the subject (e.g. a positive diagnosis of cancer is made), then an additional step of treating the cancer by therapy or surgery can be performed.
A further aspect of the present invention provides the antigen binding proteins (e.g. antibodies) or immunoconjugates of the invention for use in therapy, in particular for use in the treatment or prevention of cancer. In other embodiments, the nucleic acid molecules, expression vectors, host cells or viruses of the invention can also be used in the therapeutic methods described herein.
By “therapy” as used herein is meant the treatment of any medical condition. Such treatment may be prophylactic (i.e. preventative), curative (or treatment intended to be curative), or palliative (i.e. treatment designed merely to limit, relieve or improve the symptoms of a condition).
The cancer is typically in a subject, i.e. a subject suffering therefrom. The cancer is HLA-A*02:01/PRAME425'433 positive. The term “cancer” encompasses cancerous cells of any type, including both tumours (e.g. solid tumours) and hematological or blood cancers. Thus, “cancer cells” may be tumour cells, or may be blood cancer cells.
In another aspect, the present invention provides immunoconjugates of the invention for use in therapy, in particular for use in the treatment or prevention of cancer.
In another aspect, the present invention provides antigen binding proteins (e.g. antibodies) or immunoconjugates of the invention, for use in killing cancer cells presenting/displaying HLA-A*02:01/PRAME425-433, i.e. HLA-A*02:01/PRAME425-433 positive cancer cells. Said killing is preferably T-cell mediated killing. Such killing may also play a role in the therapeutic treatments as discussed herein.
In another aspect, the present invention provides an antigen binding protein (e.g. antibody) of the invention in a T-cell engager format, for use in redirecting T-cell activity against a cancer cell. Preferably, the T-cell activity is cytotoxic activity. The use typically comprises the step of contacting said cancer cell with said T-cell engager antigen binding protein (e.g. antibody).
Alternatively viewed, the present invention provides an antigen binding protein (e.g. antibody) of the invention in a T-cell engager format, for use in redirecting T-cell activity against a tumour. Preferably, the T cell activity is cytotoxic activity. The use typically comprises the step of contacting said tumour with said T- cell engager antigen binding protein (e.g. antibody).
In another aspect, the present invention provides an antigen binding protein (e.g. antibody) of the invention in a T-cell engager format, for use in inducing T-cell
mediated cytotoxicity against a cancer cell or tumour. The use typically comprises the step of contacting said cancer cell with said T-cell engager antigen binding protein (e.g. antibody).
Preferred antigen binding proteins (e.g. antibodies) of the invention for use in the therapies disclosed herein, including in the treatment or prevention of cancer are in a T-cell engager format, preferably a bispecific format, preferably the scDb format (as defined herein).
Preferred T cells are as described elsewhere herein.
In accordance with the present invention the antigen binding proteins (e.g. antibodies) may target HLA-A*02:01/PRAME425'433 positive cells, e.g. cancer cells.
In one embodiment, solid tumours are treated.
In one embodiment, hematological or blood cancers are treated.
In some embodiments, a tumour or cancer that is characterized by expressing or over-expressing HLA-A*02:01/PRAME425'433 is treated.
Thus, a further aspect of the present invention provides antigen binding proteins (e.g. antibodies) or immunoconjugates as defined herein for use in the treatment or prevention of a cancer (e.g. a tumour or blood cancer) that is HLA- A*02:01/PRAME425'433 positive.
Preferred cancers are as described elsewhere herein. Preferred cancers include non-small cell lung cancer (NSCLC, e.g. squamous NSCLC), small cell lung cancer (e.g extensive stage disease small cell lung cancer), melanoma (e.g. metastatic melanoma such as BRAF negative metastatic melanoma, or multiple melanoma), lymphoma (e.g. acute T-cell lymphoma, Hodgkin lymphoma, nonHodgkin lymphoma or chronic lymphocytic lymphoma), leukemia (e.g acute myeloid leukemia, acute lymphoblastic leukemia or myelodysplastic syndrome), renal cell cancer (RCC, e.g clear cell renal cancer), colorectal cancer, urothelial bladder cancer, urethral cancer, head and neck cancer (e.g. recurrent or metastatic head and neck squamous cell cancer), breast cancer (e.g. metastatic HER-2 negative breast cancer) advanced liver cancer, brain cancer (e.g. glioblastoma or astrocytoma), stomach cancer, oesophageal cancer, pancreatic carcinoma, adenocarcinomas, mesothelioma, peritoneal cancer, fallopian tube cancer, cervical cancer, ovarian cancer, sarcomas, e.g. metastatic sarcoma, hematological neoplasms, thyroid cancer, salivary cancer, laryngeal (larynx) cancer, neuroblastoma, retinoblastoma, and testis (testicular) cancer.
Preferred “cancer cells” are described elsewhere herein, and include cells from the above-mentioned preferred cancers. Cancer cells include cells selected from the group consisting of lung cancer cells, melanoma cells, and monocytes.
Without wishing to be bound by theory, it is believed that antigen binding proteins (e.g. antibodies) of the present invention may be superior to prior art antibodies in terms of their therapeutic efficacy (via T-cell mediated cytotoxicity) in particular in terms of the concentration of antigen binding protein (e.g. antibody) required to achieve marked T-cell mediated cytotoxicity. Low concentrations/doses of the antigen binding proteins (e.g. antibodies) of the invention have been shown to induce significant T-cell mediated cancer cell killing, notably in assays comprising a T-cell effector cell to target cell ratio (E:T) of only 1 :1 , and notably in disparate cancer types. Efficacy of antigen binding proteins (e.g. antibodies) of the present invention in initiating T-cell mediated cytotoxicity in both solid tumour and blood cancer cells has been demonstrated at low concentrations/doses after only a single application.
The antigen binding proteins (e.g. antibodies) of the present invention may also be superior to prior art antibodies in terms of their specificity, in particular in terms of their demonstrated lack of binding to/ cross-reactivity with many HLA- A*02:01 restricted peptides with high and very high sequence identity to the target peptide PRAME425'433; and in terms of their demonstrated thermal stability.
The antigen binding proteins (e.g. antibodies) of the present invention may also be superior to prior art antibodies in terms of their preferentially redirection of T- cell activity against HLA-A*02:01/PRAME425'433 positive cells as compared to HLA- A*02:01 positive, PRAME425'433 negative cells, and as compared to cells displaying HLA-A*02:01 restricted genomic risk peptides with high sequence identity to the target peptide PRAME425'433.
The antigen binding proteins (e.g. antibodies) of the present invention may also be superior to prior art antibodies in terms of their preferential induction of activation, differentiation and proliferation of T cells directed against HLA- A*02:01/PRAME425'433 positive cells as compared to against HLA-A*02:01 positive, PRAME425'433 negative cells.
The antigen binding proteins (e.g. antibodies) of the present invention may also be superior to prior art antibodies in terms of their preferential induction of T-cell mediated cytotoxicity against HLA-A*02:01/PRAME425'433 positive cells as compared to against HLA-A*02:01 positive, PRAME425'433 negative cells.
The antigen binding proteins (e.g. antibodies) of the present invention thus represent an exciting development in the field of anti-cancer therapeutic agents.
The administration of the antigen binding proteins (e.g. antibodies) in the therapeutic methods and uses of the invention is carried out in pharmaceutically, therapeutically, or physiologically effective amounts, to subjects (e.g. animals, e.g.
human or non-human mammals) in need of treatment. Thus, said methods and uses may involve the additional step of identifying a subject in need of treatment.
Appropriate and effective concentrations/doses to be administered can readily be determined by a person skilled in the art.
Treatment of diseases or conditions in accordance with the present invention (for example treatment of pre-existing disease) includes cure of said disease or condition, or any reduction or alleviation of disease, e.g. reduction in disease severity, or symptoms of disease.
The therapeutic methods and uses of the prevent invention are suitable for prevention of diseases as well as active treatment of diseases (for example treatment of pre-existing disease). Thus, prophylactic treatment is also encompassed by the invention. For this reason in the methods and uses of the present invention, treatment also includes prophylaxis, or prevention where appropriate.
Such preventative (or protective) aspects can conveniently be carried out on healthy or normal or at risk subjects and can include both complete prevention and significant prevention. Similarly, significant prevention can include the scenario where severity of disease or symptoms of disease is reduced (e.g. measurably or significantly reduced) compared to the severity or symptoms which would be expected if no treatment is given.
In some aspects, a therapeutic method or therapeutic use of the invention may further comprise an initial step of selecting a subject (e.g. a human subject) at risk of developing cancer, or at risk of the occurrence of cancer, or suspected of having (or developing) cancer, or potentially having (or developing) cancer. Subjects may be selected on the basis that, for example, the subject (or sample, e.g. tissue biopsy, from the subject) is positive for one or more cancer markers or risk factors.
Suitable subjects for treatment in accordance with the present invention thus include any type of animal that can suffer from cancer, and more specifically cancers comprising cells that express HLA-A*02:01/PRAME425'433.
Thus, the in vivo methods and uses as described herein are generally carried out in a mammal. Any mammal may be treated, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, horses, cows and monkeys. Preferably, however, the mammal is a human.
Thus, the term "animal" or "patient" or “subject” as used herein includes any mammal, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, horses, cows and monkeys. Preferably, however, the animal or patient or subject is a human.
Thus, subjects or patients treated in accordance with the present invention will preferably be humans.
In another embodiment the subject is a subject having, or suspected of having (or developing), or potentially having (or developing) the disease or condition in question as described above.
In some embodiments, the antibodies (or binding proteins) of the invention can be used in monotherapy. In other embodiments they can be used in combination with other standard cancer therapeutics.
Alternatively viewed, the present invention provides a method of treating or preventing cancer, which method comprises administering to a patient in need thereof a therapeutically effective amount of an antigen binding protein (e.g. antibody) or immunoconjugate of the invention.
Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to the of the invention described herein.
In another aspect, the present invention provides a method of treating or preventing cancer which method comprises administering to a patient in need thereof a therapeutically effective amount of an immunoconjugate of the invention.
In another aspect, the present invention provides a method of killing cancer cells presenting/displaying HLA-A*02:01/PRAME425-433, i.e. HLA-A*02:01/PRAME425- 433 positive cancer cells, which method comprises administering to a patient in need thereof a therapeutically effective amount of an antigen binding protein (e.g. antibody) or immunoconjugate of the invention. Said killing is preferably T-cell mediated killing. Such killing may also play a role in the therapeutic treatments as discussed herein.
In another aspect, the present invention provides a method of redirecting T- cell activity against a cancer cell, which method comprises the step of contacting said cancer cell with an antigen binding protein (e.g. antibody) of the invention in a T-cell engager format. Preferably, the T-cell activity is cytotoxic activity. The cancer cell is typically in a patient and said method typically comprises administering to said patient the T-cell engager antigen binding protein (e.g. antibody).
Alternatively viewed, the present invention provides a method of redirecting T- cell activity against a tumour, comprising the step of contacting said tumour with an antigen binding protein (e.g. antibody) of the invention in a T-cell engager format. Preferably, the T-cell activity is cytotoxic activity. The tumour is typically in a patient and said method typically comprises administering to said patient the T-cell engager antigen binding protein (e.g. antibody).
In another aspect, the present invention provides a method of inducing T-cell mediated cytotoxicity against a cancer cell or tumour comprising the step of contacting said cancer cell or tumour with an antigen binding protein (e.g. antibody) of the invention in a T-cell engager format. The cancer cell or tumour is typically in a patient and said method typically comprises administering to said patient the T-cell engager antigen binding protein (e.g. antibody).
Preferred T cells, cancers and tumours are as described elsewhere herein.
A further aspect of the present invention provides a method of treating or preventing of a cancer (e.g. a tumour or blood cancer) that is HLA- A*02:01/PRAME425'433 positive.
Preferred therapeutic methods comprise the administration or use of antigen binding proteins (e.g. antibodies) of the invention in a T-cell engager format, preferably a bispecific format, preferably the scDb format (as defined herein).
By “therapeutically effective amount” is meant an amount sufficient to show benefit to the condition of the subject. Whether an amount is sufficient to show benefit to the condition of the subject may be determined by the subject him/herself or a physician; preferably it is determined by clinical assessment and can be readily monitored. Preferred cancer therapies are as described elsewhere herein.
Further alternatively viewed, the present invention provides the use of an antigen binding protein (e.g. antibody) of the invention as defined herein in the manufacture of a medicament for use in therapy. Preferred therapy is the treatment or prevention of cancer as described elsewhere herein. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.
Further alternatively viewed, the present invention provides the use of an antigen binding protein (e.g. antibody) of the invention as defined herein for the treatment of cancer. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.
The invention further includes kits comprising one or more of the antigen binding proteins (e.g. antibodies), or compositions of the invention, or one or more of the nucleic acid molecules encoding the antigen binding proteins (e.g. antibodies) of the invention, or one or more expression vectors comprising the nucleic acid sequences of the invention, or one or more host cells or viruses comprising the expression vectors or nucleic acid sequences of the invention. Preferably said kits are for use in the methods and uses as described herein, e.g. the therapeutic, diagnostic or imaging methods as described herein, or are for use in the in vitro assays or methods as described herein. The antigen binding protein (e.g. antibody)
in such kits may be a conjugate as described elsewhere herein, e.g. may be conjugated to a detectable moiety or may be an immunoconjugate. Preferably said kits comprise instructions for use of the kit components. Preferably said kits are for diagnosis or imaging, or treating or preventing diseases or conditions as described elsewhere herein, e.g. cancer, and optionally comprise instructions for use of the kit components to diagnose, image, treat or prevent such diseases or conditions.
The antigen binding proteins (e.g. antibodies) of the invention as defined herein may also be used as molecular tools for in vitro or in vivo applications and assays, for example binding assays or diagnostic assays. As the antigen binding proteins (e.g. antibodies) comprise at least one antigen binding site that binds to HLA-A*02:01/PRAME425'433, these can be used in any assay where a HLA- A*02:01/PRAME425'433 binding member is required.
Thus, yet further aspects of the invention provide a reagent that comprises an antigen binding protein (e.g. antibody) of the invention as defined herein and the use of such antigen binding proteins (e.g. antibodies) as molecular tools, for example in in vitro or in vivo assays, for example for the detection of HLA-A*02:01/PRAME425' 433, e.g. in a sample of interest.
As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. Therefore, an "antibody", as used herein, means "at least a first antibody".
In addition, where the terms “comprise”, “comprises”, “has” or “having”, or other equivalent terms are used herein, then in some more specific embodiments, for example in the definition of the CDR or FR sequences herein, these terms include the term “consists of” or “consists essentially of”, or other equivalent terms.
The term "decrease" or "reduce" (or equivalent terms) as described herein includes any measurable decrease or reduction when compared with an appropriate control. Appropriate controls would readily be identified by a person skilled in the art and might include non-treated or placebo treated subjects or healthy subjects, or samples or assays where no antibody (or binding protein) of the invention is present, or where a control antibody (or binding protein), for example an isotype control antibody (or binding protein), is present. Preferably the decrease or reduction will be significant, for example clinically or statistically significant.
The term "increase" (or equivalent terms) as described herein includes any measurable increase or elevation when compared with an appropriate control.
Appropriate controls would readily be identified by a person skilled in the art and might include non-treated or placebo treated subjects or healthy subjects, or samples or assays where no antibody (or binding protein) of the invention is present, or where a control antibody (or binding protein), for example an isotype control antibody (or binding protein), is present. Preferably the increase will be significant, for example clinically or statistically significant.
Preferably such increases (and indeed other increases, improvements or positive effects as mentioned elsewhere herein) or such decreases (and indeed other decreases, reductions or negative effects as mentioned elsewhere herein) are measurable increases, decreases, etc., (as appropriate), more preferably they are significant increases, decreases, etc., preferably clinically significant or statistically significant increases, for example with a probability value of <0.05 or <0.05, when compared to an appropriate control level or value.
Methods of determining the statistical significance of differences between test groups of subjects or differences in levels of a particular parameter are well known and documented in the art. For example herein a decrease or increase in level of a particular parameter or a difference between test groups of subjects is generally regarded as statistically significant if a statistical comparison using a significance test such as a Student t-test, Mann-Whitney II Rank-Sum test, chi-square test or Fisher's exact test, one-way ANOVA or two-way ANOVA tests as appropriate, shows a probability value of <0.05 or <0.05.
TABLES OF AMINO ACID SEQUENCES DISCLOSED HEREIN AND THEIR SEQUENCE IDENTIFIERS (SEQ ID NOs)
All amino acid sequences are recited herein from the N-terminus to the C- terminus in line with convention in this technical field.
All nucleotide sequences are recited herein 5' to 3' in line with convention in this technical field.
Table A : sequences of specific antibodies of the invention that bind to, or specifically bind to, HLA-A*02:01/PRAME425'433.
The constant regions of 5-A05, 5-A05/1-C04, PAM22, and NGS17 shown respectively within SEQ ID NOs: 109 and 110, within SEQ ID Nos: 153 and 110, within SEQ ID Nos: 164 and 110 and within SEQ ID Nos: 153 and 171 in Table A are human IgG 1 antibody sequences well known and described in the art. Such light chain constant regions, and parts of such heavy chain constant regions, are also present in the 5-A05, 5-A05/1-C04 and PAM22 antibodies in the Fab format, which comprise the Fab VH/CH1 and Fab VL/CL sequences shown respectively in SEQ ID NOs: 202 and 203, in SEQ ID NOs: 204 and 205, and in SEQ ID NOs: 206 and 207 in Table A.
Table B : other sequences disclosed herein
The invention will now be further described in the following non-limiting Examples with reference to the following drawings:
Figure 1. Structural representation of the PRAME425'433 T cell epitope modeled by DockTope (http://tools.iedb.org/docktope/) and visualized using Pymol V2.4.3.
Figure 2. (A) Clone 5-A05 epitope specificity was assessed by positional Ala scanning mutagenesis across PRAME425'433. The individual peptide variants were loaded onto recombinant biotinylated HLA-A*02:01 (pHLAs). The pHLAs were captured on NeutrAvidin coated ELISA plates and incubated with 5-A05 phages at 109 phages/well before detection with an anti-M13 antibody. (B) pH LA capture levels were controlled with the HLA-A2 specific mAb BB7.2.
Figure 3. Principle of T-cell engager function in cancer immunotherapy. T cells eradicate target cells, such as cancer cells, by virtue of specifically recognizing intracellularly expressed antigens found as pH LA complexes displaying antigenic peptides on the target cell surface using their antigen-specific T-cell receptor (TCR). Upon productive TCR-pHLA interactions, the CD3 signaling units of the TCR
activates the T cells and induce target cell killing. TCR-Like antibodies in the form of T-cell engagers (TCEs), such as the scDb, are (at least) bi-specific molecules recognizing both specific-pHLA and the CD3 signaling unit of T cells. Thus, TCEs generically redirect T cells towards target cells expressing specific pH LA and mediate polyclonal T-cell mediated target cell killing in a TCR independent manner activating the T cells through their naturally occurring CD3 signaling machinery.
Figure 4. 5-A05 was reformatted to full length hlgG 1 (A) and scDb with (B) and without (C) a C-terminal linked His-tag. Soluble PRAME425'433-pHLA was captured on NeutrAvidin coated ELISA wells and incubated with 5-A05 hlgG 1 or scDb at serial dilutions starting from 250 nM (hlgG1) or 385 nM (scDb) before detection with protL-HRP. Dotted lines indicate the EC50 value (concentration at which half of maximal binding signal is achieved).
Figure 5. Biotinylated pMHC was captured on NeutrAvidin-coated sensor chips followed by injection of a 2-fold dilution series from 0.76 pM (A) or 1 pM (B) of 5-A05 scDb with (A) and without (B) a C-terminally linked His-tag. The equilibrium response was plotted against concentration to derive the equilibrium dissociation constant (kD) of the 5-A05. The indicated kDs are based on extrapolations to response saturations.
Figure 6. Melting temperatures of 5-A05 scDb was assessed by use of NanoDSF. The well-performing therapeutic antibody Trastuzumab (in the form of Fab) was included for comparison.
Figure 7. TCE 5-A05 mediated T cell redirection in co-culture of effector cells with 5-A05 and PRAME425'433 pulsed T2 cells assessed by (A) pan-T cell mediated cytotoxicity and (B) Jurkat NFAT mediated Luminescence.
Figure 8. (A) TCE 5-A05 off-target profile assessed by reactivity towards PRAME425'433 peptide and a panel of risk peptides extracted from US patent 10,800,832 B2. Activation was measured by Luminescence after 16h co-culture of Jurkat NFAT, 5-A05 and peptide pulsed T2 cells (100nM peptide). The dotted line represents baseline activity as determined by Jurkat NFAT activation following coculture non-pulsed T2s. Statistical analysis was completed using a one-way ANOVA (***p<0.001). (B) Statistically significant (***p<0.001) hits from (A) were analyzed further in an identical assay by peptide titration (3-fold titration starting at 10pM to 4nM).
Figure 9. 5-A05 was reformatted to scDb. (A) Jurkat activation assessed by Luminescence at serial dilutions of the target peptide (5-fold dilutions from 10pM to 600pM) was measured after 16h co-culture of Jurkat NFAT, 5-A05 and peptide pulsed T2 cells. (B) Jurkat activation assessed by Luminescence at serial dilutions of
TCE (100nM to 0.01 nM) measured after 18h co-culture of Jurkat NFAT, 5-A05 and target positive (NCI-H1703) and target negative (HCT116 and NCI-H441) cells.
Figure 10. 5-A05 mediated re-direction of T cells was assessed by 72h coculture of pan-T cells, 5-A05 (10-fold dilutions from 1000ng/ml to 0.01 ng/ml), and target positive (NCI-H1703, A375 or THP1) or target negative (NCI-H441 or Boleth) tumor cell lines. Following 72h of co-culture the specific lysis of tumor cell lines representing solid tumor (A) or liquid tumor (B) was assessed using 7-AAD by flow cytometry analysis.
Figure 11. TCE 5-A05/1-C04 (A), PAM22 (B) and PAM23 (C) and NGS55 (D), off-target profile assessed by reactivity towards PRAME425'433 peptide and a panel of peptides matching a regex motif based on the ala-scan in Figure 2. Activation was measured by Luminescence after 18h co-culture of Jurkat NFAT, TCE and peptide pulsed T2 cells (100nM peptide). The dotted line represents baseline activity as determined by Jurkat NFAT activation following co-culture non-pulsed T2s. Statistical analysis was completed using a one-way ANOVA (***p<0.001).
EXAMPLES
Throughout the Examples below, reference is made to Assays 1 to 8, which are described in detail above. The specific assay conditions and steps used in the Examples below are the specific conditions and steps disclosed in the above descriptions of Assays 1 to 8.
Example 1 : Binding Footprint and Specificity of HLA-A*02:01/ PRAME425'433 antibodies
Materials and Methods
Antibodies
The nucleotide and amino acid sequences of the heavy and light variable domains of preferred HLA-A*02:01/PRAME425'433 antibodies of the invention are shown in Table A. The antibodies designated 5-A05, 5-A05/1-C04, NGS55 and NGS17 were obtained via phage display.
An optimized version of a previously reported fully human naive scFv library1 was used for panning against specific soluble peptide-human leukocyte antigen (pHLA) complexes with the aim of isolating antibody (Ab) clones with specific binding towards the validated HLA-A*02:01/PRAME425'433 T-cell epitope (Figure 1).
Three rounds of phage display selection were performed essentially as described2, before the libraries were screened for specific target binding hits.
Screening was done using clonal phage capture ELISA against specific and unspecific pH LA targets. The response ratio of specific vs unspecific target binding was calculated to identify favourable primary clones, which were subsequently sequenced to reveal clonal identity.
These clones were subjected to further analysis and testing to look for appropriate and advantageous properties, and the clones, 5-A05, 5-A05/1-C04, NGS55 and NGS17 were identified as performing particularly well.
The CDR and framework regions of the light and heavy variable domains of 5-A05, 5-A05/1-C04, NGS55 and NGS17 are shown in Table A. These antibodies, in the scFV format, comprised the VH and VL domains connected via a peptide linker (SEQ ID NO: 28).
Further preferred HLA-A*02:01/PRAME425'433 antibodies of the invention shown in Table A are PAM6, PAM8, PAM 17, PAM 18, PAM22, and PAM23. To increase target affinity of the 5-A05/1-C04 antibody, CDRs were targeted for diversification. NNK degenerate codons were used for amino acid mutagenesis of five of the six CDR loops (LCDR1 , LCDR3, HCDR1 , HCDR2, HCDR3), either by unbiased randomization of the whole loops or by rational randomization of particular residues informed by structure models. A total of 11 CDR libraries were constructed using custom designed genes containing degenerate codons. Libraries were transformed into electrocom petent cells and packaged as (Hoydahl et al, 2016). The selection and screening of the CDR libraries were performed essentially as described for the primary selection. Of all of the antibodies subjected to further analysis and testing to look for appropriate and advantageous properties, PAM6, PAM8, PAM 17, PAM 18, PAM22, and PAM23 were identified as performing particularly well.
Peptide synthesis and HLA-A2 peptide loading
Peptides were synthesized at Genscript. Empty biotinylated HLA-A2 monomers (Tetramer Shop) were diluted in PBS to a concentration of 0.5 mg/ml and incubated with peptide (200 pM) on ice for 30 min. The peptide loaded HLA molecules were further used in ELISA and SPR target binding assessments.
Single clone phage ELISA
Assay 1 described above was performed. ELISA plates were coated with 5 pg/mL NeutrAvidin in PBS and incubated 16h/4°C (step (a)) and blocked with 5% skim milk powder in PBST for 1 h/ room temperature (RT) (step (c)). Biotinylated pHLA variants were captured for 1 h/RT (step (e)), followed by addition and incubation of 109 phages or dilution series starting at 385 nM (for scDb) or 250 nM (for IgG) (step (g)). Bound phage particles were detected with an anti-Fd antibody
(anti-M13-HRP (Amersham Biosciences)), whereas bound lgG1 and scDb was detected with HRP conjugated protein L (Genscript) (step (i)). Levels of immobilized pHLA were detected with the anti HLA-A2 specific mAb (Clone BB7.2 (AbCam)). pHLA, phage samples, lgG1, scDb, antibodies and protein L were all diluted in PBSTM. Plates were developed with TMB solution and read at 450 nm using a microplate reader (step (k)). Between each layer, the plates were washed 3x with PBST (steps (b), (d), (f), (h), and Q)).
Results
Specificity - Footprint
To examine the fine-specificity of clone 5-A05 towards the PRAME425'433 peptide, positional alanine (Ala) scanning was performed using pHLA phage capture ELISA (as described above) towards the individual variants (Table C):
Table C: Positional alanine scanning peptide sequences
As shown in Figure 2, clone 5-A05 exhibited peptide dependent binding as specific mutations affected the binding sensitivity. Specifically, the Ab reactivity was abrogated with peptide variants P2, P3, P5, P6, P7 and P9, indicating that amino acids at positions 2, 3, 5, 6, 7 and 9 of WT PRAME425'433 (S EQ ID NO: 19) were important for antibody binding.
Some peptide variants affect pHLA stability, the so-called anchor residues, whereas solvent exposed positions may be directly involved in the Ab reactivity. These exposed positions correspond to amino acid positions 4, 5, 7, and 8 in the PRAME425'433 peptide sequence (SEQ ID NO: 19) (based on a HLA-A*02:01/ PRAME425'433 pHLA modeling using DockTope). Thus, the overall, result showed that clone 5-A05 exhibited restricted positional binding dependency towards 2 of the
4 solvent exposed residues (those at positions 5 and 7 of SEQ ID NO: 19) suggesting a central binding mode and high degree of specificity.
The same analysis was performed using each of the antibodies disclosed in Table A, with the peptide-dependent binding observed being essentially the same as 5-A05, and with some of the antibodies also exhibiting restricted positional binding dependency towards the amino acid at position 8 of PRAME425'433 (SEQ ID NO: 19) (data not shown).
Example 2 : Functional characteristics of HLA-A*02:01/PRAME425'433 antibodies in T-cell engager (TCE) format
Materials and Methods
Antibodies
For targeting of T cells to the HLA-A*02:01/PRAME425'433 antigen, the present inventors designed bispecific T-cell engager antibodies in the form of a single chain diabody (scDb) based on each of the specific clones of Table A and which includes a CD3 binding domain (Figure 3).
Briefly, the scDb comprises a first antigen binding domain with a first specificity for PRAME425'433 pH LA comprising an antigen variable light (VLA) and a variable heavy (VHA) domain, and a second antigen binding domain with a second specificity for CD3 comprising an antigen variable light (VLB) and variable heavy (VHB) domain, wherein the domains are fused by peptide linkers and are in the following order: VLA - VHB - VLB - VHA - His-tag. The CD3 antigen binding domain possess the same specificity as the anti-human CD3 antibody UCHT1 , as described34.
Synthetic genes encoding the scDb were constructed and cloned into expression vector for transient expression in CHO cells. The recombinant scDb and hlgG1 was expressed and purified by affinity chromatography on a HisTrap™ FF Crude column. After concentration and dialysis against PBS buffer, content and purity of the purified protein was assessed by SDS-PAGE and analytical size exclusion chromatography.
The scDb without the His-tag was also prepared (in the same manner, though purified by affinity chromatography on Protein L resin). The human lgG1 format was also constructed by linking the VH and VL domains to the IgG 1 constant domains. Human IgG 1 was expressed as described for the scDb and purified by affinity chromatography on Protein A.
By way of example, the amino acid sequence of the scDb 5-A05 molecule (without the His-tag) is shown in SEQ ID NO: 30, which consists of the following sequences reading in the N to C direction: Table D: scDb 5-A05
In the scDb format (without the His-tag) of each of the other antibodies of the invention of Table A that were prepared (i.e. scDb 5-A05/1-C04, PAM6, PAM8, PAM 17, PAM 18, PAM22, PAM23, NGS55 and NGS17), the same long and short linker sequences were present (those shown in Table D), and the same VHB and VLB sequences (vs. CD3) were present (those shown in Table D). The VHA and VLA CDR and framework sequences that were present are those shown in Table A for the given antibody.
For instance the amino acid sequence of the scDb 5-A05/1-C04 molecule (without the His-tag) is shown in SEQ ID NO: 152, which consists of the following sequences reading in the N to C direction:
Table D (Continued): scDb 5-A05/1-C04
Binding analysis by SPR
Assay 6 above was performed. SPR was conducted using a Biacore S200 (GE Healthcare). NeutrAvidin (10 pg/mL in 10 mM sodium acetate, pH 5.0) was coupled onto a CM3 series S sensor chip to 500 response units (Rll) by amine coupling before capture of 20-100 Rll of soluble, recombinant, biotinylated pHLA (1 pg/mL). Kinetics and affinity of scDb molecules was determined using single cycle kinetics method. All samples were diluted in PBS and run at 30 pL/min at 25°C. Data was fitted to a 1:1 Langmuir binding model after buffer subtraction and NeutrAvidin- reference-cell subtraction using the S200 Evaluation Software v1.0.
Thermostability analysis by nanoDSF
Assay 7 above was performed. Purified Fab and scDb molecules were diluted to 0.04 - 0.46 mg/ml in PBS and 10 pL were transferred to glass capillaries (NanoTemper) in triplicates. Samples were subjected to a temperature from 20 °C to 95 °C, with 1 °C/min increments using a Prometheus nanoDSF (NanoTemper). An excitation wavelength of 295 nm was used, and emission was measured at 330 nm and 350 nm. Data was collected and analyzed using AB-Protein PR.ThermControl V2.12 to determine melting temperatures.
Results
Comparison of Ab Format Binding
For validation of the functional folding and integrity of the scDb antibodies, pHLA target binding was assessed by ELISA and compared with the 5-A05 hlgG 1 (as described above in Example 1 - Assay 1).
As shown in Figure 4A and 4B, the full length hl gG 1 and scDb 5-A05 bound to the biotinylated HLA-A*02:01/PRAME425'433 complex in a concentration dependent manner with a EC50 value of 1 .6 nM and 128 nM, respectively. As the His-tag, which was linked C-terminally to the VH domain of the 5-A05 molecule, potentially could interfere with the target binding activity, an untagged version of the scDb was made and compared head-to-head with the His-tagged molecule for target binding ability by ELISA. Results showed that both 5-A05 scDb variants bound towards the pHLA target with activities in the same range (Figure 4B and 4C), but with increased target sensitivity for the molecule without the His-tag (15.2 nM) as compared to the His- tagged variant.
The assay was repeated with the full suite of antibodies shown in Table A, each in the scDb format described above. The data is from a separate experiment where the intraexperimental difference was within an acceptable range. The results are shown in Table G.
Table G: scDb antibody EC50 value (concentration at which half of maximal soluble target binding signal is achieved)
Affinity
Assay 6 above was performed. To determine the apparent affinity of the 5- A05 candidate for HLA-A*02:01/PRAME425'433, surface plasmon resonance (SPR) binding analysis was used in which the biotinylated pHLA complex was captured on
NeutrAvidin-coated SPR sensor chips followed by injection of various dilutions of 5- A05 scDb. As shown in Figure 5, the sensorgrams of SPR analysis revealed similar affinities for both scDb 5-A05 molecules (i.e. with (A) and without (B) the C-terminal linked His-tag). Again, the molecule without the His-tag showed a slightly increased affinity of 1187 nM compared to the his-tagged molecules with an affinity of 1759 nM. Significantly lower binding was observed to HLA-A2 loaded with two irrelevant peptides.
The above assay was preliminary, in which the H LA-PRAM E425'433 (pHLA) used was prepared by the present inventors by loading the PRAME425-433peptide onto HLA molecules. For accuracy, the present inventors repeated the assay of 5- A05 scDb (without his-tag) using a new batch of pHLA molecules, which were purchased in pre-loaded form. After several repeats of the assay, it was determined that the accurate value for the affinity (KD) of 5-A05 (without the His tag) is 254 nM, rather than the 1187 nM determined above in the preliminary assay.
The assay (with purchased pre-loaded pHLA) was repeated with the following antibodies in the Fab format, with the following affinities determined: 558 nM (5-A05), 4656 nM (5-A05/1-C04), 113 nM (PAM22) and 231 nM (NGS17).
The assay (with purchased pre-loaded pHLA) was repeated with the 5-A05/1- C04 antibody in the scDb format (without a His-tag), and an affinity of 155 nM was determined.
Stability
Assay 7 above was performed. The thermostability of 1-5-A05 scDb was assessed by determining its melting temperature (Tm) using nanoDSF (Figure 6). The melting temperature is measured by thermal unfolding and is defined as the temperature where 50% of the molecules is unfolded. The Tm value of 5-A05 scDb was determined to be 66.7 °C. The antigen binding Fab unit of the well-performing therapeutic antibody Trastuzumab was included as control, in a format (Fab) which normally has higher thermostability due to the CH1-CL pair5 6.
The same analysis was performed using the antibody 5-A05/1-C04, and a similar thermal stability (67.9°C) was observed as for 5-A05 (data not shown).
Example 3 : Specific redirection of T-cell activity
Materials and Methods
Cancer cell lines and culture conditions
Cancer cell lines were purchased from cell banks and cultured as per recommended conditions and media. Table E details the source of cancer cell lines and the media in which cells were cultured.
Table E: Details of cancer cell lines and culture media used in cell based assays
PBMCs were isolated from Buffycoats from healthy donors. Briefly, the buffy coat was diluted 1:1 with PBS/2% FBS, layered onto Lymphoprep and centrifuged at 500g for 30 minutes. Following centrifugation, PBMCs were harvested from the interface between Lymphoprep and plasma and washed with PBS. Stocks of PBMCs where generated and stored in liquid nitrogen.
Pan T cells were isolated from whole PBMCs using ‘EasySep™ Human T cell isolation kit’. Briefly, PBMCs were resuspended in StemCell buffer kit at a concentration of 50 million/ml and 50pl of isolation cocktail was added per 1ml of cell suspension. Cells were gently mixed and then incubated for 5 minutes. Following the incubation, 40pl of pre-vortexed RapidSpheres™ were added per 1ml of cell suspension and gently mixed. Stem cell buffer was then added to the cell suspension to ensure a final volume of 2.5ml. The cell suspension was then docked to the magnetic stand and incubated at room temperature for 3 mins. Following the 3 minutes incubation, the supernatant was removed and transferred to a new tube containing 10ml assay media (RPMI/10% FBS/1% P/S). The supernatant was when centrifuged to pellet the pan T cells contained within. The purity of the pan T cell population was assessed by flow cytometry using anti-human CD3 antibody.
The assay media in all assays containing PBMCs or pan T cells was RPMI/10% FBS/1% P/S
Risk Peptide Panels
Peptides from human genomic origin having high level of sequence similarity to PRAME425'433 were identified from US 10,800,832 B2 and may potentially represent risk peptides to the extent that they can be presented on HLA-A2 and induce TCE mediated T-cell activation. Risk peptide affinities towards HLA-A*02:01 were predicted using https://tools.iedb.org/mhci/. Risk peptide sequences and their predicted affinities are shown in Table F below. Peptides were synthesized as described above for use in risk assessment analysis. Table F: to PRAME425'433 related risk peptides
For a more physiologically relevant panel of risk peptides, with more relevant off-target potential, it was determined to produce peptides based on the binding profile of 5-A05 in the PRAME peptide ala-scan (Table C, Figure 2), positional degenerate peptide search motifs were constructed, which were based on the PRAME425’433 sequence with positional allowed variation. All of the Ala substitutions were used as basis for the motifs.
The first motif xx[L]x[H][L][l][G]x allowed for any amino acid (represented by “x”) in position 1 and 4, and amino acids with similar biophysical properties in position 2, 3, 5, 6, 7, 8 and 9 (shown in brackets). In the second motif, xx[L]x[H][L][l][G]x, variations in position 1 , 2, 4 and 9 were introduced, whereas position 3, 5, 6, 7 and 8 were locked, again “x” represents any amino acid.
These motifs were used for scanning for amino acid sequence matches against the protein sequence data bases using ScanProsite (https://prosite.expasv.org/scanprosite/). Default settings were used, and taxonomy filters were restricted to Homo sapiens. All hits were further applied to The Immune Epitope Database (IEDB) epitope analysis by scanning the peptide sequences for amino acid patterns indicative of HLA-A*02:01 binding. Peptide hits with the best predicted affinities (rank 1-32), determined using https://tools.iedb.org/mhci/), shown in Table K below, were synthesized for use in risk assessment analysis.
Table K: PRAME425’433 related degenerate risk peptides
TCE mediated cytotoxicity assay
Assay 5 above was performed. The capacity of the TCE (i.e. scDb) antibodies of the invention disclosed in Table A to induce T-cell mediated cytotoxicity was assessed using co-cultures of target cells (“T”): CFSE (Biolegend) labelled target positive (NCI-H1703, A375 and/or THP1) or negative (HCT116 and Boleth) cancer cell lines or peptide pulsed T2 cells (50pM peptide overnight (16h) pulsing, excess peptides washed away prior to co-culture) with effector cells (“E”): pan T cells
isolated from PBMCs, at an E:T ratio of 1 :1. Solutions of scDb covering the concentration range of 0.01-1 OOOng/ml were added to relevant wells.
Following 72h co-culture, assay supernatants and cells were harvested from the assay plates and 7-AAD viability staining solution (BioLegend) was added. Samples were then analysed by flow cytometry.
Jurkat NFAT activation assay
Assay 2 above was performed. The capacity of TCE 5-A05 (i.e. scDb 5- A05), 5-A05/1-C04, PAM22, PAM23 and NGS55 to induce CD3-mediated activation of Jurkat NFAT cells was assessed using co-cultures of Jurkat NFAT cells, TCE antibody and either target positive or negative cancer cell lines or peptide pulsed T2 cells. In brief, T2 cells or cancer (i.e. “target”) cells were cultured harvested, washed, re-suspended in cell culture medium, diluted and seeded at 50000 cells/well on day 0 (cancer cell lines) or 25000 cells/well on day 1 (T2) in cell culture medium in round bottom 96-well cell culture plates (steps (a) to (f)). Subsequently on day 1 , Jurkat NFAT reporter cells were cultured harvested, washed, re-suspended in cell culture medium, diluted (steps (g) to (j)), then added to the target cells at a E:T ratio of 1 :1 (step (k)). 5-A05 was diluted in culture medium and added to the co-culture (0.2nM to 200nM) - controls comprised culture medium alone - to a final volume of 200pl per well (steps (I) and (m). When Jurkat NFAT cells were added to T2 cells, the following diluted peptides were added to the co-culture: (PRAME425-433: 50pM (Figure 7 A&B), PRAM E425'433 and other peptides: 100 nM ((Figure 8A and 11A to D), PRAME425' 433: range 600pM to 10pM (Figure 8B and Figure 9A)) (steps (n) to (p)).
Cells were incubated for 16h at 37°C in a humidified incubator (step (q)). D-Luciferin, Monopotassium Salt (Promega) was diluted at 1 :10 in Milli-Q water (step (r)).
In wells of a white 96-well plate, the D-Luciferin Monopotassium Salt (Promega) and cell co-culture were prepared at a ratio of 1 :3 (step (s)). Where cells were seeded in step e) in white 96-well plates, this comprised adding 50pl of luciferase substrate to the 150 pl of co-culture. Where non-white 96-well plates were used in step e) this comprised instead, in step (q), pelleting the co-culture plates at 300g for 5min, discarding excess cell culture supernatant, and re-suspending pelleted cells in remaining cell culture supernatant (1 OOpI) then in step (s), adding 25pl per well of D-Luciferin Monopotassium Salt (Promega) and 75pl per well of cell co-culture.
Luminescence was detected using Varioscan LUX (Thermo Fisher) (step (t)).
Results
To initially explore the ability of 5-A05 scDb to mediate T-cell re-directed target cell killing and T-cell activation, 5-A05 scDb was co-cultured with PRAME425' 433 peptide pulsed T2 cells. Both dose-dependent killing of PRAME425'433 pulsed T2 cells assessed via 7-AAD flow cytometry analysis (Figure 7A) and activation of NFAT Jurkat cells (Figure 7B) was observed. No response was observed when 5- A05 was co-cultured with non-pulsed T2 cells.
Further, 5-A05 mediated cytotoxicity (Figure 7A) and Jurkat activation (Figure 7B) in co-culture with peptide pulsed T2 cells exhibit the same sensitivity as seen by the dose responses in Figure 9A and 9B, validating the Jurkat NFAT activation assay a good proxy of T-cell activity/ killing.
Peptides from human genomic origin having high level of sequence similarity to PRAME425'433 were identified from US 10,800,832 B2 and may represent risk peptides to the extent that they can be presented on HLA-A2 and induce TCE mediated T-cell activation. To further determine the specificity of 5-A05, Assay 2 above was performed. The risk peptides were ordered and pulsed onto T2 cells. Following 16h co-culture of peptide pulsed T2 cells, 5-A05 and Jurkat NFAT cells, Jurkat activation was only observed when T2 cells were pulsed with PRAME425'433, SACS and TRGV10-2 peptides (***p<0.001) with the remaining risk peptides comparable to non-pulsed T2 cells (Figure 8A).
The three peptides activating Jurkat cells in Figure 8A (PRAME425'433, SACS and TRGV10-2 peptides) were analyzed further by 3-fold peptide titration, and T2 cells pulsed with SACS or TRGV10-2 peptides only activated Jurkat NFAT cells at 10pM peptide, but not lower peptide concentrations (Figure 8B). 10pM is a non- physiological concentration. Even the next dilution, ~3.5pM is non physiological, and neither SACS nor TRGV10-2 induced Jurkat activation at this peptide concentration. Further, the sensitivity window between PRAME and SACS/TRGC10-2 is in the range of 2-3 logs. Additionally, in silico assessment of these peptides in terms of HLA binding and processing ability strongly indicated lack of both key features to represent true epitopes (data not shown). Both peptides received a negative total score for TAP and MHC binding using an MHC-p processing prediction program, i.e. it is predicted that in vivo, neither peptide is processed into 9-mers, transported and presented on HLA-A2. NetMHCpan 4.1 predicts both SACS and TRGV10-2 as weak binders to HLA-A2, and neither peptide is predicted as a T-cell epitope using NetCTLpan. These data, combined with the very low T cell activation potential of these two alternative peptides, excludes them as potential cross-reactivity risks of further relevance.
Based on the Ala-scan in Figure 2, regex motifs were created and used for scanning for matches against the prosite protein sequence data base (restricted to human proteins). Sequences matching the regex motifs may potentially represent risk peptides to the extent that they can be presented on HLA-A2 and induce TCE mediated T-cell activation. To further determine the specificity of the antibodies of the invention 5-A05/1-C04, PAM23, PAM23 and NGS55, Assay 2 above was performed. Risk peptides matching the regex motif were ordered and pulsed onto T2 cells. Following 16h co-culture of peptide pulsed T2 cells, antibody and Jurkat NFAT cells, Jurkat activation was only observed when T2 cells were pulsed with PRAME-A4425' 433 peptide (***p<0.001), with all risk peptides comparable to non-pulsed T2 cells (Figure 11A to D).
Jurkat NFAT cell activation was assessed following co-culture with peptide pulsed T2 cells or cancer cell lines and 5-A05 (Assay 2 above). Dose response curves were measured following 16h co-culture of Jurkat cells, 5-A05 on T2 cells pulsed with different doses of PRAME425'433 peptide (Figure 9A). Similarly, activation was observed with co-culture with Jurkat NFAT, titrations of 5-A05, and target positive (NCI-H1703) cancer cell lines, but not on target negative (HCT116 and NCI- H441) cancer cell lines (Figure 9B).
Example 4: T-cell mediated cytotoxicity
Materials and Methods
TCE mediated cytotoxicity assay
Assay 5 above was performed to assess the capacity of each of the antibodies of Table A (scDb format) to induce T-cell mediated cytotoxicity against target-positive cancer cells (i.e. target cells).
The capacity of the antibodies to induce T-cell mediated cytotoxicity was assessed using co-cultures of target cells (“T”): CFSE (Biolegend) labelled target positive (NCI-H1703, A375, THP1 SKM1 cell line) with effector cells (“E”): PBMCs at an E:T ratio of 1:1. Solutions of antibody covering the concentration range of 0.01- 1000ng/ml were added to relevant wells, Following 72h co-culture (37°C), assay supernatants and cells were harvested from the assay plates and 7-AAD viability staining solution (BioLegend) was added. Samples were then analysed by flow cytometry
Results
The ability of 5-A05 (scDb) to induce re-directed T cell specific target killing was further assessed via 7-AAD flow cytometry analysis using a range of HLA-
A*02:01/PRAME425'433 positive (NCI-H1703, A375, THP1 and SKM1) and target negative (NCI-H441 and Boleth) tumor cell lines. Following 72h co-culture of tumor cell lines, pan T cells isolated from PBMCs and TCE, 5-A05 was able to induce redirected T cell specific killing in a dose-dependent manner, only when cultured with target positive tumor cells (Figure 10A and B). This was the case both in tumor cell lines representing solid tumors (Figure 10A) and liquid tumors (Figure 10B).
EC50 values were determined as follows for 5-A05: EC50 = 14.2 pM as measured in the cancer cell line NCI-H1703; EC50 = 22.2 pM as measured in the cancer cell line A375; EC50 = 13.2 pM as measured in the cancer cell line THP1 ; and EC50 = 6.9 pM was determined as measured in the cancer cell line SKM1.
The ability of antibodies 5-A05/1-C04, PAM6, PAM8, PAM 17, PAM 18, PAM22, PAM23, NGS55, and NGS17 (scDb) to induce re-directed T-cell specific target killing was assessed using HLA-A*02:01/PRAME425'433 positive (NCI-H1703) cancer (“target”) cell lines (Assay 5 above). Following 72h co-culture, each of the antibodies was able to induce re-directed T-cell specific killing in a dose-dependent manner, and at advantageously low concentrations (Table H, below).
Table H: Ability of antibodies of the invention to induce T-cell specific killing vs. NCI-H1703
The ability of antibodies 5-A05/1-C04, PAM22, PAM23, NGS55, and NGS17 (scDb) to induce re-directed T-cell specific target killing was further assessed via 7- AAD flow cytometry analysis, using HLA-A*02:01/PRAME425'433 positive (THP-1) cancer (“target”) cell lines (Assay 5 above). Following 72h co-culture, each of the antibodies was able to induce re-directed T-cell specific killing in a dose-dependent manner, and at advantageously low concentrations (Table I, below).
Table I Ability of antibodies of the invention to induce T-cell specific killing vs.
THP-1
Example 5 : T-cell Cytokine Release (IFNy)
Cytotoxic T cells release cytokines such as IFN-y, which induce the increased expression of MHC class I and other molecules involved in peptide loading in infected cells, which in turn increases the chance that infected cells will be recognized as target cells for cytotoxic attack. IFN-y also activates macrophages, recruiting them to sites of infection both as effector cells and as antigen-presenting cells. The ability of an antigen binding protein, particularly at low concentrations, to induce the release of IFN-y from T-cells when co-cultured with HLA- A*02:01/PRAME425'433 positive target cells would therefore be advantageous, and demonstrative of the cytotoxic and therapeutic activity of the molecules.
Assay 8 above was performed to assess the capacity of each of the antibodies of Table A (scDb format) to induce CD3-mediated cytokine (IFNy) release from T cells. Briefly, co-cultures of target positive cancer cell lines (target “T” cells) and effector cells (PBMCs comprising T cells, “E”), at an E:T ratio of about 1:1, were co-cultured in the presence of the antigen binding protein (e.g. antibody) to be tested, over a concentration range (of about 0.1-10,000pM), for 24h at about 37°C. Supernatant was harvested and 100 pl was added to ELISA plates previously coated with IFNy capture antibody, washed, blocked and washed, then incubated for about 2 hours at room temperature. After washing, the plates were incubated with 10OpI of secondary antibody for 1 hour at room temperature, washed, then incubated for 30 minutes at room temperature with 100pl of 1 :1000 diluted avidin-HRP. After washing, wells of the plate were incubated for up to 5 minutes, in the dark, with 100pl of TMB solution (3,3',5,5'-Tetramethylbenzidine), with immediate addition of 100 pl of 1M HCI to stop the reaction. Absorbance was measured at 450nM, and the concentration of IFNy in each of the wells was determined using a standard curve.
Following 24h co-culture, each of the antibodies was able to induce IFNy release from PBMCs, more specifically T cells within the PBMCs, in a dosedependent manner, and at advantageously low concentrations (Table J, below).
Table J: T-cell Cytokine release (IFNy)
In summary, the present inventors have demonstrated the following:
The antibodies of the invention bind PRAME425'433 presented on HLA-A2 with a centrally focused binding footprint.
The TCE antibodies of the invention redirect T-cell activity in a specific manner, i.e. against HLA-A*02:01/PRAME425'433 positive cells, and not against HLA- A*02:01 positive, PRAME425'433 negative cells, nor cells displaying HLA-A*02:01 restricted genomic risk peptides with high sequence identity to PRAME425'433.
TCE antibodies of the invention preferentially induce T-cell mediated cytotoxicity in a specific manner against HLA-A*02:01/PRAME425'433 positive cells as compared to against HLA-A*02:01 positive, PRAME425'433 negative cells.
The antibodies of the invention have demonstrated advantageous thermal stability.
TCE antibodies of the invention mediate T-cell re-directed target cell killing with surprisingly advantageous efficacy; cytotoxicity is demonstrated at very low concentrations (low EC50 values), in assays with an effector cell: target cell ratio of only 1 :1. This is surprising given the binding affinity. Further, it is well known that increasing Effector cell :Target cell ratios (e.g. from 1 :1 to 10:1) greatly increases measured TCE potency7 8. The present inventors have, surprisingly, shown
advantageous levels of killing with a low effector to target ratio, i.e. a ratio of 1 :1. This has been demonstrated in a variety of different target-positive cancers.
TCE antibodies of the invention induce release of IFN-gamma from T-cells in a targeted manner and with surprisingly advantageous efficacy; low EC50 values are demonstrated, surprisingly and advantageously in assays with an effector cell: target cell ratio of only 1 :1.
The antibodies of the invention achieve the above-mentioned effects, e.g. efficacy (cytotoxicity and induced IFN-gamma release), whilst having relatively low binding activities/ strength, meaning that the antibodies of the invention have an advantageous cytotoxicity/safety profile.
References
1 Hoydahl, L. S. et al. Multivalent pIX phage display selects for distinct and improved antibody properties. Sci. Rep. 6, 39066, doi:10.1038/srep39066 (2016).
2 Frick, R. et al. Affinity maturation of TCR-like antibodies using phage display guided by structural modeling. Protein Engineering, Design and Selection 35, doi:10.1093/protein/gzac005 (2022).
3 Douglass, J. et al. Bispecific antibodies targeting mutant RAS neoantigens. Science Immunology 6, eabd5515, doi:10.1126/sciimmunol.abd5515 (2021).
4 Volkel, T., Korn, T., Bach, M., Muller, R. & Kontermann, R. E. Optimized linker sequences for the expression of monomeric and dimeric bispecific singlechain diabodies. Protein Engineering, Design and Selection 14, 815-823, doi:10.1093/protein/14.10.815 (2001).
5 Webber, K. O., Reiter, Y., Brinkmann, II., Kreitman, R. & Pastan, I.
Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog. Mol Immunol 32, 249-258, doi:10.1016/0161-5890(94)00150-y (1995).
6 Jager, M. & Pluckthun, A. Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen. J Mol Biol 285, 2005-2019, doi:10.1006/jmbi.1998.2425 (1999).
7 Dao, T. et al. Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1. Nat Biotech 33, 1079-1086 (2015).
8 Qi, J. et al. Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1. Proceedings of the National Academy of Sciences 115, E5467-E5476, doi:doi:10.1073/pnas.1719905115 (2018).
Claims
1 . An antigen binding protein comprising at least one antigen binding domain which binds to HLA-A*02:01/PRAME425'433, said antigen binding domain comprising a heavy chain variable domain (VH domain) that comprises three complementarity determining regions (CDRs), and a light chain variable domain (VL domain) that comprises three CDRs, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111), or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; or wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GYTFTGYY (SEQ ID NO:5), or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of INPNSGGT (SEQ ID NO:6), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDRWELLGSSDDY (SEQ ID NO:7), or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NQ:10), or a sequence substantially homologous thereto.
2. The antigen binding protein of claim 1 , wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111) or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113) or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9) or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
3. The antigen binding protein of claim 1 or claim 2, wherein the sequence substantially homologous to the given VH CDR1 or VH CDR2 sequence is a sequence containing up to 2 amino acid substitutions as compared to the given CDR sequence; the sequence substantially homologous to the given VH CDR3 sequence is a sequence containing up to 3 amino acid substitutions as compared to the given CDR sequence; the sequence substantially homologous to the given VL CDR1 or VL CDR3 sequence is a sequence containing up to 4 amino acid substitutions, preferably up to 3 or up to 2 amino acid substitutions, as compared to the given CDR sequence; and the sequence substantially homologous to the given VL CDR2 sequence is a sequence containing only 1 amino acid substitution as compared to the given CDR sequence.
4. The antigen binding protein of any one of claims 1 to 3, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto;
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), DWISGW (SEQ ID NO:116), AFISSW (SEQ ID NO:117), EFISQW (SEQ ID NO:118), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or QQANSFPFT (SEQ ID NO:121), or a sequence substantially homologous thereto.
5. The antigen binding protein of any one of claims 1 to 4, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of
GGTFSSYA (SEQ ID NO: 111), or a sequence substantially homologous thereto;
(b) a VH CDR 2 that comprises the amino acid sequence of
IIPIFGTA (SEQ ID NO: 112), LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto; and
(c) a VH CDR 3 that comprises the amino acid sequence of
ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of
QGVSNW (SEQ ID NO:8), EFVSSW (SEQ ID NO: 119), or QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a variable light VL CDR2 that comprises the amino acid sequence of
AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of
QQYNRLPLT (SEQ ID NO: 10), or a sequence substantially homologous thereto.
6. The antigen binding protein of claim 5, wherein i) the VH CDR 2 comprises the amino acid sequence of LVVGRA (SEQ ID NO: 114), or MQVGPTRHPLWA (SEQ ID NO: 115), or a sequence substantially homologous thereto, and the VL CDR1 comprises the amino acid sequence of QGVSNW (SEQ ID NO:8) or a sequence substantially homologous thereto; or ii) the VH CDR 2 comprises the amino acid sequence of IIPIFGTA (SEQ ID NO: 112) or a sequence substantially homologous thereto, and the VL CDR1
comprises the amino acid sequence of EFVSSW (SEQ ID NO:119), or QGISSW (SEQ ID NO:120), or a sequence substantially homologous thereto.
7. The antigen binding protein of any one of claims 1 to 6, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111), or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO: 113), or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10), or a sequence substantially homologous thereto.
8. The antigen binding protein of any one of claims 1 to 6, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111), or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of LVVGRA (SEQ ID NO:114), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113), or a sequence substantially homologous thereto; and/or wherein
said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10) or a sequence substantially homologous thereto.
9. The antigen binding protein of any one of claims 1 to 6, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111), or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of MQVGPTRHPLWA (SEQ ID NO:115), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113), or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGVSNW (SEQ ID NO:8), or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10), or a sequence substantially homologous thereto.
10. The antigen binding protein of any one of claims 1 to 6, wherein said heavy chain variable domain comprises:
(a) a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTFSSYA (SEQ ID NO:111), or a sequence substantially homologous thereto;
(b) a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:112), or a sequence substantially homologous thereto; and
(c) a VH CDR3 that comprises the amino acid sequence of ARDGYNYGQFDY (SEQ ID NO:113), or a sequence substantially homologous thereto; and/or wherein said light chain variable domain comprises:
(d) a variable light (VL) CDR1 that comprises the amino acid sequence of QGISSW (SEQ ID NQ:120), or a sequence substantially homologous thereto;
(e) a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:9), or a sequence substantially homologous thereto; and
(f) a VL CDR3 that comprises the amino acid sequence of QQYNRLPLT (SEQ ID NO: 10), or a sequence substantially homologous thereto.
11. The antigen binding protein of any one of claims 1 to 7, wherein said heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:3 or 132, or a sequence having at least 74% sequence identity thereto, and/or wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 74% sequence identity thereto.
12. The antigen binding protein of claim 7, wherein the heavy chain variable domain and the light chain variable domain comprise, respectively, the following amino acid sequences:
VH domain VL domain
SEQ ID NO:3 SEQ ID NO:4
SEQ ID NO:132 SEQ ID NO:4
SEQ ID NO:132 SEQ ID NO:134
SEQ ID NO:132 SEQ ID NO:136
SEQ ID NO:132 SEQ ID NO:138
SEQ ID NO:132 SEQ ID NQ:140
SEQ ID NO:142 SEQ ID NO:4
SEQ ID NO:144 SEQ ID NO:4
SEQ ID NO:132 SEQ ID NO:147 or
SEQ ID NO:132 SEQ ID NQ:150
13. The antigen binding protein of any one of claims 1 to 12, wherein said antigen binding protein is an antibody.
14. The antigen binding protein of any one of claims 1 to 13, wherein said antigen binding protein is a bispecific antibody in which said antigen binding domain that binds to HLA-A*02:01/PRAME425'433 is a first antigen binding domain, and wherein said antigen binding protein further comprises a second antigen binding domain that binds to a T-cell antigen present on the surface of T cells, preferably wherein said T- cell antigen is CD3.
15. The antigen binding protein of any one of claims 1 to 14, wherein said antigen binding protein is a single chain bispecific diabody (scDb).
16. The antigen binding protein of claim 15, wherein said scDb comprises the amino acid sequence set out in SEQ ID NO: 30, 152, 155, 157, 159, 161, 163, 166, 168 or 170.
17. The antigen binding protein of any one of claims 1 to 16, wherein said antigen binding protein preferentially binds to HLA-A*02:01/PRAME425'433 as compared to a HLA-A*02:01 restricted peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 35 and as compared to a HLA-A*02:01 restricted peptide comprising or consisting of the amino acid sequence of SEQ ID NO:37.
18. The antigen binding protein of any one of claims 14 to 17, wherein said antigen binding protein exhibits an EC50 of less than 5 x 10'11 M as measured in cancer cells.
19. An immunoconjugate comprising the antigen binding protein of any one of claims 1 to 18, operatively attached to at least one other therapeutic or diagnostic agent.
20. One or more nucleic acid molecules comprising nucleotide sequences that encode the antigen binding protein or immunoconjugate of any one of claims 1 to 19.
21. One or more expression vectors comprising the one or more of the nucleic acid molecules of claim 20.
22. One or more host cells or viruses comprising said expression vectors of claim 21, or said nucleic acid molecules of claim 20, or expressing the antigen binding protein or immunoconjugate of any one of claims 1 to 19.
23. A method of producing the antigen binding protein or immunoconjugate of any one of claims 1 to 19, said method comprising the steps of (i) culturing a host cell comprising the expression vectors of claim 21 or the nucleic acid molecules of claim 20, under conditions suitable for the expression of the encoded antigen binding protein or immunoconjugate; and optionally (ii) isolating or obtaining the antigen binding protein or immunoconjugate from the host cell or from the growth medium/supernatant.
24. A composition comprising the antigen binding protein of any one of claims 1 to 18, the immunoconjugate of claim 19, the one or more nucleic acid molecules of claim 20, the one or more expression vectors of claim 21 , or the one or more host cells or viruses of claim 22; and a diluent, carrier or excipient.
25. The antigen binding protein of any one of claims 1 to 18, the immunoconjugate of claim 19, the one or more nucleic acid molecules of claim 20, the one or more expression vectors of claim 21 , or the one or more host cells or viruses of claims 22, for use in therapy.
26. The antigen binding protein of any one of claims 1 to 18, the immunoconjugate of claim 19, the one or more nucleic acid molecules of claim 20, the one or more expression vectors of claim 21 , or the one or more host cells or viruses of claims 22, for use in the treatment or prevention of cancer.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB2219294.2 | 2022-12-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2024133584A2 true WO2024133584A2 (en) | 2024-06-27 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107955071B (en) | Human anti-human CD47 antibody and coding gene and application thereof | |
| US11555077B2 (en) | 4-1BB antibody and preparation method and use thereof | |
| WO2016154047A2 (en) | Monoclonal antigen-binding proteins to intracellular oncogene products | |
| DK2814842T3 (en) | ANTIBODIES BINDING PEPTIDOGLYCAN RECOGNITION PROTEIN 1 | |
| CN110229232B (en) | Bispecific antibodies and uses thereof | |
| JP2021533203A (en) | A novel fusion protein specific for CD137 and PD-L1 | |
| CN108948194B (en) | Novel CTLA-4 monoclonal antibody | |
| JP2019512207A (en) | T cell receptor-like antibody specific for FOXP3-derived peptides | |
| EP4101867A1 (en) | Anti-cd3 and anti-cd123 bispecific antibody and use thereof | |
| TWI714895B (en) | Anti-csf-1r antibody, antigen-binding fragment thereof and pharmaceutical use thereof | |
| KR20230169944A (en) | MAGE-A4 peptide-MHC antigen binding protein | |
| CA3148735A1 (en) | Anti-hk2 chimeric antigen receptor (car) | |
| WO2024133584A2 (en) | Antigen binding proteins | |
| KR20230042090A (en) | Anti-CD22 Single Domain Antibodies and Therapeutic Constructs | |
| WO2024133573A1 (en) | Antigen binding proteins | |
| EP3529275B1 (en) | Proteins and uses | |
| CN115298216A (en) | Antibody or antigen binding fragment thereof, preparation method and medical application thereof | |
| JP2023116826A (en) | antigen binding molecule | |
| WO2022117032A1 (en) | Anti-cd22 nano antibody and use thereof | |
| TW202315889A (en) | Antibodies | |
| JP2024513446A (en) | GUCY2C binding polypeptide and its uses | |
| WO2023110918A1 (en) | Dual mhc-targeting t cell engager | |
| AU2022413444A1 (en) | Dual mhc-targeting t cell engager | |
| AU2022292159A1 (en) | Anti-cd307e single-domain antibodies, uses thereof in car t-cell and for the treatment of diseases | |
| KR20240099382A (en) | Bispecific CD16A binding agent |