CN117247444A - Interleukin-2 (IL-2) mutants and uses thereof - Google Patents
Interleukin-2 (IL-2) mutants and uses thereof Download PDFInfo
- Publication number
- CN117247444A CN117247444A CN202310711032.1A CN202310711032A CN117247444A CN 117247444 A CN117247444 A CN 117247444A CN 202310711032 A CN202310711032 A CN 202310711032A CN 117247444 A CN117247444 A CN 117247444A
- Authority
- CN
- China
- Prior art keywords
- mutant
- amino acid
- fusion protein
- acid sequence
- mutations
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010002350 Interleukin-2 Proteins 0.000 title claims abstract description 772
- 102000000588 Interleukin-2 Human genes 0.000 title abstract description 728
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 271
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 271
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 133
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 120
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 120
- 239000013598 vector Substances 0.000 claims abstract description 106
- 238000000034 method Methods 0.000 claims abstract description 87
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 49
- 230000035772 mutation Effects 0.000 claims description 208
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 202
- 241000282414 Homo sapiens Species 0.000 claims description 144
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 106
- 230000027455 binding Effects 0.000 claims description 89
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 70
- 229920001184 polypeptide Polymers 0.000 claims description 67
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 58
- 108010021466 Mutant Proteins Proteins 0.000 claims description 55
- 201000010099 disease Diseases 0.000 claims description 51
- 102000008300 Mutant Proteins Human genes 0.000 claims description 49
- 239000012636 effector Substances 0.000 claims description 31
- 208000023275 Autoimmune disease Diseases 0.000 claims description 28
- 206010025135 lupus erythematosus Diseases 0.000 claims description 21
- 230000002829 reductive effect Effects 0.000 claims description 20
- 102220484783 Norrin_S75P_mutation Human genes 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 18
- 102200001692 rs72552726 Human genes 0.000 claims description 17
- 208000027866 inflammatory disease Diseases 0.000 claims description 14
- 108010075254 C-Peptide Proteins 0.000 claims description 12
- 102220570645 Hepatic leukemia factor_R83N_mutation Human genes 0.000 claims description 11
- 102220274636 rs144712084 Human genes 0.000 claims description 11
- 102220050506 rs193920950 Human genes 0.000 claims description 11
- 102220058225 rs375451955 Human genes 0.000 claims description 11
- 208000012657 Atopic disease Diseases 0.000 claims description 7
- 208000009329 Graft vs Host Disease Diseases 0.000 claims description 7
- 208000005176 Hepatitis C Diseases 0.000 claims description 7
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 7
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 7
- 206010047115 Vasculitis Diseases 0.000 claims description 7
- 208000006673 asthma Diseases 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 208000035475 disorder Diseases 0.000 claims description 7
- 208000024908 graft versus host disease Diseases 0.000 claims description 7
- 201000006417 multiple sclerosis Diseases 0.000 claims description 7
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 7
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 7
- 206010003827 Autoimmune hepatitis Diseases 0.000 claims description 6
- 208000009137 Behcet syndrome Diseases 0.000 claims description 6
- 208000011231 Crohn disease Diseases 0.000 claims description 6
- 206010018364 Glomerulonephritis Diseases 0.000 claims description 6
- 102220474982 Histidine-tRNA ligase, cytoplasmic_R83A_mutation Human genes 0.000 claims description 6
- 201000004681 Psoriasis Diseases 0.000 claims description 6
- 102220631456 Putative uncharacterized protein H1-10-AS1_N71S_mutation Human genes 0.000 claims description 6
- 206010047642 Vitiligo Diseases 0.000 claims description 6
- 208000004631 alopecia areata Diseases 0.000 claims description 6
- 208000007475 hemolytic anemia Diseases 0.000 claims description 6
- 206010028417 myasthenia gravis Diseases 0.000 claims description 6
- 201000003068 rheumatic fever Diseases 0.000 claims description 6
- 102220250393 rs553900705 Human genes 0.000 claims description 6
- 102220079367 rs797045802 Human genes 0.000 claims description 6
- 102200057423 rs867410737 Human genes 0.000 claims description 6
- 206010043778 thyroiditis Diseases 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 4
- 239000000539 dimer Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 2
- 206010053177 Epidermolysis Diseases 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 8
- 210000004027 cell Anatomy 0.000 description 230
- 108090000623 proteins and genes Proteins 0.000 description 120
- 102000004169 proteins and genes Human genes 0.000 description 88
- 235000001014 amino acid Nutrition 0.000 description 80
- 235000018102 proteins Nutrition 0.000 description 78
- 229940024606 amino acid Drugs 0.000 description 67
- 150000001413 amino acids Chemical class 0.000 description 66
- 210000003289 regulatory T cell Anatomy 0.000 description 65
- 102000005962 receptors Human genes 0.000 description 45
- 108020003175 receptors Proteins 0.000 description 45
- 125000005647 linker group Chemical group 0.000 description 43
- 239000000178 monomer Substances 0.000 description 42
- 238000006467 substitution reaction Methods 0.000 description 39
- 230000014509 gene expression Effects 0.000 description 38
- 210000001744 T-lymphocyte Anatomy 0.000 description 37
- 230000006870 function Effects 0.000 description 36
- 125000000539 amino acid group Chemical group 0.000 description 28
- 229940079593 drug Drugs 0.000 description 27
- 239000003814 drug Substances 0.000 description 27
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 25
- 239000000203 mixture Substances 0.000 description 25
- 108010087819 Fc receptors Proteins 0.000 description 24
- 102000009109 Fc receptors Human genes 0.000 description 24
- 230000000694 effects Effects 0.000 description 24
- 102000001712 STAT5 Transcription Factor Human genes 0.000 description 23
- 108010029477 STAT5 Transcription Factor Proteins 0.000 description 23
- 239000003446 ligand Substances 0.000 description 23
- 238000002474 experimental method Methods 0.000 description 21
- 239000013604 expression vector Substances 0.000 description 21
- 238000011282 treatment Methods 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 20
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 20
- 230000026731 phosphorylation Effects 0.000 description 20
- 238000006366 phosphorylation reaction Methods 0.000 description 20
- 210000002966 serum Anatomy 0.000 description 19
- 108010076504 Protein Sorting Signals Proteins 0.000 description 18
- 241000588724 Escherichia coli Species 0.000 description 17
- 230000004913 activation Effects 0.000 description 17
- 239000000872 buffer Substances 0.000 description 17
- 238000001727 in vivo Methods 0.000 description 17
- 239000002609 medium Substances 0.000 description 17
- 238000001514 detection method Methods 0.000 description 16
- 230000001965 increasing effect Effects 0.000 description 16
- 210000000822 natural killer cell Anatomy 0.000 description 16
- 239000012634 fragment Substances 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 108091006020 Fc-tagged proteins Proteins 0.000 description 14
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 14
- 108060003951 Immunoglobulin Proteins 0.000 description 14
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 14
- 238000003556 assay Methods 0.000 description 14
- 102000018358 immunoglobulin Human genes 0.000 description 14
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 13
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 13
- 230000035755 proliferation Effects 0.000 description 13
- 230000003213 activating effect Effects 0.000 description 12
- 239000003623 enhancer Substances 0.000 description 12
- 230000001939 inductive effect Effects 0.000 description 12
- 210000002381 plasma Anatomy 0.000 description 12
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 11
- 235000018417 cysteine Nutrition 0.000 description 11
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 11
- 230000006698 induction Effects 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 238000004113 cell culture Methods 0.000 description 10
- 238000012217 deletion Methods 0.000 description 10
- 230000037430 deletion Effects 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 238000013518 transcription Methods 0.000 description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 9
- 102000035195 Peptidases Human genes 0.000 description 9
- 108091005804 Peptidases Proteins 0.000 description 9
- 230000007423 decrease Effects 0.000 description 9
- 238000010494 dissociation reaction Methods 0.000 description 9
- 230000005593 dissociations Effects 0.000 description 9
- 230000013595 glycosylation Effects 0.000 description 9
- 238000006206 glycosylation reaction Methods 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 210000004962 mammalian cell Anatomy 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 8
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 8
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 8
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 8
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000004071 biological effect Effects 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 230000004927 fusion Effects 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 8
- 230000036470 plasma concentration Effects 0.000 description 8
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 7
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 7
- 229960002433 cysteine Drugs 0.000 description 7
- 108020001096 dihydrofolate reductase Proteins 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 238000000855 fermentation Methods 0.000 description 7
- 230000004151 fermentation Effects 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 239000013603 viral vector Substances 0.000 description 7
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 6
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 6
- 229910052770 Uranium Inorganic materials 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- -1 e.g. Substances 0.000 description 6
- 210000003527 eukaryotic cell Anatomy 0.000 description 6
- 239000000833 heterodimer Substances 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000001990 intravenous administration Methods 0.000 description 6
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 210000001236 prokaryotic cell Anatomy 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 5
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 5
- 229940072221 immunoglobulins Drugs 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 230000019491 signal transduction Effects 0.000 description 5
- 239000007790 solid phase Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 4
- 150000008574 D-amino acids Chemical class 0.000 description 4
- 206010014989 Epidermolysis bullosa Diseases 0.000 description 4
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 108010073807 IgG Receptors Proteins 0.000 description 4
- 102000009490 IgG Receptors Human genes 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 241000288906 Primates Species 0.000 description 4
- 241000283984 Rodentia Species 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000005888 antibody-dependent cellular phagocytosis Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 230000008030 elimination Effects 0.000 description 4
- 238000003379 elimination reaction Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002523 gelfiltration Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 150000002482 oligosaccharides Chemical class 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 238000010254 subcutaneous injection Methods 0.000 description 4
- 239000007929 subcutaneous injection Substances 0.000 description 4
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 230000005030 transcription termination Effects 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 241001430294 unidentified retrovirus Species 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 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 3
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 206010061818 Disease progression Diseases 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 3
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 3
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 108090000157 Metallothionein Proteins 0.000 description 3
- 102000005431 Molecular Chaperones Human genes 0.000 description 3
- 108010006519 Molecular Chaperones Proteins 0.000 description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 3
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 3
- 229930193140 Neomycin Natural products 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 240000007019 Oxalis corniculata Species 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 241000714474 Rous sarcoma virus Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000012505 Superdex™ Substances 0.000 description 3
- 102000006601 Thymidine Kinase Human genes 0.000 description 3
- 108020004440 Thymidine kinase Proteins 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 3
- 125000000217 alkyl group Chemical group 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 3
- 229910002091 carbon monoxide Inorganic materials 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000007385 chemical modification Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 230000024203 complement activation Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 229920001577 copolymer Polymers 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000005750 disease progression Effects 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000001506 immunosuppresive effect Effects 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 229960000485 methotrexate Drugs 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 108010068617 neonatal Fc receptor Proteins 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 230000008488 polyadenylation Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 3
- 235000019419 proteases Nutrition 0.000 description 3
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 238000013456 study Methods 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 238000011191 terminal modification Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical compound C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 description 2
- 101710154825 Aminoglycoside 3'-phosphotransferase Proteins 0.000 description 2
- 244000105975 Antidesma platyphyllum Species 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000282326 Felis catus Species 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
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 2
- 208000009889 Herpes Simplex Diseases 0.000 description 2
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 108091082332 JAK family Proteins 0.000 description 2
- 102000042838 JAK family Human genes 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- 150000008575 L-amino acids Chemical class 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 102000003792 Metallothionein Human genes 0.000 description 2
- 101100515942 Mus musculus Nbl1 gene Proteins 0.000 description 2
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- 241001505332 Polyomavirus sp. Species 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 244000000188 Vaccinium ovalifolium Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000008228 bacteriostatic water for injection Substances 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 125000003636 chemical group Chemical group 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000009615 deamination Effects 0.000 description 2
- 238000006481 deamination reaction Methods 0.000 description 2
- 230000022811 deglycosylation Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 235000009424 haa Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 238000005734 heterodimerization reaction Methods 0.000 description 2
- 229920001519 homopolymer Polymers 0.000 description 2
- 238000001597 immobilized metal affinity chromatography Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229960003444 immunosuppressant agent Drugs 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- QDLAGTHXVHQKRE-UHFFFAOYSA-N lichenxanthone Natural products COC1=CC(O)=C2C(=O)C3=C(C)C=C(OC)C=C3OC2=C1 QDLAGTHXVHQKRE-UHFFFAOYSA-N 0.000 description 2
- 150000002632 lipids Chemical group 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000000284 resting effect Effects 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002047 solid lipid nanoparticle Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 125000000341 threoninyl group Chemical class [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 231100000607 toxicokinetics Toxicity 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 108010087967 type I signal peptidase Proteins 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 229920003169 water-soluble polymer Polymers 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- KUHSEZKIEJYEHN-BXRBKJIMSA-N (2s)-2-amino-3-hydroxypropanoic acid;(2s)-2-aminopropanoic acid Chemical compound C[C@H](N)C(O)=O.OC[C@H](N)C(O)=O KUHSEZKIEJYEHN-BXRBKJIMSA-N 0.000 description 1
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- WNXJIVFYUVYPPR-UHFFFAOYSA-N 1,3-dioxolane Chemical compound C1COCO1 WNXJIVFYUVYPPR-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- BCHZICNRHXRCHY-UHFFFAOYSA-N 2h-oxazine Chemical compound N1OC=CC=C1 BCHZICNRHXRCHY-UHFFFAOYSA-N 0.000 description 1
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 1
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 102100036664 Adenosine deaminase Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102100021266 Alpha-(1,6)-fucosyltransferase Human genes 0.000 description 1
- 102000003730 Alpha-catenin Human genes 0.000 description 1
- 108090000020 Alpha-catenin Proteins 0.000 description 1
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 1
- 238000012815 AlphaLISA Methods 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- 108010014223 Armadillo Domain Proteins Proteins 0.000 description 1
- 102000016904 Armadillo Domain Proteins Human genes 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101100136076 Aspergillus oryzae (strain ATCC 42149 / RIB 40) pel1 gene Proteins 0.000 description 1
- 241000713842 Avian sarcoma virus Species 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 229920002799 BoPET Polymers 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 101100228196 Caenorhabditis elegans gly-4 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 102000003813 Cis-trans-isomerases Human genes 0.000 description 1
- 108090000175 Cis-trans-isomerases Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000016736 Cyclin Human genes 0.000 description 1
- 108050006400 Cyclin Proteins 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 241000289632 Dasypodidae Species 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 108700022150 Designed Ankyrin Repeat Proteins Proteins 0.000 description 1
- BVTJGGGYKAMDBN-UHFFFAOYSA-N Dioxetane Chemical compound C1COO1 BVTJGGGYKAMDBN-UHFFFAOYSA-N 0.000 description 1
- 206010061819 Disease recurrence Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 208000010975 Dystrophic epidermolysis bullosa Diseases 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
- 241000196324 Embryophyta Species 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 101100173535 Escherichia coli (strain K12) fepE gene Proteins 0.000 description 1
- 101100409165 Escherichia coli (strain K12) prc gene Proteins 0.000 description 1
- 101000867232 Escherichia coli Heat-stable enterotoxin II Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 101100508941 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) ppa gene Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000819490 Homo sapiens Alpha-(1,6)-fucosyltransferase Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101100534458 Homo sapiens STAT5A gene Proteins 0.000 description 1
- 101000934996 Homo sapiens Tyrosine-protein kinase JAK3 Proteins 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 102000018297 Immunoglobulin subtype Human genes 0.000 description 1
- 108050007411 Immunoglobulin subtype Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108010032774 Interleukin-2 Receptor alpha Subunit Proteins 0.000 description 1
- 102000007351 Interleukin-2 Receptor alpha Subunit Human genes 0.000 description 1
- 230000004163 JAK-STAT signaling pathway Effects 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- 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 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 241001590997 Moolgarda engeli Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100178822 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) htrA1 gene Proteins 0.000 description 1
- 241000713883 Myeloproliferative sarcoma virus Species 0.000 description 1
- 239000005041 Mylar™ Substances 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 238000007126 N-alkylation reaction Methods 0.000 description 1
- 101100407828 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) ptr-3 gene Proteins 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 241001057811 Paracoccus <mealybug> Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010087702 Penicillinase Proteins 0.000 description 1
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 1
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 108090000316 Pitrilysin Proteins 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 102100037935 Polyubiquitin-C Human genes 0.000 description 1
- 241000677647 Proba Species 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 101710127332 Protease I Proteins 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 241000589180 Rhizobium Species 0.000 description 1
- 101100277437 Rhizobium meliloti (strain 1021) degP1 gene Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 101000677856 Stenotrophomonas maltophilia (strain K279a) Actin-binding protein Smlt3054 Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 1
- 101710137710 Thioesterase 1/protease 1/lysophospholipase L1 Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108010056354 Ubiquitin C Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 241000863000 Vitreoscilla Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000005257 alkyl acyl group Chemical group 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 230000006229 amino acid addition Effects 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 229940126574 aminoglycoside antibiotic Drugs 0.000 description 1
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 101150042295 arfA gene Proteins 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 229940000635 beta-alanine Drugs 0.000 description 1
- 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 1
- 238000013357 binding ELISA Methods 0.000 description 1
- 238000012925 biological evaluation Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 101150031021 birA gene Proteins 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- BGECDVWSWDRFSP-UHFFFAOYSA-N borazine Chemical compound B1NBNBN1 BGECDVWSWDRFSP-UHFFFAOYSA-N 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000000298 carbocyanine Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 208000029664 classic familial adenomatous polyposis Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 description 1
- 229940099500 cystamine Drugs 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 101150018266 degP gene Proteins 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 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 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 101150071678 dsbB gene Proteins 0.000 description 1
- 101150015101 dsbC gene Proteins 0.000 description 1
- 101150078200 dsbD gene Proteins 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 208000004298 epidermolysis bullosa dystrophica Diseases 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 208000024711 extrinsic asthma Diseases 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000033581 fucosylation Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000002343 gold Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 210000005256 gram-negative cell Anatomy 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 102000049912 human JAK3 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000014726 immortalization of host cell Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 108091008042 inhibitory receptors Proteins 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 125000001909 leucine group Chemical class [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000000670 ligand binding assay Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000012931 lyophilized formulation Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 210000003519 mature b lymphocyte Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000005541 medical transmission Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009061 membrane transport Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000006151 minimal media Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000007908 nanoemulsion Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 101150087557 omcB gene Proteins 0.000 description 1
- 101150115693 ompA gene Proteins 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229940043515 other immunoglobulins in atc Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 101150040383 pel2 gene Proteins 0.000 description 1
- 101150050446 pelB gene Proteins 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229950009506 penicillinase Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 101150058164 phoE gene Proteins 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 108060006184 phycobiliprotein Proteins 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920000191 poly(N-vinyl pyrrolidone) Polymers 0.000 description 1
- 229920005575 poly(amic acid) Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 108010043383 protease V Proteins 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 229920005604 random copolymer Polymers 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000005316 response function Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 235000019722 synbiotics Nutrition 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 101150003509 tag gene Proteins 0.000 description 1
- CYFJIBWZIQDUSZ-UHFFFAOYSA-N thioglycine Chemical compound NCC(S)=O CYFJIBWZIQDUSZ-UHFFFAOYSA-N 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 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
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 125000001493 tyrosinyl group Chemical class [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 231100000402 unacceptable toxicity Toxicity 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 239000001018 xanthene dye Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2013—IL-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
To therapeutic uses of IL-2 mutants, fusion proteins comprising IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same, as well as IL-2 mutants, fusion proteins comprising IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same. And also relates to IL-2 mutant and its fusion protein preparing process.
Description
The contents of the sequence listing submitted below are incorporated herein by reference in their entirety, file names: IL-2-420230516.Xml, date of record: 2023.05.16, size: 78 KB).
Technical Field
The present application relates to interleukin-2 (IL-2) mutants, fusion proteins comprising said IL-2 mutants, nucleic acid molecules encoding IL-2 mutants or fusion proteins, vectors and host cells comprising the nucleic acid molecules, and pharmaceutical compositions and therapeutic uses comprising the same, as well as methods for the preparation of IL-2 mutants and fusion proteins thereof.
Background
Interleukin-2 (IL-2), also known as T Cell Growth Factor (TCGF), is a bundle type I cell growth factor consisting of 4 alpha helices (a, B, C, D), which is produced primarily by antigen activated T cells (anti-activated T cells) and promotes proliferation, differentiation and survival of mature T cells and B cells, as well as cytolytic activity of natural killer cells (NK cells) in innate immune defenses (k.a. Smith, science 240,1169 (1988); B.H.Nelson, D.M.Willerford, adv.Immunol.70,1 (1998)).
IL-2 performs its function by binding to IL-2 receptors on the surface of target cells. IL-2 has three receptor subunits: alpha chain (IL-2Rα or CD 25), beta chain (IL-2Rβ or CD 122) and general cytokine receptor gamma chain (γc or IL-2Rγ or CD 132) (W.J. Leonard et al, nature 311,626 (1984); T.Nikaido et al, nature 311,631 (1984); D.Cosman et al, nature 312,768 (1984); M.Hatakeyama et al, science 244,551 (1989); T.Takeshita et al, science 257,379 (1992)).
Depending on the binding subunits and affinity for IL-2, IL-2 receptors can be divided into the following classes: the first type is low affinity receptor, consisting of the IL-2Rα subunit alone (Kd value about 10 nM), and IL-2 binding to such receptor is unable to signal and regulate cells due to the lack of intracellular signaling regions (H.M.Wang, K.A.Smith, J.Exp.Med.166,1055 (1987)). The second type is the medium affinity receptor, consisting of IL-2Rβ and yc subunits (Kd value of about 1 nM), which is expressed mainly on natural killer cells, macrophages and T cells in resting state, and activates signaling pathways at higher IL-2 concentrations (B.H.Nelson, D.M.Willerford, adv.Immunol.70,1 (1998)). Although the affinity of IL-2Rβ alone for IL-2 is very low (Kd value of about 100 nM), binding to IL-2 is hardly detected by yc alone (M.Rickert, M.J.Boulanger, N.Goriatcheva, K.C) Garcia, j.mol.biol.339,1115 (2004)), but a complex consisting of IL-2rβ and yc (IL-2rβγ) is essential for efficient cell signaling of IL-2 (y.nakamura, et al, nature 369,330 (1994); nelson, et al, nature 369,333 (1994)). The third type is a high affinity receptor consisting of three subunits, IL-2Rα, IL-2Rβ and yc (Kd value about 10 pM), typically in activated lymphocytes and CD4 + CD25 + Foxp3 + Expression on regulatory T cells (Treg) (T.Takeshita et al, science257,379 (1992); boyman O, et al, nat Rev immunol.2012Feb 17;12 (3): 180-90).
Since IL-2 can enhance the functions of effector T cells and natural killer cells, it can be used as an immune activator for treating diseases associated with lymphocyte proliferation. In recent years, IL-2 has been found to be a critical cytokine for differentiation, function and survival of Treg cells, and to be important in achieving the induction of the function, development and suppressive characteristics of Treg cells. Treg cells are generally defined by expression of CD4, CD25 and transcription factor fork P3 (Foxp 3), which are capable of suppressing autoreactive lymphocytes and regulating innate and adaptive immune responses, resulting in immunosuppression. Treg cells act as one of the key effector cells for immunosuppression and play a critical role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors, and are of great significance for the development and progression of autoimmune diseases. (Malek TR, et al, nat Rev Immunol 2004;4:665-74;Nelson BH.J Immunol 2004;172:3983-8;Piccirillo CA,et al, semin Immunol 2004; 16:81-8).
Based on the wide range of biological effects of IL-2, there remains a need in the art to develop novel IL-2 molecules with improved properties by altering the selectivity or preference of IL-2 for different receptors, allowing IL-2 to selectively activate target cells, to reduce the toxicity and/or increase the efficacy of IL-2 therapy, to increase the safety window of IL-2 for treating a particular disease, and to increase the safety and efficacy of IL-2 for treating a disease.
The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.
Summary of The Invention
One aspect of the present application relates to an IL-2 mutant comprising a mutation of R83 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of R83 is R83L, R a or R83N. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In some embodiments, the IL-2 mutants described herein further comprise a mutation in D20 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of D20 is D20N. In some embodiments, the IL-2 mutants described herein, which comprise R83L and D20N mutations relative to the amino acid sequence of human wild-type IL-2.
In some embodiments, the IL-2 mutants described herein further comprise mutations in H16 and N71 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of H16 and N71 are H16N and N71R, respectively. In some embodiments, the IL-2 mutants described herein comprise R83L, H N and N71R mutations relative to the amino acid sequence of human wild-type IL-2.
In some embodiments, the IL-2 mutants described herein further comprise mutations in N71 and N88 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of N71 and N88 are N71S and N88D, respectively. In some embodiments, the IL-2 mutants described herein comprise R83A, N S and N88D mutations relative to the amino acid sequence of human wild-type IL-2.
In some embodiments, the IL-2 mutants described herein further comprise mutations in L66 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of L66 and V91 are L66F and V91E, respectively. In some embodiments, the IL-2 mutants described herein comprise R83N, L F and V91E mutations relative to the amino acid sequence of human wild-type IL-2.
Another aspect of the present application relates to an IL-2 mutant comprising mutations in E67 and S75 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of E67 is E67I or E67G, and the mutation of S75 is S75P. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1. In some embodiments, the IL-2 mutants described herein, which comprise E67I and S75P mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein, which comprise E67G and S75P mutations relative to the human wild-type IL-2 amino acid sequence.
Another aspect of the present application also relates to an IL-2 mutant comprising mutations in L17 and P82 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L17 and P82 are L17V and P82L, respectively. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In some embodiments, the IL-2 mutants described herein further comprise a mutation relative to the human wild-type IL-2 amino acid sequence C125 in addition to the mutations described above. In some embodiments, the mutation of C125 is C125S or C125A.
In some embodiments, IL-2 mutants described herein comprise an amino acid sequence set forth in any of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with an amino acid sequence set forth in any of SEQ ID NOs 5-11.
In some embodiments, the IL-2 mutants described herein have reduced binding affinity to IL-2Rβγ and are capable of maintaining binding to IL-2Rα as compared to wild-type IL-2.
Another aspect of the present application relates to fusion proteins comprising the IL-2 mutants. In some embodiments, the fusion protein comprises Fc. In some embodiments, the Fc comprises a mutation capable of altering effector function, and/or a mutation capable of extending half-life, and/or a mutation capable of promoting dimer formation of a heterologous polypeptide. In some embodiments, the Fc is derived from a human. In other embodiments, the Fc is derived from human IgG, including IgG1, igG2, igG3, or IgG4, preferably IgG1 or IgG4.
In some embodiments, the Fc is derived from human IgG1 comprising mutations L234A and L235A; and/or N297G; and/or N297A; and/or L234A, L235A and P331S; and/or L234A, L235E, G237A, A S and P331S mutations, wherein the numbering is the EU numbering system.
In some embodiments, the Fc is derived from human IgG4 comprising mutations S228P, F234A and L235A mutation, wherein the numbering is the EU numbering system.
In some embodiments, the Fc further comprises S354C and T366W mutations; and/or Y349C, T366S, L368A, and Y407V mutations, wherein the numbering is the EU numbering system.
In some embodiments, a fusion protein as described herein, wherein the IL-2 mutant is linked to Fc. In some embodiments, wherein the IL-2 mutant is linked to the Fc via a linker peptide. In some embodiments, the connecting peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20-51. In some embodiments, the connecting peptide comprises the amino acid sequence GGGGS (SEQ ID NO: 29).
In some embodiments, the fusion proteins of an IL-2 mutant and an Fc described herein, wherein the IL-2 mutant is located at the N-terminus and/or the C-terminus of the Fc.
In some embodiments, fusion proteins described herein comprise an amino acid sequence set forth in any one of SEQ ID NOs:53-59, or a variant thereof, that has at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to an amino acid sequence set forth in any one of SEQ ID NOs: 53-59.
Also, the present application relates to isolated nucleic acids encoding any of the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein, vectors comprising the nucleic acids, host cells (e.g., CHO cells, HEK 293 cells, hela cells, or COS cells) comprising the nucleic acids or vectors, compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein. Also disclosed are methods for treating inflammatory or autoimmune diseases (e.g., lupus, graft versus host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophy epidermolysis bullosa, and Behcet's disease) in an individual in need thereof using any of the IL-2 mutants described herein or fusion proteins thereof or pharmaceutical compositions comprising the same.
Drawings
FIG. 1A is a schematic diagram showing an exemplary structure of a bivalent fusion protein of an IL-2 mutant and Fc, which comprises two identical monomers, the IL-2 mutant molecule in each monomer being linked to the N-terminus of the Fc. FIG. 1B is a schematic diagram showing an exemplary structure of a bivalent fusion protein of an IL-2 mutant and Fc, which comprises two identical monomers, the IL-2 mutant molecule in each monomer being linked to the C-terminus of the Fc.
FIG. 2 is a schematic diagram showing an exemplary structure of a monovalent fusion protein of an IL-2 mutant and Fc comprising two different monomers, one of which comprises an IL-2 mutant molecule linked to the C-terminus of Fc knob and the other of which is an Fc hole molecule.
FIG. 3A shows a bivalent fusion protein of IL-2 mutant and Fc: results of Fc-IL2-mut1, fc-IL2-mut4 and Fc-IL2-mut6 induced STAT5 phosphorylation in Treg cells.
FIG. 3B shows a bivalent fusion protein of IL-2 mutant and Fc: fc-IL2-mut1, fc-IL2-mut4 and Fc-IL2-mut6 are in CD8 + Results of induction of STAT5 phosphorylation in T lymphocytes.
FIG. 3C shows a bivalent fusion protein of IL-2 mutant and Fc: results of Fc-IL2-mut3 induced STAT5 phosphorylation in Treg cells.
FIG. 3D shows a bivalent fusion protein of IL-2 mutant and Fc: fc-IL2-mut3 in CD8 + Results of induction of STAT5 phosphorylation in T lymphocytes.
FIG. 4A shows that the bivalent fusion protein Fc-IL2-mut1 of IL-2 mutant and Fc stimulated proliferation of Treg cells in mice at a dose of 1 mg/kg.
FIG. 4B shows CD8 in mice at a dose of 1mg/kg for the bivalent fusion protein Fc-IL2-mut1 of IL-2 mutant and Fc + Effect of T lymphocyte proliferation.
Detailed description of the present application
The present application relates to IL-2 mutants, fusion proteins comprising IL-2 mutants, and the like. The IL-2 mutants described herein are capable of selectively activating Treg cells, stimulating proliferation of Treg cells, and have a higher safety window (e.g., in HEK-Blue) in the treatment of certain specific diseases (e.g., inflammatory or autoimmune diseases) than wild-type IL-2 TM In IL-2 cell line selection experiments, the cell line was selected by HEK-Blue as compared to wild-type IL-2 TM CD122/CD132 cell line for detecting EC50 value of IL-2 mutant combined with IL-2Rβγ and HEK-Blue TM IL-2 cell lines detect a higher ratio between the EC50 values of IL-2 mutants and IL-2Rαβγ binding). Further, the fusion proteins of IL-2 mutants and Fc described herein, by engineering the Fc fragment, reduce or minimize antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) to avoid undesirable immune responses during treatment, and increase the in vivo circulation half-life of IL-2 mutants, thereby reducing the frequency and total number of administrations, providing convenience to the patient and reducing the cost of treatment.
The present application also relates to nucleic acids encoding the IL-2 mutants or fusion proteins comprising IL-2 mutants, vectors comprising the nucleic acids, host cells comprising the nucleic acids or vectors, as well as methods of producing the above IL-2 mutants or fusion proteins, pharmaceutical compositions and articles of manufacture comprising the same, and methods of treating diseases (e.g., lupus, graft versus host disease, hepatitis C induced vasculitis, type i diabetes, type ii diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma or inflammatory bowel disease) using the IL-2 mutants or fusion proteins or pharmaceutical compositions comprising IL-2 mutants.
I. Definition of the definition
The practice of the present application will employ, unless the context clearly indicates otherwise, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described in detail below for illustration. Such techniques are well explained in the literature. See Current Protocols in Molecular Biology or Current Protocols in Immunology, john Wiley & Sons, new York, n.y. (2009); ausubel et al Short Protocols in Molecular Biology,3rd ed., john Wiley & Sons,1995; sambrook and Russell, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); maniatis et al Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol.I & II (D.Glover, ed.); oligonucleotide Synthesis (n.gait, ed., 1984); nucleic Acid Hybridization (b.hames & s.higgins, eds., 1985); transcription and Translation (b.hames & s.higgins, eds., 1984); animal Cell Culture (r.freshney, ed., 1986); perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.
As used herein, the term "wild-type IL-2" or "WT IL-2" refers to naturally occurring IL-2, which may be derived from any natural IL-2 of vertebrate origin, such as mammals, including but not limited to livestock (e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., humans and non-human primates, such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters). The term includes unprocessed IL-2 forms (e.g., comprising a signal peptide) and processed mature IL-2 forms (e.g., not comprising a signal peptide). The term also includes naturally occurring IL-2 allelic variants and splice variants, isoforms, homologs, and species homologs. The term also includes naturally occurring IL-2 variants and the like. For example, the naturally occurring IL-2 variant has at least 95% (e.g., 95%,96%,97%,98%, or 99%) sequence homology to native IL-2. In some embodiments, an exemplary human wild-type IL-2 amino acid sequence is shown in SEQ ID NO. 1. In some embodiments, unprocessed human wild-type IL-2 additionally comprises a N-terminal 20 amino acid signal peptide, an exemplary amino acid sequence of which is shown in SEQ ID NO. 4.
In some embodiments, wild-type IL-2 may further comprise one or more amino acid mutations that do not alter binding to the IL-2 receptor, e.g., the introduction of a serine mutation (C125S) at amino acid residue 125 of wild-type human IL-2 can prevent cysteine-induced mismatches or aggregates, an exemplary amino acid sequence of which is shown in SEQ ID NO. 2; the introduction of an alanine mutation at position 125 (C125A) can promote efficient folding and improve expression, and an exemplary amino acid sequence is shown in SEQ ID NO. 3.
"mutation" of amino acids as described herein includes substitutions, deletions and insertions. Any combination of substitutions, deletions, and insertions may be made to obtain a construct with the desired properties (e.g., reduced binding affinity to IL-2rβγ). In some embodiments, the deletion or insertion of an amino acid comprises a deletion or insertion in a polypeptide sequence. In some embodiments, the amino acid substitutions may be conservative amino acid substitutions. In other embodiments, the amino acid substitutions may be non-conservative amino acid substitutions, i.e., one amino acid is replaced with another amino acid having a different structure and/or chemical property. Amino acid substitutions also include substitution with non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). In some embodiments, the mutation of an amino acid includes any combination of substitutions, deletions, and insertions of amino acids, e.g., within a region of the polypeptide sequence, including both substitutions and deletions of amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.
An "IL-2 mutant" as described herein, includes a wild-type IL-2 amino acid sequence (e.g., SEQ ID No. 1) comprising one or more substitutions, deletions, insertions, or any form combination thereof. In some embodiments, the "IL-2 mutants" described herein are capable of reducing their binding affinity to IL-2Rβγ and maintaining binding to IL-2Rα.
In IL-2 mutants, the amino acid mutation position is based on the wild type IL-2 amino acid position determination. In some embodiments, the amino acid sequence of wild-type IL-2 is shown in SEQ ID NO.1, and the position corresponding to the mutation of the amino acid in the IL-2 mutant can be identified by amino acid sequence alignment (e.g., using BLAST) with SEQ ID NO. 1. In some embodiments, when describing, for example, S75, the amino acid residue representing position 75 relative to the wild-type IL-2 amino acid sequence is serine (S). "mutation of S75" means that the serine residue (S) at position 75 is mutated. In some embodiments, when describing IL-2 mutants, the following description is made. For example, "amino acid substitution" is denoted as "original amino acid residue/amino acid position where substitution occurs/amino acid residue after substitution", e.g., "S75P" represents substitution of serine residue (S) at position 75 relative to the wild-type IL-2 amino acid sequence (e.g., SEQ ID No. 1) with proline residue (P). As used herein with respect to the combination scheme of mutation sites "+" means that mutation occurs simultaneously at a plurality of specific positions, e.g., "E67I+S75P" means that glutamic acid residue (E) at position 67 is replaced with isoleucine residue (I) and serine residue (S) at position 75 is replaced with proline residue (P) relative to the wild-type IL-2 amino acid sequence (e.g., SEQ ID NO. 1).
As used herein, the expression "selectively activating Treg cells" with IL-2 mutants refers to the presence of other T cell subsets (e.g., CD4 + T cells, CD8 + T cells) or NK cells, which are able to target Treg cells more specifically, activate Treg cells. The ability of IL-2 mutants to "activate Treg cells" can be determined by methods well known in the art or by methods disclosed in the examples of the present application, including but not limited to: IL-2 mutants induce IL-2 receptor signaling in Treg cells or induce proliferation of Treg cells. In some embodiments, IL-2 receptor signaling may be defined by STAT5 phosphorylation levels. STAT5 phosphorylation is an essential step in the IL-2 signaling pathway, and therefore the level of STAT5 phosphorylation in Treg cells (pSTAT 5) is considered to be a reflection of IL-2 activated Treg cells. Induction of proliferationThe proliferation can be defined by a change in the number of Treg cells following IL-2 stimulation, for example by measuring the increase in the number of Treg cells in a mixed population of cells by flow cytometry or by measuring the increase in expression of proliferation-related cyclin (e.g. Ki-67) in Treg cells.
As used herein, an "autoimmune disease" refers to a non-malignant disease or condition that originates from and is directed against an individual's own tissues, where the immune system attacks its own proteins, cells and tissues. For a list and review of autoimmune diseases, reference may be made to The Autoimmune Diseases (Noel Rose, ian Mackay,2014,Academic Press). In some embodiments, examples of autoimmune diseases include, but are not limited to, lupus, graft versus host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophy epidermolysis bullosa, and Behcet's disease.
As used herein, a "treatment" or "treatment" is a method for achieving a beneficial or desired result, including clinical results. For the purposes of this application, such beneficial or desired results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by a disease, reduction of the extent of a disease, stabilization of a disease (e.g., preventing or delaying progression of a disease), prevention or delay of disease transmission, prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of the disease state, alleviation of a disease (in part or in whole), reduction of the dose of one or more other drugs required to treat a disease, delay of disease progression, improvement of quality of life, and/or prolongation of survival. Also, "treating" includes reducing the pathological consequences of the disease. The methods of the present application contemplate any one or more aspects of these treatments. For example, a patient is considered to be "treated" if one or more symptoms associated with the disease are alleviated or eliminated, including, but not limited to, reducing symptoms caused by the disease, improving the quality of life of the patient with the disease, reducing the dosage of other medications required to treat the disease, and/or extending the survival of the individual.
The term "prevention" and similar terms, such as "prevention", "prevention" and the like, mean a method of preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a disease or disorder. It also refers to delaying the onset or recurrence of a disease or disorder. As used herein, the terms "prevent," "preventing" and the like also include reducing the intensity, impact, symptoms and/or burden of a disease or disorder prior to its occurrence or recurrence, or reduce the likelihood of occurrence or recurrence of the disease or condition.
As used herein, "delay" of progression of a disease means delay, impediment, slowing, stabilizing, and/or delaying the progression of the disease. The delay time may vary depending on the history of the disease and/or the individual being treated. A method of "slowing" the progression of a disease refers to a method of reducing the probability of disease progression over a given time frame and/or reducing the extent of the disease over a given time frame, as compared to the absence of the method. In some embodiments, such comparisons may be based on animal trials, observations and/or statistics in individual animals. In other embodiments, such comparison may be based on clinical studies using statistically significant numbers of individuals.
As used herein, the term "effective amount" refers to a dosage of a drug or dosage of a pharmaceutical composition sufficient to treat a particular disorder, condition, or disease, such as to ameliorate, reduce, attenuate, and/or delay one or more symptoms. In some embodiments, the effective amount is an amount sufficient to delay the progression of the disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay the onset or recurrence of the disease. The effective amount may be administered in one or more administrations. In some embodiments, an effective amount of a drug or pharmaceutical composition refers to an amount capable of increasing the number of Treg cells in diseased tissue of a patient.
As used herein, "individual" or "subject" refers to a mammal, including but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, the individual is a human.
The term "constant region" refers to a portion of an immunoglobulin molecule (Ig) that includes a "variable region" of an antigen binding site relative to another portion of the immunoglobulin molecule. The constant region has a more conserved amino acid sequence, comprising a heavy chain constant region (C H ) Comprising C H 1、C H 2 and C H 3 domain and light chain constant region (C L ). According to the immunoglobulin heavy chain constant region (C H ) Immunoglobulins can be assigned to different classes or subtypes. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM, the heavy chains are α, δ, ε, γ and μ, respectively. According to C H The relatively small differences in sequence and function further divide γ and α into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA1 and IgA2.
As used herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulin defined by the chemical and antigenic properties of the constant region. Immunoglobulins are largely divided into five major classes: igA, igD, igE, igG and IgM, and a plurality thereof may be further divided into subclasses (subtypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. Heavy chains corresponding to different immunoglobulin classes are named using the greek letters α, δ, epsilon, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and described in detail in Abbas et al, fourth edition of cell and molecular immunology (w.b. samundrs, co., 2000).
As used herein, the terms "Fc," "Fc domain," "Fc fragment," "Fc region," or "crystallizable fragment" are used to define the C-terminal region of an immunoglobulin heavy chain, including native Fc and variant Fc. Exemplary Fc includes immunoglobulin C H 2 and C H 3 domain. Although the boundaries of the immunoglobulin heavy chain Fc may vary, a human IgG heavy chain Fc is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine of Fc (residue 447 according to the EU numbering system) may be removed, for example, during the production or purification of the protein, or by recombinant engineering of the nucleic acid encoding the protein.Suitable native sequence Fc regions for the constructs described herein are derived from, including, but not limited to, human IgG1, igG2 (IgG 2A, igG 2B), igG3, and IgG4.
"Fc receptor" or "FcR" describes a receptor that binds to an Fc region in an Fc-containing molecule (e.g., an antibody, an Fc-containing fusion protein). The preferred FcR is a human FcR native sequence. Furthermore, an exemplary FcR is one that binds an IgG antibody (a gamma receptor), including fcγri, fcγrii, and fcγriii receptor subclasses, as well as allelic variants and alternatively spliced forms of these receptors. Fcyrii receptors include fcyriia ("activating receptor") and fcyriib ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in the cytoplasmic domain. The activating receptor fcyriia comprises an immune receptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor fcyriib comprises an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in its cytoplasmic domain (see M. Annu.Rev.Immunol.15:203-234 (1997)). At Ravetch and Kinet, annu.Rev.Immunol 9:457-92 (1991); fcRs are reviewed in Capel et al, immunomethods 4:25-34 (1994) and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those that will be identified in the future.
The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transport of maternal IgG to the fetus. Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994). Methods for determining binding to FcRn are known in the art (see, e.g., ghetie and Ward, immunol. Today 18 (12): 592-8 (1997); ghetie et al, nature Biotechnology (7): 637-40 (1997); hinton et al, J. Biol. Chem.279 (8): 6213-6 (2004); WO 2004/92219 (Hinton et al)). The half-life of human FcRn high affinity binding polypeptides in vivo and in serum to FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides having variant Fc regions. WO 2004/42072 (Presta) details antibody variants that increase or decrease binding to FcRs. For FcRn, see Shield et al, J.biol.chem.9 (2): 6591-6604 (2001).
"Fc effector function" refers to those biological activities caused by an Fc region (native sequence Fc region or an Fc region comprising an amino acid sequence mutation) in an Fc-containing molecule (e.g., an antibody) that vary depending on the immunoglobulin subtype from which the Fc is derived. Examples of effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors) and B cell activation. By "reducing or minimizing" effector function is meant at least a 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) reduction in effector function as compared to a wild-type Fc or unmodified Fc-containing molecule (e.g., antibody). Determination of effector functions can be readily determined and measured by one of ordinary skill in the art. In a preferred embodiment, the effector functions of complement fixation, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity will all be affected. In some embodiments, the effector function is eliminated by eliminating glycosylation through mutations in the constant region, e.g., "null effector function mutations". In some embodiments, the null response function mutant is at C H The N297A or DANA mutation of region 2 (D265 A+N297A) can be found in Shields et al, J.biol.chem.276 (9): 6591-6604 (2001). In addition, other mutations that lead to reduced or eliminated effector function include: K322A and L234A/L235A (LALA). In addition, effector functions may be reduced or eliminated by changes in production technology, such as expression in host cells that do not undergo glycosylation (e.g., E.coli) or host cells that result in a change in glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., shinkaw et al, J.biol. Chem.278 (5): 3466-3473 (2003)).
"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig (or ligand-Fc structure) binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer cells (NK), neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind antigen-bearing (or ligand-receptor-bearing) target cells, which are then killed by cytotoxins. Antibodies (or other Fc-containing molecules) are necessary to "arm" cytotoxic cells to kill target cells by this mechanism. The primary cells that mediate ADCC include NK cells and monocytes, where NK cells express fcyriii only and monocytes express fcyri, fcyrii and fcyriii. The expression of Fc in hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a target molecule, an in vitro ADCC assay may be performed, as described in detail in U.S. Pat. No.5,500,362 or 5,821,337. Effector cells suitable for such assays include Peripheral Blood Mononuclear Cells (PBMCs) and natural killer cells (NK). Alternatively, or in addition, ADCC activity of the target molecule can also be assessed in vivo, for example in an animal model as disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).
"complement dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1 q) to an Fc-containing molecule (of the appropriate subclass) that binds to its cognate receptor via an Fc-fused ligand. To assess complement activation, CDC assays may be performed as described in Gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996). Antibody variants with altered amino acid sequences in the Fc region to increase or decrease the C1q binding capacity are described in detail in U.S. Pat.No.6,194,551B1 and WO 99/51642. The contents of these patent publications are incorporated herein by reference. See Idusogie et al J.Immunol.164:4178-4184 (2000).
As used herein, a "regulatory T cell" or "Treg" is a class of CD4 that has significant immunosuppressive effects + T cell subsets are capable of avoiding suppression of immune responses by other cells. Treg is characterized by expressing the alpha subunit of the IL-2 receptor (CD 25) and the transcription factor fork P3 (FOXP 3) and plays an important role in maintaining the immune balance of the body and preventing autoimmune diseases, graft rejectionIs used. Treg requires IL-2 to achieve its function and development and induction of its inhibitory features.
As used herein, "effector cells" refers to a population of lymphocytes that mediate a cytotoxic effect induced by IL-2. Effector cells include mainly effector T cells such as CD8 + Cytotoxic T cells, NK cells, lymphokine Activated Killer (LAK) cells, macrophages, and the like.
As used herein, the terms "specific binding," "specific recognition," or "specific for" refer to a measurable and reproducible interaction, such as binding between a ligand and a receptor, the presence of which can be determined in the presence of heterogeneous populations of molecules, including biomolecules. For example, a ligand that specifically binds to a receptor has greater affinity, avidity, ease and/or duration in binding to the receptor of interest when compared to binding to other receptors. In some embodiments, the ligand binds to less than 10% of the receptor of interest as determined by, for example, radioimmunoassay (RIA) methods. In some embodiments, the ligand that specifically binds to the target receptor has an equilibrium dissociation constant (Kd) of 10 or less - 5 M、≤10 -6 M、≤10 -7 M、≤10 -8 M、≤10 -9 M、≤10 -10 M、≤10 -11 M or less than or equal to 10 -12 M. In some embodiments, the ligand may specifically bind to a receptor that is conserved among different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding. The binding specificity of a ligand can be determined experimentally using methods known in the art. Such as but not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM Test and peptide scan.
"affinity" or "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., ligand) and its binding partner (e.g., receptor). Unless otherwise indicated, "binding affinity" as described herein refers to an intrinsic binding affinity that can reflect a 1:1 interaction between binding pair members. Binding affinity can be achievedRepresented by Kd, koff, kon or Ka. As used herein, the term "Koff" refers to the rate constant of dissociation of a ligand from a ligand/receptor complex, as determined by a kinetic selection device, in s -1 Expressed in units. As used herein, the term "Kon" refers to the binding rate constant, M, of a ligand to receptor binding to form a ligand/receptor complex -1 s -1 Expressed in units. As used herein, the term equilibrium dissociation constant "Kd" refers to the dissociation constant at a particular ligand-receptor interaction, meaning that in a receptor solution, the ligand occupies half of all receptor binding sites and the concentration of ligand required to reach equilibrium is equal to Koff/Kon. The dissociation constant (Kd) can be used as an indicator reflecting the affinity of a ligand for a receptor. The Kd value obtained using the method is expressed in units of M (mol/L). The premise behind the determination of Kd is that all binding molecules are in solution. Where the receptor is on a cell membrane, the corresponding dissociation rate constant is expressed as an EC50 value. The EC50 value is a good approximation of Kd. Affinity constant Ka, the reciprocal of dissociation constant Kd, is M -1 Expressed in units. The dissociation constant (Kd) can be used as an indicator reflecting the affinity of a ligand for a receptor.
Semi-inhibitory concentration (IC 50) is a measure of the effectiveness of a substance (e.g., ligand) in inhibiting a particular biological or biochemical function. It indicates how much particular drug or other substance (inhibitor, e.g., ligand) is required to inhibit a given biological process by half. IC50 values are generally expressed as molar concentrations. The IC50 is comparable to the "EC50" of an agonist drug or other substance (e.g., ligand). EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. As used herein, "IC50" is used to denote the effective concentration of ligand required to neutralize 50% of the receptor biological activity in vitro. The IC50 or EC50 can be determined by biological measurement, such as by FACS analysis (competitive binding assay) to inhibit ligand binding, cell-based cytokine release assay, or homogeneous enzyme-linked immunosorbent assay (AlphaLISA) to amplify luminescence.
"fusion" as used herein refers to the attachment of individual components by peptide bonds, either directly or through one or more linking peptides (otherwise referred to as "linkers"). The sequence of a "linker" may be a single amino acid or polypeptide sequence. In some embodiments, the connecting peptide (or linker) comprises or consists of a glycine-serine linker. As used herein, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly 4 Ser) n, where n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). One preferred glycine-serine linker is GGGGS.
As used herein, "covalent bond" refers to a stable bond between two atoms formed by sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, "peptide bond" refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. As used herein, "disulfide" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments bound by one or more disulfide bonds. One or more disulfide bonds between the two fragments may be formed by linking thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of the two Fc fragments. Oxidation of two thiol groups can form a disulfide bond. In some embodiments, the covalent linkage is formed by direct covalent linkage. In some embodiments, the covalent linkage is directly linked by a peptide bond or disulfide bond.
As used herein, the term "derivative" refers to a molecule having a certain protein amino acid sequence or analog thereof, but having additional modifications at one or more of the amino acid groups, alpha carbon atoms, amino terminus, or carboxyl terminus. Modifications, as described herein, include, but are not limited to, chemical modifications, amino acid side group modifications, amino terminal modifications, carboxy terminal modifications. Wherein chemical modifications include, but are not limited to, adding chemical groups, creating new chemical bonds, and removing chemical groups. Modifications of the amino acid side groups include, but are not limited to, epsilon aminoacylation of lysine, N-alkylation of arginine, histidine or lysine, carboxyalkylation of glutamic acid or aspartic acid, deamination of glutamine or asparagine. Modifications at the amino terminus include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Carboxyl terminal modifications include, but are not limited to, amide, lower alkyl acyl, dialkyl amide and lower alkyl ester modifications. In some embodiments, the lower alkyl group is a C1-C4 alkyl group. Furthermore, one or more pendant or terminal groups may be protected by those skilled in the chemical arts using known protecting groups. The alpha carbon of an amino acid may be mono-or di-methylated.
"percent (%) amino acid sequence identity" or "homology" of a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to a particular polypeptide or polypeptide sequence, after sequence alignment and introduction of gaps (if necessary) to maximize the percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. To determine the percent amino acid sequence identity, various alignment schemes within the skill of the art can be used, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine suitable parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared.
As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide, the amine group of which forms a peptide bond with the carboxyl group of its adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid residue of the polypeptide, the carboxyl group of which forms a peptide bond with the amine group of its adjacent amino acid residue.
An "isolated" polypeptide refers to a polypeptide that has been identified, isolated, and/or recovered from components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is not associated with all other components in its production environment. Contaminating components of the production environment, such as those produced by recombinant transfected cells, often interfere with the study, diagnosis or treatment of the polypeptide and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In some embodiments, the polypeptide will be purified to: (1) Polypeptide content is greater than 95% by weight, as determined by Lowry method, in some embodiments, polypeptide content is greater than 99% by weight; (2) To an extent sufficient to obtain at least 15N-terminal residues or internal amino acid sequences by using a rotary cup sequencer; or (3) homogeneity by SDS-PAGE under non-reducing or reducing conditions using coomassie blue or preferably silver staining. The isolated polypeptide includes the polypeptide in situ within the recombinant cell because at least one element of the polypeptide's natural environment is absent. However, typically, an isolated polypeptide is subjected to at least one purification step.
"amino acid" is used herein in its broadest definition, including naturally occurring amino acids, as well as non-naturally occurring amino acids, including analogs and derivatives of amino acids. The latter includes molecules containing amino acid moieties. According to this broad definition, one skilled in the art will recognize that the amino acids described herein include, for example, natural L-amino acids that form proteins; d-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; natural amino acids that do not form proteins, such as norleucine, beta-alanine, ornithine, GABA, and the like; and chemically synthesized compounds having amino acid characteristics known in the art. As used herein, the term "protein-forming" refers to the amino acids of peptides, polypeptides or proteins that can be synthesized by metabolic pathways into cells.
An "isolated" nucleic acid molecule encoding a protein or polypeptide (e.g., an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein) is one that has been identified and isolated from a nucleic acid molecule that contains at least one impurity, which is typically associated with the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with all components in its production environment. An isolated nucleic acid molecule encoding a polypeptide as described herein is in a form or morphology that is not one in which it is found in nature. Thus, an isolated nucleic acid molecule differs from the nucleic acid encoding a polypeptide described herein that naturally occurs in a cell. An isolated nucleic acid comprises a nucleic acid molecule contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present at a chromosomal location that is extrachromosomal or different from its natural chromosomal location.
The term "control sequences" refers to DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes include promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
A nucleic acid is "operably linked" to another nucleic acid sequence when the nucleic acid is in a functional relationship with another nucleic acid sequence. For example, DNA encoding a pre-sequence or a secretory leader sequence is involved in the secretory process of a polypeptide expressed as a precursor protein, and is operably linked to DNA encoding the polypeptide molecule; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or operably linked to a coding sequence if the ribosome binding site is at a position that facilitates translation. In general, "operably linked" means that the DNA sequence being linked is continuous and, for the secretory leader, not only continuous but also in the reading phase. However, the enhancers do not have to be contiguous. Ligation is accomplished by ligation at appropriate restriction sites. If such sites are not present, synthetic oligonucleotide aptamers or linkers are used as usual.
As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid molecule to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that are introduced into the genome of a known host cell. Certain vectors are capable of directing expression of nucleic acids linked thereto. Such vectors are referred to herein as "expression vectors".
As used herein, the terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected", "transformed" or "transduced" cell is a cell transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary test cells and their progeny.
The terms "host cell", "host cell line", and "host cell culture" are used interchangeably to refer to cells of exogenous nucleic acid, including progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and progeny produced thereby, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid to the parent cell, e.g., may contain a mutation. Included herein are screening or selecting mutant progeny in the original transformed cell that have the same function or biological activity as it had.
The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation that is in a form that is effective for the biological activity of the active ingredient and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation is administered. Such a formulation is sterile. "sterile" formulations are sterile or free of any viable microorganisms and spores thereof.
The embodiments described herein should be understood to include embodiments consisting of … and/or consisting essentially of ….
Reference in the present application to "about" is a numerical value or parameter, including (and describing) variations on the numerical value or parameter itself. For example, a description relating to "about X" includes a description of "X".
As used herein, reference to "not" a value or parameter generally means and describes "in addition to" a value or parameter. For example, the method cannot be used to treat type X cancers, meaning that the method is generally used to treat other types of diseases in addition to type X cancers.
As used herein, the term "about X-Y" is synonymous with "about X to about Y".
As used herein and in the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
IL-2 mutants
IL-2 mutants of the present application select for IL-2 receptors andpreference for lymphocyte activation/proliferation and biology
Properties:
in some embodiments, the IL-2 mutants provided herein have reduced affinity for IL-2Rβ and/or IL-2Rβγ as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1). In some embodiments, the IL-2 mutant has at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) reduced affinity for IL-2Rbeta and/or IL-2Rbeta gamma as compared to human wild-type IL-2.
In some embodiments, the IL-2 mutants provided herein have increased affinity for IL-2Rα as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1). In some embodiments, the IL-2 mutant has at least a 1-fold (e.g., at least a 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold) increase in affinity for IL-2Rα as compared to human wild-type IL-2.
In some embodiments, the IL-2 mutants provided herein have a constant or NO significant decrease in affinity for IL-2Rα or an increase in affinity for IL-2Rα as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1).
In some embodiments, the IL-2 mutants provided herein have reduced affinity for IL-2Rβ and/or IL-2Rβγ while maintaining the affinity for IL-2Rα unchanged or without significant attenuation or increased affinity for IL-2Rα as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1).
Affinity between IL-2 mutants and receptors can be determined by conventional methods known in the art, e.g. binding ELISA experiments, HEK-Blue TM IL-2 cell line detection experiments or Surface Plasmon Resonance (SPR) and the like.
In some embodiments, the IL-2 mutants provided herein have increased safety in the treatment of certain specific diseases as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1).
For example, in HEK-Blue TM In the IL-2 cell line (InvivoGen, cat#hkb-IL 2) experiments, the cell line was isolated by HEK-Blue compared to human wild-type IL-2 TM Detection of IL-2 mutants and IL-2Rβγ by the CD122/CD132 cell line (below)Called di-receptor) bound EC50 value by HEK-Blue TM IL-2 cell lines detect a higher ratio between the EC50 values of IL-2 mutants and IL-2Rαβγ (hereinafter referred to as tri-receptor) binding, i.e., have a higher therapeutic safety window, and therefore have higher safety. In some embodiments, by HEK-Blue TM CD122/CD132 cell line assay to determine the EC50 value of IL-2 or IL-2 mutant binding to IL-2Rβγ by HEK-Blue TM IL-2 cell line detection experiments to determine the IL-2 or IL-2 mutant and IL-2Rαβγ binding of EC50 value. In some embodiments, IL-2 mutant EC50 Two receptors /EC50 Tri-receptors The ratio is the EC50 of human wild-type IL-2 Two receptors /EC50 Tri-receptors At least 2 times the ratio, for example: 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 700-fold, 1000-fold, 2000-fold, 10000-fold, 20000-fold, 30000-fold or more.
In some embodiments, the IL-2 mutants provided herein are capable of selectively activating Treg cells, reducing activation of other T cells, e.g., reducing activation of CD8, as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1) + Activation of T cells; reduces activation of NK cells.
In some embodiments, the IL-2 mutants provided herein are capable of selectively activating Treg cells as compared to human wild-type IL-2 (e.g., SEQ ID NO: 1). For example, in STAT5 phosphorylation experiments, the ability of IL-2 mutants to activate Treg cells is reflected by detecting activation of STAT5 phosphorylation signals by IL-2 or IL-2 mutants in Treg cells. Detection by flow cytometry, graphPad Prism software analyzed and calculated EC50 values for IL-2 or IL-2 mutant to activate STAT5 phosphorylation in Treg cells, which EC50 values indirectly reflect the activation ability of IL-2 or IL-2 mutant to Treg cells, indicating that IL-2 mutant is able to activate Treg cells effectively.
In some embodiments, the IL-2 mutants provided herein are capable of reducing the level of CD8 compared to human wild-type IL-2 (e.g., SEQ ID NO: 1) + Activation of T cells. For example, in STAT5 phosphorylation experiments, the IL-2 or IL-2 mutant is detected in CD8 + Activation of STAT5 phosphorylation signals in T lymphocytes to reflect IL-2 mutant pair CD8 + Activation ability of T lymphocytes. Detection by flow cytometry, graphPad Prism software analysis and calculation of IL-2 mutants at CD8 + EC50 value for activating STAT5 phosphorylation in T lymphocytes, which EC50 value indirectly reflects IL-2 or IL-2 mutant versus CD8 + Activation ability of T lymphocytes. In some embodiments, IL-2 mutants are directed against CD8 as compared to human wild-type IL-2 + The activation ability of T lymphocytes is reduced by at least 10%. For example: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
In some embodiments, the IL-2 mutants provided herein are capable of effectively expanding Treg cells in vivo or in vitro. In some embodiments, the IL-2 mutant is capable of increasing CD4 in a subject + The ratio of Treg cells in a T cell population (e.g., CD4 + CD4 in T cell populations + CD25 + Foxp3 + The cell duty cycle increases).
In some embodiments, the IL-2 mutant is capable of increasing the ratio of Treg cells to non-Treg cells (e.g., CD4 + CD25 + Foxp3 + Cell and CD8 + The ratio of cells increases). For example, in the examples herein, the number of Treg cells in the peripheral blood of a cynomolgus monkey in a test animal increases over a period of time after stimulation by an IL-2 mutant, but for CD8 + The cell number of T lymphocytes is not significantly affected. In some embodiments, treg cells in the subject are stimulated by the IL-2 mutant to be in CD4 + The ratio in T lymphocytes is increased by at least 1 fold. For example: 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, 10-fold, 15-fold, 20-fold or more.
IL-2 mutants of the present application:
in some embodiments, the IL-2 mutants described herein have at least one amino acid mutation, such as a substitution, deletion, insertion of an amino acid, or a combination comprising any of the foregoing mutant forms, as compared to wild-type IL-2 (e.g., human wild-type IL-2).
In some embodiments, the IL-2 mutants described herein comprise a mutation in R83 relative to the amino acid sequence of human wild-type IL-2 (e.g., SEQ ID NO: 1). In some embodiments, the mutation of R83 is R83L, R a or R83N.
In some embodiments, the IL-2 mutants described herein further comprise a mutation in D20 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of D20 is D20N. In some embodiments, the IL-2 mutants described herein, which comprise R83L and D20N mutations relative to the amino acid sequence of human wild-type IL-2.
In some embodiments, the IL-2 mutants described herein further comprise mutations in H16 and N71 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of H16 and N71 are H16N and N71R, respectively. In some embodiments, the IL-2 mutants described herein comprise R83L, H N and N71R mutations relative to the amino acid sequence of human wild-type IL-2.
In some embodiments, the IL-2 mutants described herein further comprise mutations in N71 and N88 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of N71 and N88 are N71S and N88D, respectively. In some embodiments, the IL-2 mutants described herein comprise R83A, N S and N88D mutations relative to the amino acid sequence of human wild-type IL-2.
In some embodiments, the IL-2 mutants described herein further comprise mutations in L66 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of L66 and V91 are L66F and V91E, respectively. In some embodiments, the IL-2 mutants described herein comprise R83N, L F and V91E mutations relative to the amino acid sequence of human wild-type IL-2.
In other embodiments, the IL-2 mutants described herein comprise mutations in E67 and S75 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). In some embodiments, the mutation of E67 is E67I or E67G, and the mutation of S75 is S75P. In some embodiments, the IL-2 mutants described herein, which comprise E67I and S75P mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein, which comprise E67G and S75P mutations relative to the human wild-type IL-2 amino acid sequence.
In other embodiments, the IL-2 mutants described herein comprise mutations in L17 and P82 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). In some embodiments, the mutations of L17 and P82 are L17V and P82L, respectively.
Mutation at other sites:
in some embodiments, the IL-2 mutants of the present application may further have one or more mutations at other positions or regions relative to the human wild-type IL-2 amino acid sequence, provided that they meet the reduced selectivity of the IL-2 mutants for the receptor IL-2Rβγ, and/or one or more beneficial properties, described herein. In some embodiments, the IL-2 mutants described herein also include mutations at other positions relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1) that do not alter affinity for the IL-2 receptor. In some embodiments, the IL-2 mutants described herein have an amino acid mutation at position 125 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1), such as C125S, C125A, C T or C125V (see, in particular, U.S. Pat. No.4,518,584). The skilled person knows how to add other additional mutations than those described in the present application.
In some embodiments, the IL-2 mutants described herein further comprise a C125S mutation relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise a C125A mutation relative to the human wild-type IL-2 amino acid sequence.
In some embodiments, IL-2 mutants described herein comprise an amino acid sequence set forth in any of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with an amino acid sequence set forth in any of SEQ ID NOs 5-11.
In some embodiments, one or more mutations at the other positions or regions comprise one or more conservative amino acid substitutions. "conservative substitution" refers to substitution with another amino acid that has the same net charge and about the same size and shape as the substituted amino acid. When the total number of carbon atoms and heteroatoms in the side chains differs by no more than 4, the amino acids with side chains of aliphatic or substituted aliphatic amino acids are about the same size. When the number of branches in the side chains differs by not more than 1, the amino acids are approximately the same shape. Amino acids having a phenyl group or a substituted phenyl group in a side chain are considered to be approximately the same in size and shape. Unless otherwise indicated, conservative substitutions preferably use the natural amino acid. Exemplary conservative substitutions are shown in table 1.
More substantial substitutions are provided under the heading "substitution example" in table 1, as further detailed below with respect to the amino acid side chain class section. Amino acids can be categorized according to the usual side chain properties: (1) hydrophobicity: norleucine (Norleucine), met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues affecting the chain direction: gly, pro; (6) aromatic: trp, tyr, phe. Non-conservative substitutions require replacement of one member of these classes with a member of another class. Amino acid substitutions can be introduced into the protein construct and the product screened for compliance with the desired activities described above.
TABLE 1 amino acid substitutions
In some embodiments, the insertion of unnatural amino acids, including synthetic unnatural amino acids or one or more D-amino acids, into IL-2 mutants or IL-2 mutant fusion proteins of the application can have a variety of benefits. The D-amino acid-containing polypeptide and the like exhibit higher stability in vitro and in vivo than the L-amino acid-containing polypeptide. Thus, the construction of polypeptides, such as by addition of D-amino acids, is particularly useful when better intracellular stability is desired. In particular, D-peptides and analogs thereof are resistant to endogenous peptidases and proteases, thereby increasing the bioavailability of the molecule and extending its life in vivo when desired. In addition, D-peptides and analogs thereof cannot be efficiently processed for limited presentation to helper T cells via the Major Histocompatibility Complex (MHC) class II and therefore are not prone to induce humoral immune responses in a subject.
In some embodiments, the IL-2 mutants described herein are modified IL-2 mutants, such as pegylated IL-2 mutants, or covalently modified IL-2 mutants, or glycosylation modified IL-2 mutants.
In some embodiments, the IL-2 mutants described herein are full length sequences. In other embodiments, the N-terminus of the IL-2 mutant comprises a signal peptide, either from a different molecule, or from wild-type IL-2.
In some embodiments, IL-2 mutants described herein comprise an amino acid sequence set forth in any of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with an amino acid sequence set forth in any of SEQ ID NOs 5-11.
The amino acid sequences of exemplary wild-type IL-2 and its C125 mutants are shown in Table 2, and the amino acid sequences of exemplary IL-2 mutants are shown in Table 3.
Table 2 exemplary wild-type IL-2 and C125 mutants
TABLE 3 exemplary IL-2 mutants
TABLE 4 exemplary Fc mutant sequences
/>
Table 5 exemplary linker peptide sequences
/>
TABLE 6 bivalent fusion protein sequences of exemplary IL-2 mutants and Fc
/>
/>
Fusion proteins comprising IL-2 mutants
The present application also provides fusion proteins comprising IL-2 mutants. IL-2 mutants as described herein may be fused to other proteins to increase circulatory half-life by increasing molecular size and/or decreasing renal clearance. In some preferred embodiments, the IL-2 mutants described herein are fused to Fc. In other preferred embodiments, the IL-2 mutants described herein are fused to Human Serum Albumin (HSA). In other preferred embodiments, the IL-2 mutants described herein are fused to some short peptide (e.g., XTEN).
Fusion protein of IL-2 mutant with Fc:
fc fusion proteins can be produced by recombinant DNA techniques in which the translational reading frame of the Fc domain of a mammalian immunoglobulin (e.g., igG) is fused to another protein to produce a new single recombinant polypeptide. Fc fusion proteins can generally be produced as disulfide-linked dimers, linked together by disulfide bonds located in the hinge region in the Fc domain.
IL-2 mutants may be fused to the N-and/or C-terminus of Fc. Preferably, the IL-2 mutant is fused to the C-terminus of Fc. In some embodiments, the IL-2 mutant and Fc are directly fused by peptide bonds. In some embodiments, the IL-2 mutant and Fc between containing connecting peptide.
In some embodiments, the Fc described herein is from any of IgA, igD, igE, igG and IgM and subclasses thereof. Of all immunoglobulins, igG is the highest in serum content and the half-life is the longest. Unlike other immunoglobulins, igG is efficiently recovered after binding to Fc receptors (FcRs). In some preferred embodiments, the Fc is from an IgG (e.g., igG1, igG2, igG3, or IgG 4). In some embodiments, the Fc is from a human IgG. In some embodiments, the Fc comprises C H 2 and C H 3 domain. In some embodiments, the Fc further comprises all or part of a hinge region. In some embodiments, the Fc is from human IgG1 or human IgG4. In some embodiments, two subunits of an Fc dimerize via one or more (e.g., 1, 2, 3, 4, or more) disulfide bonds. In some embodiments, each subunit of Fc comprises a full-length Fc sequence. In some embodiments, each subunit of Fc comprises an N-terminal truncated Fc sequence, e.g., truncated Fc contains fewer N-terminal cysteines, to reduce mismatch of disulfide bonds during dimerization. In some embodiments, the Fc is truncated at the N-terminus, e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, the complete immunoglobulin Fc is deleted, 9. 10 or even more amino acids. In some embodiments, the Fc comprises one or more mutations, such as insertions, deletions, and/or substitutions.
In some embodiments, the Fc described herein has reduced or eliminated Fc effector function. In some embodiments, the Fc has reduced Fc-mediated effector functions, such as reduced ADCC, CDC, and/or ADCP effector functions.
Fc-containing fusion proteins can activate complement and interact with Fc receptors (FcRs). In some cases, this inherent immunoglobulin property has been considered disadvantageous because such fusion proteins may target cells expressing Fc receptors, rather than the preferred cells expressing IL-2 receptors, which may cause undesired cytotoxicity. And further considering that Fc fusion proteins have long half-lives, they are difficult to apply in therapy or have limited therapeutic applications due to systemic toxicity. Thus, in some embodiments, the Fc is engineered (e.g., comprises one or more amino acid mutations) to alter its binding to FcR, in particular to alter binding to fcγ receptor (responsible for ADCC) and/or to alter effector functions, such as altering antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce binding to FcRn receptor (related to half-life).
In some embodiments, fc (e.g., that of human IgG 1) is mutated to remove one or more effector functions, such as ADCC, ADCP, and/or CDC, hereinafter referred to as "no effect" or "almost no effect" Fc. For example, in some embodiments, the Fc is a null human IgG1 Fc comprising mutations L234A and L235A. In some embodiments, the human IgG1 Fc comprises one or more of the following mutations (e.g., in each Fc subunit): L234A, L235E, G237A, A S and P331S. As known to those skilled in the art, the combination of K322A, L234A and L235A in IgG1 Fc is sufficient to completely eliminate fcγr and C1q binding (Hezareh et al, J Virol 75,12161-12168,2001). MedImmune found that Fc' S containing a panel of three mutations L234F/L235E/P331S had very similar effects (Oganesman et al, acta crystal graphic 64,700-704,2008). In some embodiments, the Fc comprises a glycosylation modification on the IgG1 Fc region N297, which is known to be necessary for producing optimal FcR interaction. The modification of Fc may be any suitable engineered IgG Fc mentioned by Wang et al ("IgG Fc engineering to modulate antibody effector functions," Protein cell.2018Jan;9 (1): 63-73), the contents of which are incorporated herein by reference in their entirety.
In some embodiments, the fusion protein of the IL-2 mutant and Fc does not have ADCC and/or CDC, or does not have detectable ADCC and/or CDC, as described herein. In some embodiments, as described herein, the fusion protein of an IL-2 mutant with Fc results in at least a 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) decrease in ADCC and/or CDC as compared to a fusion protein comprising the same IL-2 mutant portion but fused to a wild-type or unmodified Fc.
Glycosylation variants
In some embodiments, the degree of glycosylation of the construct is increased or decreased by altering the Fc or the fusion protein of the IL-2 mutant to Fc. Glycosylation sites can be added or deleted in the Fc by altering the amino acid sequence to create or remove one or more glycosylation sites.
Natural Fc-containing proteins produced by mammalian cells typically comprise a branched chain double-antennary oligosaccharide, which is typically linked to Fc C via an N-bond H 2 at Asn297 of domain. See Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc on the "stem" of a double-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the Fc can be modified to produce certain improved properties.
In some embodiments, the Fc or IL-2 mutant and Fc fusion proteins as described herein have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, in such Fc or IL-2 mutant fusion proteins with Fc, the fucose content may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. As described in WO 2008/077546, the content of fucose is determined by MALDI-TOF mass spectrometry of the average content of fucose within the sugar chain attached to Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g. complex, hybrid and high mannose structures). Asn297 refers to the asparagine residue at position 297 of the Fc region (the EU numbering system for the Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in the Fc region. Such fucosylated variants may have enhanced ADCC function. See US Patent Publication nos. US 2003/0157108 (Presta, l.), US 2004/0093621 (Kyowa Hakko Kogyo co., ltd). Examples of publications related to antibody variants that are "defucosylated" or "lack of fucose" include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/015614; US 2002/0164328; US 2004/0093621; US 2004/013321; US 2004/010704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; okazaki et al, J.mol. Biol.336:1239-1249 (2004); yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated Fc-containing proteins include Lec13 CHO cells lacking the fucosylation function of the protein (Ripka et al, arch. Biochem. Biophys.249:533-545 (1986); US Pat Appl No US 2003/0157108A1, presta, L; and WO 2004/056312 A1,Adams et al, especially example 11), and knockout cell lines such as alpha-1, 6-fucosyltransferase genes, FUT8 knockout CHO cells (see Yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004); kanda, Y. Et al, biotechnol. Bioeng.,94 (4): 680-688 (2006), and WO 2003/085107).
Effector function variants
In some embodiments, the present application contemplates an Fc that has some but not all Fc effector functions, making it an ideal candidate for an application in which the half-life of the fusion protein of an IL-2 mutant with Fc is important in vivo, but some effector functions (e.g.CDC and ADCC) are unnecessary or detrimental. Cytotoxicity assays may be performed in vitro or in vivo to determine a reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that Fc or fusion proteins comprising such Fc and IL-2 mutants lack fcγr binding (and thus may lack ADCC activity), but retain FcRn binding capacity. The primary cells mediating ADCC, natural killer cells (NK), express fcγriii only, whereas monocytes express fcγri, fcγrii and fcγriii. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, annu. Rev. Immunol.9:457-492 (1991). U.S. patent No.5,500,362 (see helstrom, i.et al, proc.nat 'alacat.sci.usa 83:7059-7063 (1986)) and helstrom, i.et al, proc.nat' alacat.sci.usa 82:1499-1502 (1985); non-limiting examples of in vitro assays for assessing ADCC activity of a target molecule are detailed in U.S. Pat. No.5,821,337 (see Bruggemann, M.et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive detection methods (see ACTI for flow cytometry TM Non-radioactive toxicity test (CellTechnology, inc.Mountain View, calif.) and CytotoxNon-radioactive toxicity test (Promega, madison, wis.). Effector cells suitable for such assays include Peripheral Blood Mononuclear Cells (PBMCs) and NK cells. Furthermore, ADCC activity of the target molecule can also be assessed in vivo, for example, in animal models as disclosed in Clynes et al, proc.Nat' l Acad.Sci.USA 95:652-656 (1998). A C1q binding assay may also be performed to determine that fusion proteins of IL-2 mutants with Fc are unable to bind C1q and thus lack CDC activity. See WO 2006/029879 and WO 2005/100402 for C1q and C3C binding enzyme-linked immunosorbent assays. CDC assays can be performed to assess complement activity (see Gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996); cragg, M.S. et al, blood 101:1045-1052 (2003) and Cragg, M.S. and M.J. Glennie, blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can be performed using methods known in the art (see Petkova, s.b. et al., int' l.immunol.18 (12): 1759-1769 (2006)).
Fc with reduced effector function comprises substitutions of one or more residues in positions 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. patent No.6,737,056). Such Fc mutants include substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with alanine (US patent No.7,332,581). Certain antibody variants that enhance or reduce binding to FcRs are described in detail (see U.S. patent No.6,737,056; WO 2004/056312, and thields et al, j. Biol. Chem.9 (2): 6591-6604 (2001): in some embodiments, the Fc region is engineered to alter (i.e., increase or decrease) C1q binding and/or CDC, e.g., as described in U.S. patent No.6,194,551, WO 99/51642, and Idusogie et al, j. Immunol.164:4178-4184 (2000).
In some embodiments, the Fc comprises one or more amino acid substitutions that increase half-life and/or enhance binding to neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to neonatal FcRn are responsible for transporting maternal IgGs to the fetus (Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994)), and are described in detail in US2005/0014934A1 (Hinton et al). Those comprising an antibody with one or more substituted Fc regions thus increase the binding of the Fc region to FcRn. Such Fc variants include those with one or more substitutions of an Fc region residue, e.g., substitution of an Fc region 434 residue (US patent No.7,371,826).
See Duncan and Winter, nature 322:738-40 (1988); U.S. patent nos. 5,648,260; U.S. patent nos. 5,624,821; and WO 94/29351 relates to other examples of Fc variants.
Cysteine engineered variants
In some embodiments, it may be desirable to create a cysteine engineered Fc or fusion protein comprising such Fc with an IL-2 mutant, wherein one or more residues of the Fc are substituted with cysteine residues. In some embodiments, the substitution residue occurs at a site accessible on the Fc or fusion protein of the IL-2 mutant with Fc. By replacing these residues with cysteines, active thiol groups are thus located at accessible sites of the Fc or fusion protein of the IL-2 mutant with Fc, and can be used to conjugate molecules with other moieties, such as drug moieties or linker-drug moieties, to create conjugates of the fusion proteins of the IL-2 mutant with Fc. In some embodiments, any one or more of the following residues may be substituted with a cysteine: heavy chain a118 (EU numbering system) and heavy chain Fc domain S400 (EU numbering system). Cysteine engineered molecules may be produced as described in U.S. patent No.7,521,541.
In some embodiments, the Fc is from an IgG1 Fc. In some embodiments, the Fc is from a human IgG1 Fc. In some embodiments, the Fc is from a human wild-type IgG1 Fc. In some embodiments, the Fc does not comprise a hinge region of an IgG1 Fc. In some embodiments, the Fc comprises one or more non-effector mutations and/or deglycosylation mutations.
In some embodiments, the Fc comprises one or more mutations in amino acid residue L (L234) and amino acid residue L (L235) at position 234 and amino acid residue L (L235) relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations of L234 and L235 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations L234A and L235A relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID No. 12 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO. 12.
In some embodiments, the Fc comprises a mutation at amino acid residue N (N297) at position 297 relative to the amino acid sequence of a human wild-type IgG1 Fc. In some embodiments, fc comprises the mutation N297G or N297A relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID No. 13 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO 13. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID No. 14 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO. 14.
In some embodiments, the Fc comprises one or more mutations in amino acid residue L (L234), amino acid residue L (L235) at position 235, and amino acid residue P (P331) at position 331 relative to the amino acid sequence of a human wild-type IgG1 Fc. In some embodiments, the Fc comprises mutations of L234, L235, and P331 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations L234A, L235A and P331S relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID No. 15 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO. 15.
In some embodiments, the Fc comprises one or more mutations in amino acid residue L (L234), amino acid residue L (L235), amino acid residue G (G237), amino acid residue a (a 330), and amino acid residue P (P331) at position 234, 237, relative to the amino acid sequence of a human wild-type IgG1 Fc. In some embodiments, the Fc comprises mutations of L234, L235, G237, a330, and P331 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises the mutations L234A, L235E, G237A, A S and P331S relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID No. 16 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO. 16.
In some embodiments, the Fc is from an IgG4 Fc. In some embodiments, the Fc is from a human IgG4 Fc. In some embodiments, the Fc is from a human wild-type IgG4 Fc. In some embodiments, the Fc does not comprise a hinge region of IgG 4. In some embodiments, the Fc comprises one or more non-effector mutations and/or deglycosylation mutations.
In some embodiments, the Fc comprises one or more mutations in amino acid residue S228 (S228), amino acid residue F234 (F234), and amino acid residue L235 (L235) relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc comprises S228, F234, and L235 mutations relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc comprises mutations S228P, F234A and L235A relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID No. 17 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO 17.
KIH (Knob-into-Hole) technique:
additional mutations that may be made to Fc include those that promote heterodimer formation between Fc polypeptides. In some embodiments, the problem of heterodimer assembly is solved by the KIH (Knob-into-Hole) technique, KIH technique is described in C H Introduction of an asymmetric mutant structure into the 3 domain ("knob" mutation means at C H 3 with a large amino acid residue to replace a smaller residue, whereas "hole" mutation refers to the use of a small amino acid residue to replace a larger residue). Engineered fcs are more prone to heterodimerization than homodimerization due to steric effects (Ridgway J B, et al, "Knobs-into-legs" engineering ofantibody C H 3 domains for heavy chain heterodimerization[J]Protein Eng.1996,9 (7): 617-621). I.e. C H Substitution of threonine residue (T) at position 366 of the 3-domain with tryptophan residue (W) forms a "knob" structure and pairs the other C H Substitution of threonine residue (T) at position 366 with serine residue (S), substitution of leucine residue (L) at position 368 with alanine residue (a), substitution of tyrosine residue (Y) at position 407 with valine residue (V) forms a "mortar" (holes) structure while introducing two cysteines that form a stabilized disulfide bridge
Mutation of residue (C) (S354C on the "knob" side and Y349C on the "hole" side) enhances the stability of KIH, wherein the numbering is according to the EU index as Kabat (Kuglstatter A, et al Structure differences between glycosylated, disfig-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products [ J ]. Protein Eng Des Sel.,2017,30 (9): 649-656).
Exemplary Fc mutant sequences are shown in table 4.
In some embodiments, the fusion protein of an IL-2 mutant of the present application with Fc is in a bivalent form, comprising 2 IL-2 mutant molecules in the fusion protein. For example, a homodimeric form consisting of 2 fusion polypeptide chain monomers as described above. In some embodiments, an exemplary structure thereof is shown in fig. 1A and 1B.
In some embodiments, the fusion protein of an IL-2 mutant of the present application with Fc is in a monovalent form, and in this fusion protein, only 1 IL-2 mutant molecule is included. For example, a heterodimeric form consisting of 1 fusion polypeptide chain monomer as described above and 1 Fc subunit. In some embodiments, an exemplary structure thereof is shown in fig. 2. In other embodiments, the fusion protein of an IL-2 mutant of the present application with Fc is in a monovalent form, consisting of 1 fusion polypeptide chain monomer as described above.
Joint
In some embodiments, the IL-2 mutant and Fc can be linked by a linker (e.g., a linker peptide, a non-linker peptide). In some embodiments, the joint is a flexible joint. In some embodiments, the linker is a stable linker. In general, the ideal linker will not affect or will not significantly affect the proper folding and conformation of the fusion proteins of IL-2 mutants and Fc described herein. Preferably, the linker confers flexibility to the fusion protein of the IL-2 mutant with Fc, retains or enhances the biological function of the IL-2 mutant, and/or does not affect or does not significantly affect the half-life and/or stability of the fusion protein of the IL-2 mutant with Fc in vivo. In some embodiments, the linker is a stable linker (e.g., is not cleavable by proteases, particularly MMPs).
In some embodiments, the linker is a linker peptide. The linker peptide may be of any length. In some embodiments, the linker peptide is 1 to 10 amino acids, 3 to 18 amino acids, 1 to 20 amino acids, 10 to 20 amino acids, 21 to 30 amino acids, 1 to 30 amino acids, 10 to 30 amino acids, 1 to 50 amino acids, 5 to 40 amino acids, 12 to 18 amino acids, 4 to 25 amino acids in length. In some embodiments, the linker peptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the linker peptide is 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the linker peptide is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, the linker peptide is no longer than necessary to prevent undesired domain interactions and/or optimize biological function and/or stability. In some embodiments, the connecting peptide is up to 30 amino acids in length, e.g., up to 20 amino acids, or up to 15 amino acids. In some embodiments, the linker peptide is 5 to 30 amino acids in length, or 5 to 18 amino acids in length.
The linker peptide may have a naturally occurring sequence or may have a non-naturally occurring sequence. For example, sequences from the heavy chain hinge region of an antibody may be used as linkers. See, for example, WO1996/34103. In some embodiments, the connecting peptide is a human IgG1, igG2, igG3, or IgG4 hinge region. In some embodiments, the connecting peptide is a mutated human IgG1, igG2, igG3, or IgG4 hinge region. In some embodiments, the joint is a flexible joint. Typical flexible linkers include, but are not limited to, glycine polymer (G) n (SEQ ID NO: 20), glycine-serine polymers (including, for example, (GS) n (SEQ ID NO: 21), (GGS) n (SEQ ID NO: 22), (GGGS) n (SEQ ID NO: 23), (GGGGS) n (SEQ ID NO: 24), or (GGS) m (GGGS) n (SEQ ID NO: 25), where m and n are integers of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as a neutral chain between components. Glycine has more phi-psi space than alanine and is less restricted than residues with longer side chains (see Scheraga, rev. Computer chem.11173-142 (1992)). Examples of flexible linkers include, but are not limited to, the amino acid sequences shown in table 5. In general, those skilled in the art will appreciate that contemplated fusion proteins of IL-2 mutants with Fc may include all or part of a flexible linker such that the linker may include one flexible linker moiety and one or more moieties that provide less flexible structure to provide the structure and function of a desired fusion protein of IL-2 mutant with Fc. In some embodiments, the connecting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs 20-51 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with an amino acid sequence set forth in any one of SEQ ID NOs 20-51.
Exemplary linker peptide sequences are shown in table 5.
Other considerations regarding linkers include the effect on the physical or pharmacokinetic properties of the resulting fusion protein of the IL-2 mutant with Fc, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and planned degradation), rigidity, flexibility, immunogenicity, binding of the IL-2 mutant to IL-2 receptor, binding capacity of a colloid or liposome, and the like.
In some embodiments, fusion proteins of IL-2 mutants described herein with Fc include bivalent and monovalent forms. In other embodiments, the bivalent form, which comprises two IL-2 mutant molecules, may consist of two identical or different monomers, e.g., the monomer is a fusion polypeptide chain of an IL-2 mutant and Fc. In other embodiments, the monovalent form, which comprises one IL-2 mutant molecule, may be composed of two different monomers, for example, one of which is a fusion polypeptide chain of an IL-2 mutant and one Fc subunit, and the other monomer is an Fc subunit. In other embodiments, the monovalent form comprises an IL-2 mutant molecule consisting of only one monomer, e.g., the monomer is a fusion polypeptide chain of an IL-2 mutant and an Fc subunit.
In some embodiments, the IL-2 mutants described herein, which comprise a mutation in R83 relative to the amino acid sequence of human wild-type IL-2, in a fusion protein of the IL-2 mutant with Fc (e.g., a bivalent or monovalent fusion protein form). In some embodiments, the mutation of R83 is R83L, R a or R83N. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In some embodiments, the IL-2 mutants described herein further comprise a mutation in D20 relative to the human wild-type IL-2 amino acid sequence in a fusion protein of the IL-2 mutant with Fc (e.g., a bivalent or monovalent fusion protein form). In some embodiments, the mutation of D20 is D20N. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of R83L and D20N mutation.
In some embodiments, the IL-2 mutants described herein in fusion proteins with Fc (e.g., bivalent or monovalent fusion protein forms), the IL-2 mutants, in some embodiments, further comprise mutations of H16 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of H16 and N71 are H16N and N71R, respectively. In some embodiments, the IL-2 mutant, which comprises relative to the wild-type IL-2 amino acid sequence of R83L, H N and N71R mutation.
In some embodiments, the IL-2 mutants described herein further comprise mutations in N71 and N88 relative to the human wild-type IL-2 amino acid sequence in a fusion protein of the IL-2 mutant with Fc (e.g., a bivalent or monovalent fusion protein form). In some embodiments, the mutations of N71 and N88 are N71S and N88D, respectively. In some embodiments, the IL-2 mutant, which comprises relative to the wild-type IL-2 amino acid sequence of R83A, N S and N88D mutation.
In some embodiments, the IL-2 mutants described herein further comprise mutations in L66 and V91 relative to the human wild-type IL-2 amino acid sequence in a fusion protein of the IL-2 mutant with Fc (e.g., a bivalent or monovalent fusion protein form). In some embodiments, the mutations of L66 and V91 are L66F and V91E, respectively. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of R83N, L F and V91E mutation.
In other embodiments, the IL-2 mutants described herein comprise mutations in E67 and S75 relative to the amino acid sequence of human wild-type IL-2 in a fusion protein of the IL-2 mutant with Fc (e.g., a bivalent or monovalent fusion protein form). In some embodiments, the mutation of E67 is E67I or E67G, and the mutation of S75 is S75P. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of E67I and S75P mutation. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of E67G and S75P mutation. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In other embodiments, the IL-2 mutants described herein comprise mutations in L17 and P82 relative to the amino acid sequence of human wild-type IL-2 in a fusion protein of the IL-2 mutant with Fc (e.g., a bivalent or monovalent fusion protein form). In some embodiments, the mutations of L17 and P82 are L17V and P82L, respectively. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In some embodiments, the IL-2 mutants described herein further comprise a mutation of C125 relative to the amino acid sequence of human wild-type IL-2 in addition to the above-described mutations in the fusion proteins of IL-2 with Fc (e.g., bivalent or monovalent fusion protein forms). In some embodiments, the mutation of C125 is C125S or C125A.
In some embodiments, the IL-2 mutants described herein comprise an amino acid sequence as set forth in any of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to an amino acid sequence as set forth in any of SEQ ID NOs 5-11 in a fusion protein (e.g., a bivalent or monovalent fusion protein form) with an Fc.
The amino acid sequences of exemplary IL-2 mutants and Fc bivalent fusion proteins are shown in Table 6.
In some embodiments, in the bivalent fusion protein form of IL-2 mutants and Fc described herein, each monomer comprises from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) IL-2 mutant and (ii) Fc.
In some embodiments, in the bivalent fusion protein form of IL-2 mutants and Fc described herein, each monomer comprises from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) an IL-2 mutant, (ii) a linker peptide, and (iii) Fc.
In some embodiments, the IL-2 mutant described herein is in the form of a bivalent fusion protein with an Fc, the Fc is an IgG1 Fc, and the Fc comprises L234A and L235A (LALA) mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO:12.
In some embodiments, the divalent fusion protein form of an IL-2 mutant described herein with an Fc, the Fc is an IgG1 Fc, and the Fc comprises the N297G mutation. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO:13.
In some embodiments, the divalent fusion protein form of an IL-2 mutant described herein with an Fc, the Fc is an IgG1 Fc, and the Fc comprises the N297A mutation. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO. 14.
In some embodiments, the IL-2 mutant described herein is in the form of a bivalent fusion protein with Fc, the Fc is an IgG1 Fc, and the Fc comprises L234A, L235A and P331S mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO. 15.
In some embodiments, the IL-2 mutant described herein is in the form of a bivalent fusion protein with Fc, the Fc is an IgG1 Fc, and the Fc comprises L234A, L235E, G237A, A S and P331S mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO. 16.
In some embodiments, the IL-2 mutant described herein is in the form of a bivalent fusion protein with Fc, the Fc is an IgG4 Fc, and the Fc comprises S228P, F234A and L235A mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO:17.
In some embodiments, in the bivalent fusion protein form of IL-2 mutants and Fc described herein, each monomer comprises from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID NOs:5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence set forth in any one of SEQ ID NOs:5-11 and (ii) Fc comprising an amino acid sequence as set forth in any one of SEQ ID NOs:12-17 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence set forth in any one of SEQ ID NOs: 12-17.
In some embodiments, in the bivalent fusion protein form of IL-2 mutants and Fc described herein, each monomer comprises from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 5-11, (ii) a linker peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs 20-51 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 12-17, and (iii) an Fc comprising an amino acid sequence as set forth in any one of SEQ ID NOs 12-17 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 96%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 12-17.
In some embodiments, the divalent fusion protein forms of IL-2 mutants and Fc described herein comprise an amino acid sequence as set forth in any of SEQ ID NOs:53-59, or a variant thereof, that has at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to an amino acid sequence set forth in any of SEQ ID NOs: 53-59.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants described herein and Fc, wherein Fc is an IgG1 Fc. In some embodiments, the Fc comprises a KIH mutation (referred to as Fc knob and Fc hole, respectively). In some preferred embodiments, the Fc comprises LALA mutations and KIH mutations (referred to as Fc LALA knob and Fc LALA hole, respectively). In some more preferred embodiments, the Fc LALA knob may comprise the mutations L234A, L235A, T366W and S354C and the Fc LALA hole may comprise the mutations L234A, L235A, T366S, L368A, Y35407V and Y349C.
In some embodiments, the IgG1 Fc LALA knob comprises the amino acid sequence of SEQ ID NO:18 and the Fc LALA hole comprises the amino acid sequence of SEQ ID NO:19.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) IL-2 mutant and (ii) Fc knob, and one monomer comprises: fc hole.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) An IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 5-11 and (ii) an Fc LALA knob comprising an amino acid sequence of SEQ ID NO 18 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO 18; and another monomer is an Fc LALA hole comprising the amino acid sequence SEQ ID NO. 19 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO. 19.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant and (ii) an Fc hole; and the other monomer comprises: fc knob.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) An IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 5-11 and (ii) an Fc LALA hole comprising an amino acid sequence of SEQ ID NO 19 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO 19; and another monomer is Fc LALA knob comprising the amino acid sequence SEQ ID NO. 18 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO. 18.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant, (ii) a linker peptide, and (iii) Fc knob; and the other monomer comprises: fc hole.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 5-11, (ii) a linker peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs 20-51 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 20-51 and (iii) an Fc la kb comprising an amino acid sequence of SEQ ID NO 18 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO 18; and another monomer is an Fc LALA hole comprising the amino acid sequence SEQ ID NO. 19 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO. 19.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant, (ii) a linker peptide, and (iii) an Fc hole, and another monomer comprises: fc knob.
In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutants and Fc described herein, one of the monomers comprises from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 5-11, (ii) a linker peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs 20-51 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs 20-51 and (iii) an Fc la hole comprising an amino acid sequence of SEQ ID NO 19 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO 19; and another monomer is Fc LALA knob comprising the amino acid sequence SEQ ID NO. 18 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO. 18.
In some embodiments, the fusion proteins of IL-2 mutants described herein with Fc have a half-life of at least 10 hours (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 hours or more) when administered to an individual (e.g., a human) by intravenous injection, intramuscular injection, or subcutaneous injection.
Binding affinity
The binding affinity of a molecule (e.g., an IL-2 mutant or fusion protein comprising an IL-2 mutant) to its binding partner (e.g., an IL-2 receptor, such as IL-2Rα, IL-2Rβ, IL-2Rγ, IL-2Rβγ, or IL-2Rαβγ) can be determined by any suitable ligand binding assay known in the art, e.g., western blot, enzyme-linked immunosorbent assay (ELISA), meso Scale Discovery (MSD) electrochemiluminescence, bead-based Multiplex Immunoassay (MIA), RIA, surface Plasmon Resonance (SPR), ECL, IRMA, EIA, biacore assay, octet assay, peptide scan, and the like. For example, by using various labeling reagents labeled IL-2 mutants or fusion proteins comprising IL-2 mutants or their receptors (e.g., IL-2Rα, IL-2Rβ, IL-2Rγ, IL-2Rβγ, or IL-2Rαβγ), or subunits thereof for simple analysis, biacoreX (Amersham Biosciences), an over-the-counter measurement kit or the like, can be used as well, which can be operated according to the user manual and experimental protocols attached to the kit.
In some embodiments, protein microarrays are used to analyze the interactions, functions, and activities of IL-2 mutants or fusion proteins comprising IL-2 mutants described herein with their receptors on a large scale. Protein microarrays have a support surface that binds to a range of capture proteins (e.g., IL-2 receptors or subunits thereof). A fluorescently labeled probe molecule (e.g., an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein) is then added to the array and interacts with the bound capture protein, releasing the fluorescent signal and reading by a laser scanner.
Binding affinity can also be measured using Biacore. For example, IL-2Rβγ or IL-2Rαβγ is coupled to the CM-5 sensor chip surface as a ligand using EDC/NHS chemistry. A series of dilutions of IL-2 mutants or IL-2 mutant and Fc fusion proteins described herein are then used as analytes for binding to ligands coupled to the chip, and IL-2 binding to or dissociation from IL-2Rβγ or IL-2Rαβγ can be monitored in real time. Affinity (Kd) can be determined by kinetic analysis using BIA assessment software.
In some embodiments, biacore experiments can be used to determine the Kd value of the IL-2 mutants or fusion proteins of IL-2 mutants and Fc provided herein to IL-2rβγ to determine binding affinity, and in some embodiments, the fusion proteins of IL-2 mutants and Fc provided herein have lower binding affinity to IL-2rβγ relative to wild-type fusion proteins of IL-2 and Fc. In some embodiments, the affinity of the fusion protein of the IL-2 mutant with Fc to IL-2rβ and/or IL-2rβγ is reduced by at least 5% (e.g., any of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) relative to the fusion protein of the wild-type IL-2 with Fc. In some embodiments, the IL-2 mutant and Fc fusion proteins described herein have increased or maintained or no significant decrease in affinity for IL-2Rα relative to a wild-type IL-2 and Fc fusion protein (e.g., human wild-type IL-2). In some embodiments, the binding force of the IL-2 mutant and Fc fusion proteins of the present application to IL-2Rαβγ is increased, maintained, or not significantly reduced relative to a wild-type IL-2 and Fc fusion protein.
In some embodiments, the Kd value of binding between an IL-2 mutant or IL-2 mutant and Fc fusion protein described herein and a medium affinity receptor (IL-2Rβγ) is greater than the Kd value of binding between wild-type IL-2 or wild-type IL-2 and Fc fusion protein and the same receptor. In some embodiments, the Kd value for binding between an IL-2 mutant or fusion protein of an IL-2 mutant and an Fc described herein and its high affinity receptor (IL-2rαβγ) is close to (e.g., equal to, less than, or slightly greater than) the Kd value for binding between the wild-type and the same receptor (IL-2rαβγ).
Pharmacokinetic (PK)
Pharmacokinetic (PK) refers to absorption, distribution, metabolism, and excretion of a drug (e.g., an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein) following administration to a subject. Pharmacokinetic parameters useful for determining clinical utility include, but are not limited to, serum/plasma concentration, time-varying serum/plasma concentration, maximum serum/plasma concentration (C max ) Time to reach maximum concentration (T max ) Half-life (T) 1/2 ) Area under concentration-time curve (AUC) within dosing interval τ ) Etc.
Techniques for obtaining PK profiles for drugs (e.g., IL-2 mutants or fusion proteins comprising IL-2 mutants described herein) are known in the art. See Heller et al Annu RevAnal Chem,11,2018; and Ghandforoush Sattari et al, J Amino Acids, arc ID 346237,Volume 2010. In some embodiments, the PK profile of an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein is measured in a blood, plasma, or serum sample of an individual. In some embodiments, the PK profile of an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein in an individual is measured using mass spectrometry techniques (e.g., LC-MS/MS or ELISA). PK analysis can be performed on PK curves by any method known in the art, for example, non-interventricular analysis, using PKSolver V2 software (Zhang y et al, "PKSolver: an add-in program for pharmacokinetic andpharmacodynamic data analysis in Microsoft Excel," Comput Methods Programs biomed.2010;99 (3): 306-1). Example method see example 7.
"C" means a drug in the plasma, serum, or any suitable body fluid or tissue of a subject (e.g., IL-2 mutant or comprising an IL-2 mutationBulk fusion protein) concentration, typically expressed as mass per unit volume, e.g., nanograms per milliliter. For convenience, the concentration of the drug in serum or plasma is referred to herein as "serum concentration" or "plasma concentration". Serum/plasma concentrations at any time after administration (e.g., intravenous, intraperitoneal or subcutaneous injection of IL-2 mutant or fusion protein polypeptide comprising IL-2 mutant) are referred to as C time Or C t . The maximum serum/plasma drug concentration during administration is called C max ;C min Refers to the minimum serum/plasma drug concentration at the end of the dosing interval; c (C) ave Mean concentration during the dosing interval.
The term "bioavailability" refers to the degree or rate at which a drug (e.g., an IL-2 mutant or fusion protein comprising an IL-2 mutant) passes through the systemic circulation, thereby entering the site of action.
"AUC" is the area under the serum/plasma concentration-time curve, which is considered the most reliable measure of bioavailability, such as the area under the concentration-time curve (AUC tau) over the dosing interval, "total exposure" or "total drug exposure over a period of time" (AUC 0-∞ ) Area under the concentration-time curve at time t after administration (AUC 0-t ) Etc.
Peak time of serum/plasma concentration (T max ) Is that serum/plasma concentration (C) is achieved after administration (e.g., IL-2 mutant or fusion protein comprising IL-2 mutant) max ) The time of the peak.
Half-life (T) 1/2 ) Refers to the time required for the concentration of a drug (e.g., an IL-2 mutant or fusion protein comprising an IL-2 mutant) measured in plasma or serum (or other biological matrix) to drop to half its concentration or amount at a particular point in time. For example, after intravenous administration, the concentration of the drug in the plasma or serum decreases due to the distribution and elimination of the drug. In curves of plasma or serum drug concentration over time following intravenous administration, the first or rapid decrease phase is believed to be due primarily to distribution, whereas the decrease in later phase is generally slower, primarily due to elimination, although both processes occur in both phases. Distribution is considered to be sufficientAfter a time. Generally, the elimination half-life is determined by the end-stage or elimination (major) phase of the plasma/serum concentration-time curve. See Michael Schrag and Kelly Regal, "chapter 3 of the comprehensive guidelines for preclinical drug development toxicology," pharmacokinetics and toxicology, "2013.
In some embodiments, a fusion protein comprising an IL-2 mutant (e.g., a bivalent fusion protein form of an IL-2 mutant and Fc) described herein has a half-life of at least 10 hours (e.g., intravenous injection, subcutaneous injection, or intramuscular injection, such as human injection), such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 hours, or more. IL-2 mutants or derivatives of fusion proteins comprising IL-2 mutants
In some embodiments, the IL-2 mutants or fusion proteins comprising IL-2 mutants referred to herein may be further modified to comprise additional non-protein portions known and readily available in the art. Exemplary non-protein moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamic acids (homopolymers or random copolymers), dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the IL-2 mutant or fusion protein comprising the IL-2 mutant may be different, if multiple polymers are attached, they may be the same or different molecules. In general, the amount and/or type of polymer used for derivatization may be determined based on considerations including, but not limited to, the particular nature or function of the IL-2 mutant or fusion protein comprising the IL-2 mutant to be modified, whether the IL-2 mutant or derivative of the fusion protein comprising the IL-2 mutant will be used in a therapy under particular conditions, and the like.
In some embodiments, the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein further comprise a tag selected from chromophores, fluorophores (e.g., coumarin, xanthene, cyanine, pyrene, borazine, oxazine and derivatives thereof), fluorescent proteins (e.g., GFP, phycobiliprotein and derivatives thereof), phosphorescent dyes (e.g., dioxetane, xanthene or carbocyanine dyes, lanthanide chelates), tandem dyes (e.g., cyanine-phycobiliprotein derivatives and xanthene-phycobiliprotein derivatives), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), haptens, enzymes (e.g., peroxidases, phosphatases, glycosidases, luciferases), and radioisotopes (e.g., 125 I、 3 H、 14 C、 32 P)。
in some embodiments, the IL-2 mutant or fusion protein comprising the IL-2 mutant may be further modified to comprise one or more other biologically active proteins, polypeptides, or fragments thereof. As used herein, "biological activity" or "biologically active" are used interchangeably to refer to exhibiting biological activity in vivo to perform a particular function. For example, it may mean binding to a particular biomolecule, such as a protein, DNA, etc., and then promoting or inhibiting the activity of that biomolecule. In some embodiments, biologically active proteins or fragments thereof include proteins and polypeptides that are administered to a patient as an active drug for the prevention or treatment of a disease or condition, as well as proteins and polypeptides for diagnostic purposes, such as enzymes for diagnostic assays or in vitro assays, and proteins and polypeptides that are administered to a patient to prevent a disease, such as a vaccine. In some embodiments, the biologically active protein or fragment thereof has immunostimulatory/immunomodulatory, membrane transport, or enzymatic activity. In some embodiments, the biologically active protein, polypeptide, or fragment thereof is an enzyme, hormone, growth factor, cytokine, or mixture thereof. In some embodiments, the biologically active protein, polypeptide, or fragment can specifically recognize a peptide of interest (e.g., an antigen or other protein).
In some embodiments, the biologically active protein or fragment thereof that can be included in an IL-2 mutant or fusion protein comprising an IL-2 mutant described herein is an antigen binding protein (e.g., an antibody). In some embodiments, the biologically active protein or fragment thereof that may be included in an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein is an antibody mimetic, which is a small-sized engineering protein comprising an antigen binding domain that reminds humans of an antibody (GGeering and Fusseneger, trends Biotechnol.,33 (2): 65-79,2015). These molecules are derived from existing human scaffold proteins and consist of a single polypeptide. Examples of antibody mimetics that can be included in an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein can be, but are not limited to, engineered ankyrin repeat proteins (DARPin; comprising 3-5 fully synthesized ankyrin repeat sequences flanked by N-terminal and C-terminal cap domains), an affinity multimer (avimer; a high affinity protein comprising multiple A domains, each domain having a lower affinity for the target), or an anticoagulant (based on a lipid scaffold, having four accessible loops, each of which can be random in sequence). In some embodiments, the biologically active protein or fragment thereof that can be included in an IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein is a armadillo-repeat protein (e.g., β -catenin, α -catenin, plague, polyposis coli Adenoma (APC)), comprising a armadillo-repeat unit (characteristic, the length of the repeat amino acid sequence is about 40 residues). Each armadical repeat unit consists of a pair of alpha helices forming a hairpin structure. Multiple repeated copies form the alpha solenoid structure. Is capable of binding different types of peptides, depending on the constant binding pattern of the peptide backbone, without the need for specific conserved side chains or interactions with the free N-or C-terminus of the peptide. The possibility of recognizing peptide residues through residues, coupled with the intrinsic modularity of the repeat proteins, makes the armadillo repeat proteins promising candidates for peptide binding to universal scaffolds.
Nucleic acids encoding IL-2 mutants or fusion proteins comprising IL-2 mutants
The present application also relates to isolated nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising an IL-2 mutant, vectors comprising nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising an IL-2 mutant. Also relates to an isolated host cell (e.g., CHO cell, HEK293 cell, hela cell or COS cell) comprising the above nucleic acid or the above vector. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding an N-terminal signal peptide of an IL-2 mutant or a fusion protein comprising an IL-2 mutant.
In some embodiments, vectors comprising nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants are suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK293 cells, hela cells, COS cells). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector, such as pTT5.
Many viral-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retrovirus vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art, and is described in detail, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York), and other virology and molecular biology manuals. Retrovirus provides a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to the engineered mammalian cells in vitro or under ex vivo conditions. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying construct protein coding sequences may be packaged using experimental methods known in the art. The resulting lentiviral vector may be used to transduce into mammalian cells using methods known in the art. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transduction, as they allow long-term, stable integration of transgenes and propagation in daughter cells. Lentiviral vectors are also low immunogenic and can transduce non-proliferating cells.
In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a pt 5 vector. In some embodiments, the vector is a transposon, such as a Sleeping Beauty (SB) transposon system or a PiggyBac transposon system. In some embodiments, the carrier is a polymer-based non-viral carrier, including, for example, poly (lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI), and dendrimers. In some embodiments, the carrier is a non-viral carrier based on cationic lipids, such as cationic liposomes, lipid nanoemulsions, and Solid Lipid Nanoparticles (SLNs). In some embodiments, the vector is a peptide-based non-viral gene vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing may be used to introduce nucleic acids encoding IL-2 mutants or fusion proteins comprising IL-2 mutants into host cells. See Yin h.et al, nature rev.genetics (2014) 15:521-555; aronovich EL et al, "The Sleeping Beauty transposon system:a non-viral vector for gene therapy," hum. Mol. Genet. (2011) R1:R14-20 and Zhao S.et al, "PiggyBac transposon vectors: the tools ofthe human gene coding." Transl. Lung Cancer Res. (2016) 5 (1): 120-125, incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding an IL-2 mutant or fusion protein comprising an IL-2 mutant described herein are introduced into a host cell (e.g., CHO, HEK 293, hela, or COS) by physical methods, including, but not limited to, electroporation, sonoporation, photopporation, magnetic transfection, aqueous poration.
In some embodiments, the vector comprises a selectable marker gene or reporter gene for selecting cells expressing an IL-2 mutant or fusion protein comprising an IL-2 mutant described herein from a population of host cells transfected with the vector (e.g., lentiviral vector, pTT5 vector). Both the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable expression in the host cell. For example, the vector may comprise transcription and translation terminators, initiation sequences, and promoters for regulating expression of the nucleic acid sequences.
Any molecular cloning method known in the art may be used, including, for example, cloning the nucleic acid into the vector using restriction enzyme sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters for gene expression in prokaryotic or eukaryotic cells (e.g., mammalian cells) have been explored and any promoter known in the art may be used in the present application. Promoters can be broadly classified as constitutive or regulated, such as inducible.
In some embodiments, the nucleic acid encoding an IL-2 mutant or fusion protein comprising an IL-2 mutant described herein is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Examples of promoters contemplated herein include, but are not limited to, the CMV promoter (CMV), human elongation factor-1 alpha (hEF 1 alpha), ubiquitin C promoter (Ubic), phosphoglycerate kinase Promoter (PGK), simian virus 40 early promoter (SV 40), chicken beta-actin promoter coupled to CMV early enhancer (CAGG), the Rous Sarcoma Virus (RSV) promoter, polyomavirus enhancer/herpes simplex thymidine kinase (MC 1) promoter, beta actin (beta-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgene expression has been widely compared in a number of studies. In some embodiments, a nucleic acid encoding an IL-2 mutant or fusion comprising an IL-2 mutant described herein is operably linked to a CMV promoter.
In some embodiments, the nucleic acid encoding an IL-2 mutant or fusion protein comprising an IL-2 mutant described herein is operably linked to an inducible promoter. Inducible promoterBelongs to the category of regulatory promoters. The inducible promoter may be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducer (i.e., an inducing agent), or a combination thereof. In some embodiments, the induction conditions do not induce expression of an endogenous gene in the host cell. In some embodiments, the induction conditions are selected from: inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, and host cell activation state. In some embodiments, the inducible promoter may be the NFAT promoter,Promoters or nfkb promoters. />
V. preparation method
The present application also relates to methods of making any of the IL-2 mutants described herein or fusion proteins comprising an IL-2 mutant. Accordingly, in some embodiments, a method of making an IL-2 mutant or a fusion protein comprising an IL-2 mutant is contemplated, comprising: (a) Culturing a host cell (e.g., CHO cell, HEK 293 cell, hela cell, or COS cell) comprising a nucleic acid or vector encoding any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants under conditions effective to express the encoded IL-2 mutants or fusion proteins comprising IL-2 mutants; and (b) obtaining from the host cell an expressed IL-2 mutant or a fusion protein comprising an IL-2 mutant. In some embodiments, the method of step (a) further comprises producing a host cell comprising a nucleic acid or vector encoding an IL-2 mutant or fusion protein comprising an IL-2 mutant described herein. The IL-2 mutants described herein or fusion proteins comprising IL-2 mutants may be prepared using any method known in the art or described herein.
In some embodiments, the IL-2 mutants described herein or fusion proteins comprising the IL-2 mutants may be expressed by eukaryotic cells, such as mammalian cells. In some embodiments, the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants may be expressed by prokaryotic cells.
1. Recombinant products of prokaryotic cells
a) Vector construction
The polynucleotide sequences encoding the protein constructs described herein can be obtained using standard recombinant techniques. Polynucleotides may be synthesized using nucleotide synthesizers or PCR techniques. Once the sequence encoding the polypeptide is obtained, it is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a prokaryotic host. Many vectors known in the art and usable are available for use in the present application. The choice of a suitable vector will depend primarily on the size of the nucleic acid into which the vector is to be inserted and the particular host cell into which the vector is to be transformed. Each vector contains various components, depending on the function of the vector (amplification or expression of the heterologous polynucleotide, or both) and the compatibility of the vector with the particular host cell in which it is located. Carrier components generally include, but are not limited to: replication initiation sites, selectable marker genes, promoters, ribosome Binding Sites (RBSs), signal sequences, heterologous nucleic acid inserts, and transcription termination sequences.
In general, plasmid vectors contain replicon and control sequences from species compatible with the host cells with which they are used. The vector typically carries a replication site, and a marker sequence capable of providing phenotypic selection in transformed cells. For example, E.coli is typically transformed with pBR322, pBR322 being a plasmid derived from E.coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides simple means for identifying transformed cells. pBR322, derivatives thereof, or other bacterial plasmids or phages may also contain or be modified to contain promoters which can be used by microorganisms to express endogenous proteins. Examples of pBR322 derivatives for expressing specific antibodies are detailed in Carter et al, U.S. Pat. No.5,648,237.
In addition, phage vectors comprising replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors with these host cells. For example, phages such as GEM TM -11 can be used to prepare recombinant vectors which can be used to transform susceptible host cells such as e.coli LE392.
The promoter is an untranslated regulatory sequence located upstream (5') of the cistron, which can regulate the expression of downstream genes. Prokaryotic promoters are generally divided into two classes, inducible and constitutive. An inducible promoter is a promoter that can initiate and increase the level of transcription of a cistron in response to a change in culture conditions (e.g., the presence or absence of nutrients or a change in temperature).
Many promoters recognized by potential host cells are well known. The promoter is removed from the source DNA by restriction enzymes and the isolated promoter sequence is inserted into the vector of the present application, the selected promoter being operably linked to the cistron DNA encoding the polypeptide. Both native promoter sequences and a number of heterologous promoters can be used to direct the amplification and/or expression of a target gene. In some embodiments, heterologous promoters are utilized because heterologous promoters generally allow for greater transcription and higher yields of expressed target genes than native target polypeptide promoters.
Promoters suitable for use in the prokaryotic host include the PhoA promoter, the-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters, such as the tac or trc promoters. However, other promoters that are functional in bacteria (e.g., other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences have been disclosed so that the skilled artisan can use the linker or aptamer to provide any desired restriction site to ligate them to cistrons encoding the light and heavy chains of interest (Siebenlist et al, (1980) Cell 20:269).
In some embodiments, each cistron within the recombinant vector comprises a secretion signal sequence component that directs the transfer of the expressed polypeptide across the membrane. In general, the signal sequence may be part of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for this application should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that are unable to recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, ipp, or thermostable enterotoxin II (STII) precursors, lamP, phoE, pelB, ompA, and MBP.
In some embodiments, production of the IL-2 mutants of the present application or fusion proteins comprising the IL-2 mutants may occur in the cytoplasm of the host cell, and thus it is not necessary that a secretion signal sequence be present within each cistron. In some embodiments, the polypeptide components are expressed, folded, and assembled to form a protein construct within the cytoplasm. Certain host strains (e.g., E.coli trxB-strain) provide cytoplasmic conditions that favor disulfide bond formation, allowing proper folding and assembly of the expressed protein subunits. See Proba and Pullthun, gene,159:203 (1995).
b) Prokaryotic host cell
Prokaryotic host cells suitable for expressing the proteins of the present application include archaebacteria and eubacteria, such as gram-negative or gram-positive bacteria. Examples of useful bacteria include E.coli (e.g., E.coli), bacillus (e.g., B.subtilis), E.coli, pseudomonas (e.g., P.aeruginosa), salmonella typhimurium, serratia marcescens, klebsiella, proteus, shigella, rhizobium, vitreoscilla or Paracoccus. In some embodiments, gram negative cells are used. In some embodiments, the E.coli cells serve as hosts for the present application. Examples of E.coli strains include strain W3110 (Bachmann, cellular and Molecular Biology, vol.2 (Washington, D.C.: american Society for Microbiology, 1987), pp.1190-1219;ATCC Deposit No.27,325) and derivatives thereof, including strain 33D3 (U.S. Pat.No.5,639, 635) having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A (nmpc fepE) degP41 kanR. Other strains and derivatives thereof, such as E.coli 294 (ATCC 31446), E.coli B, E.coli 1776 (ATCC 31537) and E.coli RV308 (ATCC 31608), are also suitable. These examples are illustrative and not limiting. Methods of constructing bacterial derivatives of any of the above mentioned known genotypes are known in the art and are described in detail, for example, in Bass et al, proteins,8:309-314 (1990). In view of the replicability of replicons in bacterial cells, it is often necessary to select suitable bacteria. For example, when a known plasmid such as pBR322, pBR325, pACYC177 or pKN410 is used to provide a copy, escherichia coli, serratia or Salmonella is suitable as a host.
In general, the host cell should secrete minimal amounts of proteolytic enzymes and additional protease inhibitors need to be added to the cell culture as appropriate.
c) Protein production
Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media, suitably modified, to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation refers to the introduction of DNA into a prokaryotic host, such that the DNA may replicate as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques suitable for such cells. Calcium treatment with calcium chloride is commonly used for bacterial cells containing a large number of cell wall barriers. Another conversion method employs polyethylene glycol/dimethyl sulfoxide. Another technique is electroporation.
Prokaryotic cells for the production of the protein constructs of the present application are grown in media known in the art and suitable for culturing the host cell of choice. Suitable media include Luriabroth (LB) and necessary nutritional supplements. In some embodiments, the medium further comprises a selection agent selected based on the structure of the expression vector to selectively allow the growth of prokaryotic cells comprising the expression vector. For example, ampicillin is added to the medium for cell growth expressing the ampicillin resistance gene.
In addition to the carbon source, nitrogen source, and inorganic phosphate source, any necessary supplements may also be added at appropriate concentrations, alone or as a mixture with other supplements or mediums (e.g., complex nitrogen sources). Alternatively, the medium may comprise one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycine, dithioerythritol and dithiothreitol. The prokaryotic host cells are cultured at a suitable temperature. For example, for E.coli growth, the preferred temperature range is 20℃to 39 ℃, more preferably 25℃to 37 ℃, even more preferably 30 ℃. The pH of the medium may be any pH between 5 and 9, depending mainly on the host organism. For E.coli, the pH is preferably from 6.8 to 7.4, and more preferably 7.0.
If an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one aspect of the application, the PhoA promoter is used to control transcription of a polypeptide. Thus, the transformed host cells are cultured in phosphate-limiting medium for induction. Preferably, the phosphate limiting medium is C.R.A.P medium (see Simmons et al, J.Immunol. Methods (2002), 263:133-147). Depending on the support structure employed, a variety of other inducers known in the art may be used, as is known in the art.
The protein constructs expressed herein are secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves destruction of microorganisms, typically by osmotic shock, sonication, or lysis. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein may be further purified by affinity resin chromatography. Alternatively, the protein may be transported to the culture medium and isolated therein. Cells can be removed from the medium, and the medium supernatant filtered and concentrated to further purify the produced protein. The expressed polypeptide can be further separated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot test.
Alternatively, proteins are produced on a large scale by fermentation processes. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. The volume of the large-scale fermentation is at least 1000 liters, preferably 1000 to 100000 liters. These fermentors use agitator impellers to dispense oxygen and nutrients, particularly glucose (carbon/energy source of choice). Small scale fermentation generally refers to fermentation in a fermenter having a volumetric volume of no more than 100 liters and ranging from 1 liter to 100 liters.
During fermentation, cells typically begin to induce protein expression after they grow to a desired density under appropriate conditions, e.g., at an OD550 of about 180-220, when the cells are in an early resting stage. Depending on the support structure employed, a variety of inducers known in the art and described above may be used. Cells can be grown for a short period of time before induction. Cells are typically induced for about 12-50 hours, although induction times that may be longer or shorter may be used.
To increase the yield and quality of the IL-2 mutants or fusion proteins comprising IL-2 mutants of the present application, various fermentation conditions may be modified. For example, to enhance proper assembly and folding of secreted polypeptides, additional vectors that overexpress chaperones may be used to co-transform host prokaryotic cells, such as Dsb protein (DsbA, dsbB, dsbC, dsbD or DsbG) or FkpA (peptide prolyl cis-trans isomerase with chaperone activity). Chaperones have been shown to help promote proper folding and solubilization of heterologous proteins produced in bacterial host cells. Chen et al, (1999) J Bio Chem 274:19601-19605; georgiou et al, U.S. Pat. No.6,083,715; georgiou et al, U.S. Pat. nos. 6,027,888; bothmann and Pluckaphun (2000) J.biol. Chem.275:17100-17105; ramm and Pluckaphun (2000) J.biol. Chem.275:17106-17113; arie et al, (2001) mol. Microbiol.39:199-210.
In order to minimize the hydrolysis of expressed heterologous proteins, particularly proteolytically sensitive proteins, certain host strains lacking proteolytic enzymes may be used in the present application. For example, the host cell strain may be modified such that a gene encoding a known bacterial protease is genetically mutated, such as protease III, ompT, degP, tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some coliprotease deleted strains can be used, described in Joly et al, (1998), supra; georgiou et al, U.S. Pat. nos. 5,264,365; georgiou et al, U.S. Pat. nos. 5,508,192; details are described in Hara et al, microbial Drug Resistance,2:63-72 (1996).
Coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperones may be used as host cells in expression systems encoding the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein.
d) Protein purification
The protein constructs produced herein are further purified to obtain a substantially homogeneous formulation for further analysis and use. Standard protein purification methods known in the art can be employed. The following procedure is an example of a suitable purification procedure: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reversed phase liquid chromatography HPLC, silica or cation exchange resin (such as DEAE) chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration, e.g., sephadex G-75.
In some embodiments, protein a immobilized on a solid phase is used for immunoaffinity purification of a protein construct comprising an Fc region as described herein. Protein a is a 42kDa surface protein from staphylococcus aureus that has a high binding affinity for Fc-containing structures, e.g., comprising an IL-2 mutant-Fc fusion protein as described herein. Lindmark et al, (1983) J.Immunol. Meth.62:1-1. The solid phase for immobilizing protein a preferably comprises a glass or silica surface column, more preferably a controlled pore glass column or a silicic acid column. In certain applications, the chromatographic column is coated with a reagent, such as glycerol, to prevent non-specific adhesion of contaminants. The solid phase is then washed to remove contaminants that do not specifically bind to the solid phase. Finally, the target protein construct is recovered from the solid phase by elution.
2. Recombination products of eukaryotic cells
For eukaryotic expression, the vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
a) Signal sequence element
The vector for eukaryotic hosts may also be an insert encoding a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence of choice is preferably a sequence recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, it is useful to obtain both mammalian signal sequences as well as viral secretion leads, e.g., herpes simplex gD signals. The DNA of the precursor region is linked in frame to DNA encoding the protein construct of the present application.
b) Origin of replication
In general, mammalian expression vectors do not require an origin of replication element (the SV40 origin is typically used only because it contains an early promoter).
c) Selection Gene element
Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode the following proteins: (a) proteins that are resistant to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic proteins, or (c) proteins that provide key nutrients that cannot be provided by complex media, such as genes encoding bacillus D-alanine racemase.
One example of an alternative is to use a drug to prevent growth of the host cell. Those cells successfully transformed with the heterologous gene produce a protein that is resistant to the drug and thus survive the selection regimen. Examples of such advantageous selections use the drugs neomycin, mycophenolic acid and hygromycin.
Another example of a selectable marker suitable for use in mammalian cells is one that recognizes cells capable of carrying nucleic acid encoding a protein construct described herein, such as DHFR, thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.
For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is the Chinese Hamster Ovary (CHO) cell line that lacks DHFR activity (e.g., ATCC CRL-9096).
Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3' -phosphotransferase (APH), particularly wild-type host containing endogenous DHFR, can be selected by cell growth in a medium containing a selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.4,965,199.
d) Promoter element
Expression and cloning vectors typically contain a promoter that is recognized by the host and is operably linked to nucleic acid encoding a desired polypeptide sequence. Almost all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream of the transcription initiation point. Another sequence found 70 to 80 bases upstream of many gene transcription initiation sites is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic organisms there is an AATAAA sequence, which may be a signal that increases the poly A tail at the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.
Polypeptide transcription in mammalian host cell vectors is controlled by a promoter, e.g., by a promoter obtained from the viral genome, such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus, and most preferably simian virus 40 (SV 40), a promoter from a heterologous mammal, e.g., an actin promoter or an immunoglobulin promoter, from a heat shock promoter, provided that such promoters are compatible with the host cell system.
The SV40 early and late promoters are conveniently available as SV40 restriction fragments that also comprise the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is readily available as a HindIII E restriction fragment. U.S. Pat. No.4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. Improvements to this system are detailed in U.S. Pat. No.4,601,978. See Reyes et al, nature 297:598-601 (1982) regarding the expression of human interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the rous sarcoma virus long terminal repeat may be used as a promoter.
e) Enhancer element
Transcription of the DNA encoding the protein constructs of the present application by higher eukaryotes is typically increased by inserting an enhancer sequence into the vector. Many enhancer sequences (globin, elastase, albumin, alpha-fetoprotein and insulin) have been found in mammalian genes. However, enhancers of eukaryotic viruses are commonly used. Examples include SV40 enhancer at the end of the replication origin (100-270 bp), cytomegalovirus early promoter enhancer, polyoma enhancer at the end of the replication origin, and adenovirus enhancers. See Yaniv, nature 297:17-18 (1982) on enhancing elements that activate eukaryotic promoters. Enhancers may be spliced into the vector 5' or 3' to the polypeptide coding sequence, but are preferably located 5' to the promoter.
f) Transcription termination element
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or other nucleated cells of multicellular organisms) also contain sequences necessary for the termination of transcription and for stabilizing mRNA. These sequences are typically available from the 5 'untranslated region, occasionally the 3' end, of eukaryotic or viral DNA or cDNA. These regions comprise nucleic acid fragments which are transcribed as polyadenylation fragments in the untranslated portion of the mRNA encoding the polypeptide. One suitable transcription termination element is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vectors disclosed therein.
g) Selection and transformation of host cells
Suitable host cells for cloning or expressing the DNA in the vectors described herein include the higher eukaryotic cells described herein, including vertebrate host cells. The culture propagation (tissue culture) of vertebrate cells has become a routine procedure. Useful mammalian host cell lines are exemplified by the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); COS fibroblast-like cell lines derived from monkey kidney tissue; human embryonic kidney lines (293 or 293 cell subclones for suspension culture growth, graham et al, J.Gen. Virol.36:59 (1977)); milk hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc.Natl. Acad.sci.usa 77:4216 (1980)); mouse Sertoli cells (TM 4, mather, biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC-ccl 2); canine kidney cells (MDCK, ATCC-ccl 34); buffalo-rat hepatocytes (BRL 3a, atcc CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,ATCC CCL51); TR1 cells (Mather et al, annals N.Y. Acad. Sci.383:44-68 (1982)); MRC5 cells; FS4 cells and human liver cancer cell lines (Hep G2).
Host cells are transformed with the above-described expression vectors or cloning vectors to produce protein structures and cultured in conventional nutrient media suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences.
h) Culturing host cells
Host cells for the production of the protein constructs of the present application can be cultured in a variety of media. Commercial media such as Ham's F (Sigma), minimal media ((MEM), sigma), RPMI-1640 (Sigma) and Dulbecco's modified Eagle's Medium ((DMEM), sigma) are suitable for culturing host cells. Furthermore, ham et al, meth.Enz.58:44 (1979), barnes et al, anal biochem.102:255 (1980), U.S. Pat.No.4,767,704;4,657,866;4,927,762;4,560,655 or 5,122,469, WO 90/03430, WO 87/00195 or U.S. Pat.Re.30,985 can be used as a medium for host cells. Any of these media may be supplemented with hormones and/or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium and phosphate), buffers (e.g., HEPE), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin) TM Drugs), trace elements (defined as the final concentration typically present in the micromolar rangeInorganic compounds of (c) and glucose or an equivalent energy source. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for expression by the host cell and will be apparent to one of ordinary skill.
VI pharmaceutical composition
The present application further relates to pharmaceutical compositions comprising any of the IL-2 mutants described herein or fusion proteins comprising an IL-2 mutant, or pharmaceutical compositions comprising nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising an IL-2 mutant, or pharmaceutical compositions comprising vectors encoding nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising an IL-2 mutant. These compositions may optionally further comprise pharmaceutically acceptable carriers and/or excipients. In some embodiments, the adjuvant comprises an excipient and/or a stabilizer. The pharmaceutical compositions can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an IL-2 mutant or a fusion protein comprising an IL-2 mutant of the desired purity as described herein with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed (1980)).
Method for treating diseases
The IL-2 mutants described herein or fusion proteins comprising IL-2 mutants or compositions (e.g., pharmaceutical compositions) comprising the same may be used for a variety of purposes, such as diagnostics, molecular detection, and therapy. In some embodiments, the present application relates to a method of treating a disease in an individual (e.g., a human) comprising administering to the individual an effective dose of any of the IL-2 mutants, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same as described herein. In some preferred embodiments, the fusion protein comprising an IL-2 mutant is a fusion protein of an IL-2 mutant with Fc. In some embodiments, the disease is associated with modulation of Treg cells in a subject. In some embodiments, the disease comprises an inflammatory disease or an autoimmune disease. In some embodiments, the IL-2 mutant or fusion protein comprising the IL-2 mutant or a pharmaceutical composition comprising the IL-2 mutant or fusion protein thereof is administered by intravenous injection, intramuscular injection, or subcutaneous injection.
In some implementations, the disease includes lupus, graft versus host disease, hepatitis C-induced vasculitis, type i diabetes, type ii diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophy epidermolysis bullosa, and behcet's disease.
For example, in some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, vectors and host cells comprising such nucleic acid, or a pharmaceutical composition comprising the same described herein. Wherein the IL-2 mutant comprises a mutation in R83 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of R83 is R83L, R a or R83N. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same, as described herein. Wherein the IL-2 mutant further comprises a mutation in D20 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of D20 is D20N. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of R83L and D20N mutation.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same, as described herein. Wherein the IL-2 mutant further comprises mutations in H16 and N71 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of H16 and N71 are H16N and N71R, respectively. In some embodiments, the IL-2 mutant, which comprises relative to the wild-type IL-2 amino acid sequence of R83L, H N and N71R mutation.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same, as described herein. Wherein the IL-2 mutant further comprises mutations in N71 and N88 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of N71 and N88 are N71S and N88D, respectively. In some embodiments, the IL-2 mutant, which comprises relative to the wild-type IL-2 amino acid sequence of R83A, N S and N88D mutation.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same, as described herein. Wherein the IL-2 mutant further comprises mutations in L66 and V91 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L66 and V91 are L66F and V91E, respectively. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of R83N, L F and V91E mutation.
In other embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same, as described herein. Wherein the IL-2 mutant comprises mutations in E67 and S75 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of E67 is E67I or E67G, and the mutation of S75 is S75P. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of E67I and S75P mutation. In some embodiments, the IL-2 mutant, which contains relative to the wild-type IL-2 amino acid sequence of E67G and S75P mutation. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In other embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same, as described herein. Wherein the IL-2 mutant comprises mutations in L17 and P82 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L17 and P82 are L17V and P82L, respectively. In some embodiments, the amino acid sequence of human wild-type IL-2 is set forth in SEQ ID NO. 1.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, or a fusion protein comprising an IL-2 mutant. Wherein the IL-2 mutant or fusion protein thereof further comprises a mutation relative to the human wild-type IL-2 amino acid sequence C125 in addition to the mutations described above. In some embodiments, the mutation of C125 is C125S or C125A.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, or a fusion protein comprising an IL-2 mutant. The IL-2 mutants comprise an amino acid sequence as set forth in any of SEQ ID NOs 5-11 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with the amino acid sequence as set forth in any of SEQ ID NOs 5-11. In some embodiments, the fusion protein comprising an IL-2 mutant is a fusion protein of an IL-2 mutant with Fc. In some embodiments, the fusion protein of the IL-2 mutant and Fc is in a bivalent form. In some embodiments, the IL-2 mutant and Fc bivalent fusion protein comprises an amino acid sequence as shown in any of SEQ ID NOs 53-59 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence shown in any of SEQ ID NOs 53-59. In some embodiments, the fusion protein of the IL-2 mutant and Fc is in a monovalent form. In some embodiments, the disease or disorder is selected from lupus, graft versus host disease, hepatitis C-induced vasculitis, type i diabetes, type ii diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, and behcet's disease.
In some embodiments, the present application relates to a method of treating an individual having an inflammatory disease or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, fusion protein comprising an IL-2 mutant, nucleic acid encoding an IL-2 mutant or fusion protein thereof, vector and host cell comprising such nucleic acid, or pharmaceutical composition comprising the same, as described herein, and further comprising administering to the individual one or more additional active ingredients that are required for the particular indication being treated, preferably with active ingredients that do not affect the therapeutic effect between each other. For example, when used to treat autoimmune diseases or to alleviate or prevent autoimmune reactions following organ transplantation, can be used in combination with immunosuppressants. In some embodiments, the immunosuppressant is selected from the group consisting of: glucocorticoids; azathioprine; cyclosporin a; methotrexate; an anti-CD 3 antibody; or an anti-TNF-alpha antibody, etc.
The administration of the IL-2 mutants, fusion proteins comprising IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same, as described herein, may be performed in any known and convenient manner, including by injection or infusion. The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus injections or prolonged infusion in an appropriate manner.
The dosage and desired drug concentration of the pharmaceutical compositions of the present application may vary depending on the particular application. Determination of the appropriate dosage or route of administration is well within the skill of the ordinary artisan. Animal experiments provide reliable guidance for determining effective dosages for human therapy. Effective dosages of the interspecies can be analogized according to the principles of Mordinti, J.and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, yacobi et al, eds, pergamon Press, new York 1989, pp.42-46.
When IL-2 mutants, fusion proteins comprising IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same are used for in vivo administration, a therapeutically effective amount may depend, for example, on the therapeutic context and goal. Those of ordinary skill in the art will appreciate that the appropriate dosage level for treatment will vary depending on the molecule being delivered, the indication for which the IL-2 mutant is being used for treatment, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. Within the scope of the present application, different formulations will be effective for different treatments and different diseases, and the manner of administration intended for treating a particular organ or tissue may be different from that intended for another organ or tissue. In addition, the dosages may be administered by one or more separate administrations or continuous infusions. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the disease symptoms reach the desired degree of inhibition. Alternatively, other dosage regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.
VIII, products and kit
The application further relates to kits, unit doses and articles of manufacture comprising any of the IL-2 mutants described herein, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same. In some embodiments, the kits comprise any of the pharmaceutical compositions described herein, and preferably provide instructions for their use, as for treating a disease described herein (e.g., an autoimmune disease).
Kits of the present application include one or more containers comprising an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, vectors and host cells comprising such nucleic acid, or pharmaceutical compositions comprising the same, e.g., for treating a disease. For example, instructions comprising instructions describing administration of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, vectors and host cells comprising such nucleic acid, or pharmaceutical compositions comprising the same to treat a disease (e.g., an autoimmune disease). The kit may further comprise a description of selecting an individual (e.g., a human) suitable for treatment based on identifying whether the individual has a disease and a disease stage. Instructions relating to the use of IL-2 mutants, fusion proteins comprising IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising the same, typically include information about the dosage, dosing schedule and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the present application are typically written instructions on labels or pharmaceutical instructions (e.g., paper included in the kit), but machine readable instructions (e.g., instructions stored on a magnetic or optical disk) are also acceptable. The kits of the present application employ suitable packaging. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed mylar or plastic bags), and the like. Packages such as infusion devices, e.g., micropumps, are also contemplated for use in connection with certain devices. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such a nucleic acid, or a pharmaceutical composition comprising the same, as described herein. The container may further comprise a second pharmaceutically active agent. The kit may optionally provide additional components such as buffers and interpretation information. Generally, a kit comprises a container and a label or a pharmaceutical instruction on or associated with the container.
For example, in some embodiments, the kit comprises: a) Comprising any of the IL-2 mutants described herein, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or fusion proteins thereof, vectors and host cells comprising the nucleic acids, or pharmaceutical compositions comprising the same, and b) at least one additional agent in an amount effective to enhance the effect (e.g., therapeutic effect) of the IL-2 mutants, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or fusion proteins thereof, vectors and host cells comprising the nucleic acids, or pharmaceutical compositions comprising the same.
In some embodiments, the kit comprises: a) A nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same comprising any of the IL-2 mutants, fusion proteins comprising an IL-2 mutant or a fusion protein thereof described herein, and b) instructions for administering to an individual a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising the same for treating an autoimmune disease (e.g., lupus).
In some embodiments, the kit comprises: a) a nucleic acid comprising any one of the IL-2 mutants described herein, a fusion protein comprising the IL-2 mutants, a nucleic acid encoding the IL-2 mutants or fusion proteins thereof, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same, and b) at least one additional agent capable of enhancing the effect (e.g., therapeutic effect) of the IL-2 mutants, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or fusion proteins thereof, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same, and c) instructions for administering the IL-2 mutants, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or fusion proteins thereof, vectors comprising the same, or pharmaceutical compositions and other substances to an individual for treating an autoimmune disease (e.g., lupus). The IL-2 mutant, fusion protein comprising the IL-2 mutant, nucleic acid encoding the IL-2 mutant or fusion protein thereof, vector and host cells comprising such nucleic acid, or pharmaceutical compositions and other substances comprising the same may be present in separate containers or in the same container. For example, the kit may comprise one particular composition or two or more compositions, wherein one composition comprises an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or fusion protein thereof, a vector and host cell comprising such a nucleic acid, or a pharmaceutical composition comprising the same, and the other composition comprises another agent.
In some embodiments, the kit comprises a nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or fusion protein thereof (e.g., a fusion protein of an IL-2 mutant with Fc). In some embodiments, the kit comprises: a) A nucleic acid (or a set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (e.g., a fusion protein of an IL-2 mutant with Fc), and b) a host cell expressing the nucleic acid (or the set of nucleic acids). In some embodiments, the kit comprises: a) A nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or fusion protein thereof (e.g., a fusion protein of an IL-2 mutant with Fc), and b) instructions for use, suitable for: i) Expressing the IL-2 mutant or fusion protein thereof in a host cell, ii) preparing a composition comprising the IL-2 mutant or fusion protein thereof, and iii) administering the composition comprising the IL-2 mutant or fusion protein thereof to the individual to treat an autoimmune disease (e.g., lupus). In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or fusion protein thereof (e.g., a fusion protein of an IL-2 mutant with Fc), b) a host cell expressing the nucleic acid (or set of nucleic acids), and c) instructions for use, suitable for: i) Expressing the IL-2 mutant or fusion protein thereof in a host cell, ii) preparing a composition comprising the IL-2 mutant or fusion protein thereof, and iii) administering the composition comprising the IL-2 mutant or fusion protein thereof to the individual to treat an autoimmune disease (e.g., lupus).
In another aspect, the present application is directed to articles, including vials (e.g., sealed vials), bottles, cans, flexible packages, and the like. The article of manufacture comprises a container and a label or a pharmaceutical instruction on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of a variety of materials, such as glass or plastic. In general, the container holds a composition that is effective in treating the diseases or disorders described herein (e.g., autoimmune diseases) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or the pharmaceutical instructions indicate that the composition is used to treat a specific condition in an individual. The label or the pharmaceutical instructions further comprise instructions for administering the composition to the subject. The tag may be directed to instructions for reconstruction and/or use. The container containing the pharmaceutical composition may be a multi-use vial, allowing repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The pharmaceutical instructions refer to instructions that are typically contained in commercial packages of therapeutic products, including indications, usage, dosages, administration, contraindications and/or warnings concerning the use of such therapeutic products. In some embodiments, the instructions indicate that the treatable condition includes lupus, graft versus host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophy epidermolysis bullosa, and Behcet's disease. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. From a commercial and user perspective, other desirable materials may be further included, including other buffers, diluents, filters, needles and syringes.
The kit or article of manufacture comprises a plurality of unit doses of the pharmaceutical composition and instructions for use, packaged in an amount sufficient for storage and use in a pharmacy, such as a hospital pharmacy and a compound pharmacy.
Detailed Description
Those skilled in the art will recognize several embodiments that are possible within the scope and spirit of the present application. The present application will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present application but should not be construed as in any way limiting its scope.
Example 1: design and expression of human wild type IL-2 and IL-2 mutants
Construction and expression of wild-type IL-2:
the nucleic acid sequence for encoding the wild type IL-2 (the amino acid sequence of which is shown as SEQ ID NO: 1) is synthesized by Nanjing Jinsri biotechnology Co., ltd, and enzyme cutting sites are respectively introduced at the 5 'end and the 3' end of the synthesized wild type IL-2 nucleic acid sequence. And (3) after the synthesized nucleic acid fragment is digested, the nucleic acid fragment is connected with a vector fragment subjected to the same double digestion, and then the wild type IL-2 expression vector is obtained. In addition, on the basis of the expression vector, a C125S mutation (the amino acid sequence is shown as SEQ ID NO:2, and the mutation can prevent cysteine mismatch or aggregation) is introduced at the 125 th position of the wild IL-2, so that the expression vector of the IL-2C125S mutant is obtained; C125A mutation (the amino acid sequence is shown as SEQ ID NO:3, the mutation can promote effective folding and improve expression) is introduced at the 125 th position of the wild IL-2, and the expression vector of the IL-2C125A mutant is obtained. The two IL-2 mutants did not alter their binding to the IL-2 receptor compared to wild-type IL-2.
Design, screening and expression of IL-2 mutants:
through research, design and FACS screening, a series of IL-2 mutants were finally obtained, the screening procedure was as follows:
firstly, preparing the biotinylated IL-2Ralpha and IL-2Rbeta gamma required in the screening process:
preparation of IL-2Rα: the extracellular region coding sequence of IL-2Rα (Uniprot ID: P01589) was codon optimized and then subjected to total gene synthesis by Nanjing Jinsri Biotechnology Co. The synthetic gene sequence was constructed into an expression vector carrying the His-tag gene and expressed in 293F cells. According to the instructions, IL-2Rα was purified using a (Ni) nickel column. Briefly, immobilized Metal Affinity Chromatography (IMAC) was performed using Ni-NTA from QIAGEN. First buffer A1 (50 mM Na 3 PO 4 0.15M NaCl,pH 7.2) equilibrated nickel column, flow rate 150cm/h. The pH of the culture supernatant was adjusted to 7.2, and the sample was applied at room temperature at a flow rate of 150cm/h. Subsequently, the column was again equilibrated with 6 column volumes of A1 buffer at a flow rate of 150cm/h. Finally, elution was performed with 10 column volumes of 50mM PB solution (containing 0.15M NaCl and 0.2M imidazole, pH 7.2) and the eluate was collected.
Preparation of IL-2Rβγ: IL-2Rβγ used in the examples of the present application is an Fc heterodimer (IL-2Rβγ -Fc heterodimer) constructed by KIH (Knob-into-Hole) technology, and the extracellular region coding sequences of IL-2Rβ (Uniprot ID: P14784) and IL-2Rγ (Uniprot ID: P31785) were cloned and fused to the N-terminus of Fc Hole molecule (SEQ ID NO: 61) and Fc Knob molecule (SEQ ID NO: 60), respectively, for the preparation of IL-2Rβγ -Fc heterodimer. IL-2Rβ -Fc hole and IL-2Rγ -Fc knob were constructed into pcDNA3.1 eukaryotic expression vectors, respectively, and 293F cells were co-transfected to collect cell culture fluids. IL-2Rβγ -Fc heterodimers with protein A affinity And chromatography and gel filtration Superdex 200 column chromatography. Briefly, the culture broth of 293F cells was purified by protein A affinity chromatography, and the protein A column was first equilibrated with 50mM PBS buffer containing 0.15M NaCl (pH 7.2) at a flow rate of 150cm/h and a volume of 6 times the column volume. The supernatant of the medium (pH adjusted to 7.2) was passed through the column at a flow rate of 150 cm/h. After further equilibration, the column was washed with 50mM sodium citrate (pH 3.5) and the eluate was collected. Then, the IL-2Rβγ -Fc heterodimer is obtained after purification by gel filtration Superdex 200 column chromatography.
Biotinylated markers for IL-2Rα and IL-2Rβγ: IL-2Rα and IL-2Rβγ were biotinylated using a biotinylated Ligase according to the Biotin-Protein Enzyme/BirA Enzyme (GeneCopoeia cat#BI001) kit protocol. Briefly, IL-2Rα and IL-2Rβγ protein solutions were added with the corresponding Biotin Ligase BufferA, buffer B and birA ligases, respectively, and incubated at 30℃for 2 hours. The biotinylation efficiency was detected by ELISA method, confirming the success of the biotin labelling.
FACS screening: the designed IL-2 mutant library is constructed into a yeast display system, and the flow cytometry fluorescence sorting technology (FACS) is used for screening mutants with reduced affinity to IL-2 Rbeta gamma but basically maintained unchanged or not obviously weakened affinity to IL-2 Ralpha from the library. FACS screening methods are well known to those skilled in the art, and first 4 rounds of FACS screening are performed using biotinylated IL-2Rβγ (Biotin-IL-2Rβγ) to enrich a yeast positive population with reduced affinity for IL-2Rβγ. Briefly, the yeast cells induced in SGCAA medium were precipitated, 1mL of PBSM was added, 14000g was centrifuged for 30sec, and the supernatant was discarded to wash the cell pellet. The yeast cells were resuspended in PBSM buffer containing Biotin-IL-2Rβγ and incubated for 1h at room temperature. After washing, cells were stained with strepavidin-PE (BD Biosciences, 554061) and anti-V5 tag antibody [ iFluor 647] (GenScript, A01805-100) and sorted. The enriched positive yeast population with reduced binding to Biotin-IL-2Rβγ was then used to screen for monoclonal antibodies with substantially unchanged or no significant reduction in affinity for biotinylated IL-2Rα (Biotin-IL-2 Rα) using the FACS method. And finally, sequencing the monoclonal obtained by screening, constructing an expression vector carrying the His tag gene, preparing a sample and performing corresponding biological evaluation.
By analyzing the sequencing results of the monoclonal, specific information of each mutant is shown in Table 7, "+" represents simultaneous mutation.
TABLE 7
Mutant | Mutation site |
IL2-mut1 | R83L+D20N+C125S |
IL2-mut2 | R83L+H16N+N71R+C125S |
IL2-mut3 | L17V+P82L+C125S |
IL2-mut4 | R83A+N71S+N88D+C125S |
IL2-mut5 | R83N+L66F+V91E+C125S |
IL2-mut6 | E67I+S75P+C125S |
IL2-mut7 | E67G+S75P+C125S |
Expression and purification of IL-2 mutants: the gene coding sequence of the IL-2 mutant is obtained through PCR, and is connected to the expression vector carrying His tag genes which are subjected to the same double enzyme digestion after enzyme digestion, so as to obtain the recombinant expression vector of each IL-2 mutant. The recombinant expression vector is transfected into 293F cells and subjected to 5% CO at 37 DEG C 2 After 5 days of culture at 120rpm, the cell culture supernatant was collected and subjected to the procedure according to the (Ni) nickel column purification protocol to complete purification of the IL-2 mutant. The specific operation steps are as described above.
Whether the finally obtained IL-2 mutants are able to selectively activate Treg cells is further examined by the following experiments. The ability of wild-type IL-2 or IL-2 mutants to "activate Treg cells" can be determined by methods well known in the art or by methods disclosed in the examples herein, including, but not limited to, for example, by comparing the EC50 values for IL-2 mutants to IL-2rβγ or IL-2rβγ, the greater the difference between the two (e.g., the greater the EC50 value for IL-2 mutants to IL-2rβγ while they remain bound to IL-2rβγ), the better the IL-2 mutants are shown to be selective for Treg cells; for another example, the determination can be made by the change in the number of Treg cells after stimulation with an IL-2 mutant, e.g., by flow cytometry to measure the change in the number of Treg cells in a mixed cell population, etc.
Example 2: determination of binding affinity of IL-2 mutants to IL-2Rαβγ and IL-2Rβγ
HEK-BLUE TM Experimental principle of cell line detection:
HEK-Blue TM the IL-2 cell line (InvivoGen, cat#hkb-IL 2) is a HEK293 cell line stably transfected with the human IL-2Rα (CD 25), IL-2Rβ (CD 122) and IL-2Rγ (CD 132) genes. Another cell line HEK-Blue TM CD122/CD132 (InvivoGen, cat#hkb-IL2 bg) expresses only IL-2Rβ (CD 122) and IL-2Rγ (CD 132), but not IL-2Rα (CD 25). In addition, both cell lines stably express human JAK3 and STAT5 genes and have complete IL-2 signal paths; in addition, the expression of SEAP reporter gene induced by STAT5 is introduced, and after IL-2 stimulation, QUANTI-Bl can be usedue TM The solution (InvivoGen, cat#rep-qbs) was tested for STAT 5-induced SEAP expression levels. Wherein HEK-Blue TM IL-2 cell lines are used to detect binding of IL-2 or mutants thereof to IL-2Rαβγ. HEK-Blue TM The CD122/CD132 cell line is used to detect binding of IL-2 or a mutant thereof to IL-2Rβγ. When IL-2 or a mutant thereof is added to the above cell line, IL-2 or a mutant thereof is capable of binding to IL-2 high affinity receptor IL-2Rαβγ or medium affinity receptor IL-2Rβγ, activating intracellular Janus family tyrosine kinase signaling cascades (JAK 1 and JAK 3), initiating signal transduction and phosphorylation of transcription activator 5 (STAT 5), thereby inducing expression of SEAP. Using the substrate QUANTI-Blue TM The expression level of SEAP is detected, that is, the OD value is read at 630nm, and the corresponding EC50 value is calculated to reflect the condition that STAT5 is activated after IL-2 or a mutant thereof binds to IL-2Rαβγ or IL-2Rβγ. Wild-type human IL-2 was used as a control in this experiment.
By HEK-Blue TM CD122/CD132 cell line to determine EC50 value of IL-2 mutant binding to IL-2Rβγ, HEK-Blue was used TM IL-2 cell lines to determine IL-2 mutants and IL-2Rαβγ binding of EC50 value, the ratio between the two, HEK-IL-2Rβγ EC50/HEK-IL-2Rαβγ EC50, defined as IL-2 selective activation of Treg cells therapeutic safety window. The safety window value can reflect the selective targeting of the IL-2 mutant to Treg cells, and the higher the value, the stronger the selective targeting of the IL-2 mutant to Treg cells.
HEK-BLUE TM Cell line detection experiments:
firstly, diluting the IL-2 mutant to be detected to 800 mug/mL by using a detection culture solution, and then diluting the IL-2 mutant to be detected to 96-well U-shaped plates (Coring) in a 4-fold gradient manner, wherein the total concentration is 12, and the IL-2 mutant is ready for use. The digested cells (HEK-Blue) TM IL-2 cell line or HEK-Blue TM CD122/CD132 cell line) suspension to 2.8x10 5 After cells/mL, 180. Mu.L of cell suspension/well was inoculated into 96-well flat bottom cell culture plates, 20. Mu.L of diluted IL-2 or IL-2 mutant was added to each well, gently shaken and mixed with the cell suspension, and then placed at 37℃with 5% CO 2 Culturing in an incubator for 24 hours. Cell culture by pipetting 20. Mu.L/well from the cell plateThe supernatant was transferred to another new 96-well flat bottom cell culture plate and 180. Mu.L of QUANTI-Blue was added per well TM The detection solution is evenly mixed, incubated for 1h at 37 ℃, and absorbance value at 620nm is read. The EC50 value of the sample to be tested was calculated by counting and calculating the EC50 value of the sample to be tested using GraphPad Prism 8.0.1 by converting the concentration value into logarithm, i.e., x=log (X), and then calculating the EC50 value of the sample to be tested according to a log (agonist) vs. response-Variable slope (four parameters) fitting curve.
HEK-BLUE TM Experimental results of cell line detection:
the results of the detection of IL-2 mutants are shown in Table 8. The results show that both IL-2 mutants of the present application are able to bind to IL-2rαβγ and that their EC50 value for binding to IL-2rβγ is increased, the affinity is reduced, and the safety window for their treatment is significantly increased compared to wild-type IL-2, indicating that these mutants have excellent Treg cell selection targeting.
TABLE 8IL-2 mutant HEK-BLUE TM Detection result
( And (3) injection: * The marked values are due to the low ability of these mutants to activate IL-2Rβγ cells, and the exact EC50 values cannot be obtained by fitting within the concentration range used in the experiments )
Example 3: expression and purification of IL-2 mutants and FC fusion proteins
The IL-2 mutant is connected with IgG1 Fc LALA through connecting peptide, and the formed fusion protein of IL-2 and Fc comprises two construction forms, namely a bivalent form and a monovalent form. Wherein IgG1 Fc LALA refers to IgG1 Fc having L234A and L235A mutations, numbered according to the EU Kabat numbering system (Kabat et al, J.biol. Chem.252:6609-6616 (1977); kabat et al, U.S. Dept. Of Health and Human Services, "Sequences ofproteins ofimmunological interest" (1991)).
A bivalent form of a fusion protein of an IL-2 mutant and Fc, comprising two IL-2 mutant molecules in total: it comprises two identical monomers, each of which has an IL-2 mutant linked to the N-or C-terminus of Fc. An exemplary structure of one such form is shown in FIG. 1A (IL-2-Fc), which consists of two identical monomers, each comprising from N-terminus to C-terminus: IL-2 mutants (SEQ ID NOs: 5-11), connecting peptides (SEQ ID NO: 29) and IgG1 Fc LALA (SEQ ID NO: 12). Another exemplary structure of this type is shown in fig. 1B (Fc-IL-2), which consists of two identical monomers, each comprising from N-terminus to C-terminus: igG1 Fc LALA (SEQ ID NO: 12), a connecting peptide (SEQ ID NO: 29) and IL-2 mutants (SEQ ID NOs: 5-11).
The IL-2 mutant and the monovalent form of the Fc fusion protein together comprise an IL-2 mutant molecule: it comprises two different monomers, in one of which an IL-2 mutant is fused to the N-or C-terminus of one of the Fc subunits, the other monomer being the other Fc subunit, the two asymmetric Fc subunits being paired with one another by means of knob and hole mutations. In a preferred form, it comprises two different monomers, in one of which the IL-2 mutant is fused to the C-terminus of one of the subunits of the Fc via a linker peptide, which monomers comprise, from N-terminus to C-terminus, igG1 Fc LALA knob (SEQ ID NO: 18), linker peptide (SEQ ID NO: 29) and IL-2 mutant (SEQ ID NOs: 5-11); and the other monomer is IgG1 Fc LALA hole (SEQ ID NO: 19). An exemplary structure of the fusion protein is shown in FIG. 2.
Specific construction procedure of IL-2 mutant and Fc fusion protein (taking the bivalent fusion protein form of IL-2 mutant and Fc as an example): the recognition sites for the restriction endonucleases HindIII and XhoI were introduced by PCR, the nucleic acid sequence encoding IgG1 Fc LALA was ligated with the nucleic acid sequence encoding IL-2 mutant by the oligonucleotide sequence encoding GGGGS and constructed into pcDNA3 1 eukaryotic expression vector, and 293F cells were transfected at 37℃with 5% CO 2 After 5 days of culture at 120rpm, the culture broth was collected and purified by protein A affinity chromatography followed by gel filtration Superdex 200 column chromatography. The specific procedure is as described in example 1.
Example 4: in vitro biological Activity assay of IL-2 mutants
When IL-2 binds to its cell surface receptor, it activates the JAK-STAT signaling pathway, phosphorylating STAT 5. STAT5 phosphorylation is an essential step in the IL-2 signaling pathway, and STAT5 is phosphorylated and then enters the nucleus to participate in downstream signaling processes. Thus, phosphorylated STAT5 signals in cells can reflect IL-2 activation.
CD8 + T lymphocytes express IL-2Rβγ on their surface, but not IL-2Rα. Treg cell surface expresses both IL-2Rβγ and IL-2Rα. Determination of CD8 by flow cytometry + Levels of STAT5 phosphorylation (pSTAT 5) in T lymphocytes and Treg cells, activating CD8 with IL-2 mutants in this indirect response + Activity of T lymphocytes and Treg cells (see, e.g., peterson LB, et al J Autoimmun.2018Dec; 95:1-14).
The specific operation steps are as follows: PBMC cells (cat#pb050C, synbiotics limited, shanghai seconds) were resuscitated one day ahead, PBMCs were collected overnight, cells were washed with PBs, and collected by centrifugation. Counting after mixing with 2% complete culture medium, adding 60 μl of cell suspension per well, about 5-6X10 5 The individual cells are inoculated in a cell culture plate and put into a cell culture box for standby. The samples to be tested included the exemplary IL-2 mutant and Fc bivalent fusion proteins Fc-IL2-mut1, fc-IL2-mut3, fc-IL2-mut4 and Fc-IL2-mut6. After the samples were respectively subjected to gradient dilution, the cells were stimulated for 30min, the cell fixative was added, the cells were washed, then anti-CD 4 antibody (BD cat# 562424), anti-CD 25 antibody (Biolegend cat# 356128) and anti-CD 8 antibody (Biolegend cat# 300932) were added, incubated at 4℃for 30min in the absence of light, and 150. Mu.L/Kong Yuleng of Phosflow was added after washing the cells TM Perm Buffer III (BD cat# 558050), cells were washed with FACS Buffer, and after addition of 100. Mu.L/well PE Mouse Anti-Stat5 (pY 694) (BD cat# 612567) diluted with FACS Buffer and Anti-Foxp 3 antibody (Biolegend cat# 320112), incubated at 4℃for 30min in the absence of light. Cells were washed with FACS buffer, centrifuged and the supernatant discarded. After resuspension of cells with 100 μl/well FACS buffer, the assay was performed on-press. CD8 + The surface marker of T lymphocytes was CD8, CD4 was used in this experiment - CD8 + Is defined as CD8 + T lymphocytes. Surface markers for Treg cells typically include CD4, CD25 and Foxp3, whereCD4 was used in the experiments + CD25 + Foxp3 + Is defined as Treg cells. Data processing pSTAT5 fluorescence values (MFI) after treatment with IL-2 mutants at different concentrations for the two cell populations described above were counted using GraphPad Prism 8.0.1 and EC50 values were calculated. The fitting method comprises converting concentration value into logarithm, i.e. X=log (X), and then fitting curve according to log (agonist) vs. response-Variable slope (four parameters), calculating Treg cells and CD8 + EC50 values of T lymphocytes.
FIGS. 3A and 3C show the results of exemplary IL-2 mutant and Fc bivalent fusion proteins inducing STAT5 phosphorylation in Treg cells, and FIGS. 3B and 3D show exemplary IL-2 mutant and Fc bivalent fusion proteins in CD8 + Results of induction of STAT5 phosphorylation in T lymphocytes.
From the above results, it can be seen that: IL-2 mutant and Fc bivalent fusion protein can induce STAT5 phosphorylation in Treg cells, but in CD8 + IL-2 mutant and Fc bivalent fusion protein did not significantly induce STAT5 phosphorylation in T lymphocytes. The above results further demonstrate that the IL-2 mutants of the invention are capable of selectively activating Treg cells without activating CD8 + T lymphocytes have good Treg cell targeting and higher safety in the human body treatment process compared with wild type IL-2.
Example 6: determination of IL-2 mutant Activity in mice
To demonstrate the selectivity of IL-2 mutants for Treg cells in rodents, blood Treg cells and CD8 cells were detected separately by single administration of Fc-IL-2 mutant bivalent fusion protein in mice + Proliferation of T lymphocytes.
The specific operation is as follows: c57LB/6 mice were purchased from Vetong Liwa and kept in an environment without specific pathogens. All animal experiments were conducted according to the protocol and guidelines of the Institutional Animal Care and Use Committee (IACUC).
Experimental animal treatment: c57BL/6 mice were 9, SPF-rated, female, and divided into 3 groups of 3 mice each, wherein the positive control group mice were injected with Fc-AMG592 (SEQ ID NO: 62), the negative control group mice were injected with PBS, and the administration group mice were subcutaneously injected with the bivalent fusion protein Fc-IL2-mut1 of the exemplary IL-2 mutant and Fc.
First, a single dose was administered at 0 day, and each mouse was dosed at 1mg/kg. 100 mu L of blood was collected intravenously before 0 day and 3 days after administration, and animal numbers were marked on EDTA anticoagulation blood collection tubes.
The detection was performed using Trye-Nulear Transcription Factor Buffer Set (Biolegend cat# 424401). Briefly, 80. Mu.L of blood sample was added to 1mL of 1 XLyse/Fix buffer (BD Cat # 558049), mixed well, placed in an incubator at 37℃for 30min, and centrifuged at 400g X8 min to discard the supernatant. Detection with flow cytometry: cells were first fixed, 1X Transcription Factor Fix working solution in 1000. Mu.L/tube was added and incubated at room temperature for 40min in the absence of light. Then cell membrane perforation is carried out, supernatant is removed after 400g×8min centrifugation, 2 mL/tube of 1×Perm buffer is added for resuspension, and supernatant is removed after 400g×8min centrifugation; the cell membrane perforation step was repeated once. Cells in the tube were transferred to a V-shaped 96-well plate. Cells were resuspended in 1 XPerm buffer and anti-CD 4 antibody (Biolegend cat# 317429), anti-CD 25 antibody (Biolegend cat# 356128), anti-Foxp 3 antibody (Biolegend cat# 320112) and anti-CD 8 antibody (Biolegend cat# 344742) were added, respectively. After incubation for 1h at room temperature, centrifugation was performed at 600 g.times.8 min and the supernatant was discarded. The supernatant was removed by centrifugation and resuspended in 100. Mu.L of FACS buffer after washing twice with 200. Mu.L/well 1% BSA and detected on-machine.
Statistics were performed using the flow software CytExpert, lymphocyte population (P1) in-gate count 30000, calculating the fraction of Treg cells to CD4, respectively + Percentage of cell population and CD8 + T lymphocytes are a percentage of total lymphocytes. CD8 + The surface marker of T lymphocyte is CD8, and CD8 is used + Is defined as CD8 + T lymphocytes. The surface markers of Treg cells are CD4, CD25 and Foxp3, CD4 will be + CD25 + Foxp3 + Is defined as Treg cells.
As a result, as shown in FIG. 4A, the bivalent fusion protein Fc-IL2-mut1 of the exemplary IL-2 mutant and Fc was obtained at a dose of 1mg/kg in mice, compared with the negative control PBS solution groupThe mice can stimulate proliferation of Treg cells in vivo, and the effect is equivalent to that of a positive control Fc-AMG592 group. In addition, as shown in FIG. 4B, fc-IL2-mut1 stimulated proliferation of Treg cells while not stimulating CD8 + Proliferation of T lymphocytes suggests the specificity of stimulating expansion of Treg cells in vivo. And compared with the positive control group, it was against CD8 + T lymphocytes have less effect.
The above results indicate that the IL-2 mutant molecules of the invention are capable of specifically activating Treg cells in rodent animals without targeting CD8 + T cells have an effect.
Example 7: PK experiments of IL-2 mutants in mice
C57LB/6 mice were purchased from Vetong Liwa and kept in an environment without specific pathogens. All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) protocol and guidelines.
The specific procedures for the in vivo PK experiments in mice were as follows: 12C 57LB/6 mice were randomly divided into 2 groups of 6 male and female halves. Each group of mice was injected with a single tail vein with 1mg/kg of the bivalent fusion protein Fc-IL2-mut1 or Fc-IL2-mut3 of the exemplified IL-2 mutant and Fc, respectively. Anticoagulated whole blood was collected at 0 hours, 15 minutes, 5 hours, 24 hours, 72 hours, 168 hours, 240 hours, 336 hours, 432 hours, 600 hours before administration, respectively, and plasma was separated. The plasma drug concentration in each sample was determined by ELISA. Pharmacokinetic parameters were calculated for each animal using the winnonlin6.0 non-compartmental model (NCA).
Exemplary IL-2 mutants and Fc bivalent fusion protein Fc-IL2-mut1 or Fc-IL2-mut3 intravenous mice at 1mg/kg, the blood concentration gradually decreased with time. The results of PK experiments show that the divalent fusion proteins of IL-2 mutants and Fc of the present invention have good pharmacokinetic properties in mice (PK curves not shown).
Claims (29)
1. An IL-2 mutant comprising a mutation of R83 relative to the amino acid sequence of human wild-type IL-2; preferably, the mutation of R83 is R83L, R a or R83N.
2. The IL-2 mutant of claim 1, wherein the human wild-type IL-2 amino acid sequence is set forth in SEQ ID No. 1.
3. The IL-2 mutant of claim 1 or 2, further comprising a mutation of D20 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutation of D20 is D20N; more preferably, the mutant comprises the mutations R83L and D20N.
4. The IL-2 mutant of claim 1 or 2, further comprising mutations of H16 and N71 relative to the amino acid sequence of human wild-type IL-2; preferably, the mutations of H16 and N71 are H16N and N71R, respectively; more preferably, the mutant comprises the mutations R83L, H N and N71R.
5. The IL-2 mutant of claim 1 or 2, further comprising mutations of N71 and N88 relative to the amino acid sequence of human wild-type IL-2; preferably, the mutations of N71 and N88 are N71S and N88D, respectively; more preferably, the mutant comprises the mutations R83A, N S and N88D.
6. The IL-2 mutant of claim 1 or 2, further comprising mutations of L66 and V91 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations of L66 and V91 are L66F and V91E, respectively; more preferably, the mutant comprises the mutations R83N, L F and V91E.
7. An IL-2 mutant comprising mutations of E67 and S75 relative to the amino acid sequence of human wild-type IL-2; preferably, the mutation of E67 is E67I or E67G, and the mutation of S75 is S75P.
8. The IL-2 mutant according to claim 7, wherein the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO. 1.
9. The IL-2 mutant of claim 7 or 8, comprising E67I and S75P or E67G and S75P mutations relative to the human wild-type IL-2 amino acid sequence.
10. An IL-2 mutant comprising mutations of L17 and P82 relative to the amino acid sequence of human wild-type IL-2; preferably, the mutations of L17 and P82 are L17V and P82L, respectively; preferably, the amino acid sequence of the human wild-type IL-2 is shown in SEQ ID NO: 1.
11. The IL-2 mutant of any one of claims 1-10, further comprising a mutation of C125 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutation of C125 is C125S or C125A.
12. An IL-2 mutant comprising an amino acid sequence as set forth in any one of SEQ ID nos. 5-11 or a variant thereof having at least about 90% sequence homology with the amino acid sequence set forth in any one of SEQ ID nos. 5-11.
13. The IL-2 mutant of any one of claims 1-12, which has reduced binding affinity to IL-2rβγ and retains binding to IL-2rα as compared to human wild-type IL-2.
14. A fusion protein comprising the IL-2 mutant of any one of claims 1-13.
15. The fusion protein of claim 14, comprising Fc; preferably, wherein the Fc comprises a mutation capable of altering effector function; and/or mutations that can extend half-life; and/or a mutation capable of promoting dimer formation of a heterologous polypeptide; more preferably, the Fc is derived from human IgG1 or human IgG4.
16. The fusion protein of claim 15, wherein the Fc comprises:
(i) L234A and L235A mutations; and/or
(ii) A N297G mutation; and/or
(iii) A N297A mutation; and/or
(iv) L234A, L a and P331S mutations; and/or
(v) L234A, L235E, G237A, A S and P331S mutations, wherein the numbering is the EU numbering system.
17. The fusion protein of claim 15, wherein the Fc comprises S228P, F a and L235A mutations, wherein the numbering is the EU numbering system.
18. The fusion protein of any one of claims 15-17, wherein the Fc further comprises:
(i) S354C and T366W mutations; and/or
(ii) Y349C, T366S, L368A, and Y407V mutations, wherein the numbering is the EU numbering system.
19. The fusion protein of any one of claims 15-18, wherein the IL-2 mutant is linked to an Fc; preferably, the IL-2 mutant is linked to Fc via a linker peptide; preferably, the linker peptide comprises the amino acid sequence shown in any one of SEQ ID NOs 20-51; more preferably, the connecting peptide comprises the amino acid sequence GGGGS (SEQ ID NO: 29).
20. The fusion protein of any one of claims 15-19, wherein the IL-2 mutant is at the N-terminus and/or C-terminus of Fc.
21. A fusion protein comprising an IL-2 mutant, wherein the fusion protein comprises SEQ ID no
An amino acid sequence as shown in any one of NOs 53-59 or a variant thereof which hybridizes with SEQ ID NOs 53-
59 has at least about 80% sequence homology.
22. A nucleic acid encoding the IL-2 mutant of any one of claims 1-13 or the fusion protein of any one of claims 14-21.
23. A vector comprising the nucleic acid of claim 22.
24. A host cell comprising the vector of claim 23.
25. A method of making an IL-2 mutant or a fusion protein comprising an IL-2 mutant, comprising:
a) Culturing the host cell of claim 24 under conditions effective to express an IL-2 mutant or a fusion protein comprising an IL-2 mutant; and is also provided with
b) The expressed IL-2 mutant or fusion protein comprising the IL-2 mutant is obtained from a host cell.
26. A pharmaceutical composition comprising the IL-2 mutant of any one of claims 1-13 or the fusion protein of any one of claims 14-21, the nucleic acid of claim 22, the vector of claim 23, the host cell of claim 24, or the IL-2 mutant or the fusion protein comprising the IL-2 mutant prepared using the method of claim 25, and a pharmaceutically acceptable carrier and/or adjuvant.
27. A method of treating and/or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the IL-2 mutant of any one of claims 1-13, or the fusion protein of any one of claims 14-21, or the nucleic acid of claim 22, or the vector of claim 23, or the host cell of claim 24, or the IL-2 mutant or the fusion protein comprising the IL-2 mutant prepared using the method of claim 25, or the pharmaceutical composition of claim 26.
28. The method of claim 27, wherein the disease or condition comprises an inflammatory disease or an autoimmune disease.
29. The method of claim 27 or 28, wherein the disease or condition comprises lupus, graft versus host disease, hepatitis C-induced vasculitis, type i diabetes, type ii diabetes, multiple sclerosis, rheumatoid arthritis, atopic disease, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophy bullous epidermolysis, and behcet's disease.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2022106949734 | 2022-06-17 | ||
CN202210694973 | 2022-06-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117247444A true CN117247444A (en) | 2023-12-19 |
Family
ID=89133859
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310711032.1A Pending CN117247444A (en) | 2022-06-17 | 2023-06-15 | Interleukin-2 (IL-2) mutants and uses thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117247444A (en) |
-
2023
- 2023-06-15 CN CN202310711032.1A patent/CN117247444A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11981717B2 (en) | Engineered IL-2 Fc fusion proteins | |
JP7516254B2 (en) | IL-15/IL-15RA HETERODIMERIC FC FUSION PROTEINS AND USES THEREOF | |
US10501543B2 (en) | IL15/IL15Rα heterodimeric Fc-fusion proteins | |
TWI687435B (en) | Interleukin-2 muteins for the expansion of t-regulatory cells | |
KR20210010862A (en) | IL-15/IL-15Rα Fc-fusion protein and PD-1 targeting heterodimer fusion protein containing PD-1 antigen binding domain and uses thereof | |
US20110123440A1 (en) | Altered Antibody FC Regions and Uses Thereof | |
EP2985294A1 (en) | Recombinant antibody molecule and its use for target cell restricted T cell activation | |
TWI828662B (en) | Combination therapies of 4-1bb agonists with anti-cd20 antibodies | |
JP2023518443A (en) | Interleukin-2 mutants and uses thereof | |
KR20230029622A (en) | APRIL and BAFF inhibitory immunomodulatory proteins and methods of use | |
JP2022528030A (en) | Multifunctional fusion protein and its use | |
JP2023509952A (en) | Novel 4-1BBL trimer-containing antigen-binding molecule | |
CN1589902A (en) | Application of anti-lymphotoxin-beta receptor antibodies in preparation of medical composition | |
US20230340054A1 (en) | Interleukin-2 muteins and uses thereof | |
CN117946281A (en) | Long-acting nerve growth factor polypeptide and application thereof | |
US20230167164A1 (en) | Immunocytokines and uses thereof | |
WO2023241653A1 (en) | Interleukin-2 (il-2) mutant and use thereof | |
CN117247444A (en) | Interleukin-2 (IL-2) mutants and uses thereof | |
CN117247443A (en) | Interleukin-2 (IL-2) mutants and uses thereof | |
WO2024031034A9 (en) | A novel il-15r alpha fc fusion protein and uses thereof | |
WO2024140625A1 (en) | Method for screening and/or identifying mutable site in ngf, and method for screening and/or identifying ngf mutant | |
RU2779938C2 (en) | HETERODIMERIC FC-FUSED PROTEINS IL15/IL15Rα | |
WO2024153183A1 (en) | Il-18 variant polypeptides | |
TW202305128A (en) | Gene transfer vectors and methods of engineering cells | |
KR20230148226A (en) | Immunomodulatory Molecules and Their Uses |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |