US20200308304A1 - Hinge modified antibody fragments and methods of making - Google Patents
Hinge modified antibody fragments and methods of making Download PDFInfo
- Publication number
- US20200308304A1 US20200308304A1 US16/852,136 US202016852136A US2020308304A1 US 20200308304 A1 US20200308304 A1 US 20200308304A1 US 202016852136 A US202016852136 A US 202016852136A US 2020308304 A1 US2020308304 A1 US 2020308304A1
- Authority
- US
- United States
- Prior art keywords
- fab
- seq
- amino acid
- composition
- antibody
- 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
- 102000008394 Immunoglobulin Fragments Human genes 0.000 title claims abstract description 175
- 108010021625 Immunoglobulin Fragments Proteins 0.000 title claims abstract description 175
- 238000000034 method Methods 0.000 title abstract description 44
- 239000000203 mixture Substances 0.000 claims abstract description 67
- 230000009257 reactivity Effects 0.000 claims abstract description 49
- 230000002829 reductive effect Effects 0.000 claims abstract description 45
- 230000027455 binding Effects 0.000 claims description 55
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 54
- 150000001413 amino acids Chemical class 0.000 claims description 48
- 239000008194 pharmaceutical composition Substances 0.000 claims description 13
- 230000004048 modification Effects 0.000 claims description 12
- 238000012986 modification Methods 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 12
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 claims description 9
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 9
- 239000003937 drug carrier Substances 0.000 claims description 9
- 238000003776 cleavage reaction Methods 0.000 description 62
- 230000007017 scission Effects 0.000 description 62
- 241000282414 Homo sapiens Species 0.000 description 55
- 238000012217 deletion Methods 0.000 description 49
- 230000037430 deletion Effects 0.000 description 49
- 235000001014 amino acid Nutrition 0.000 description 48
- 210000004027 cell Anatomy 0.000 description 42
- 229940024606 amino acid Drugs 0.000 description 38
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 32
- 239000003814 drug Substances 0.000 description 31
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 30
- 201000010099 disease Diseases 0.000 description 30
- 210000002966 serum Anatomy 0.000 description 27
- 210000004899 c-terminal region Anatomy 0.000 description 26
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 24
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 24
- 125000000539 amino acid group Chemical group 0.000 description 24
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 22
- 150000007523 nucleic acids Chemical class 0.000 description 22
- 239000004365 Protease Substances 0.000 description 21
- 108020004707 nucleic acids Proteins 0.000 description 21
- 102000039446 nucleic acids Human genes 0.000 description 21
- 238000006467 substitution reaction Methods 0.000 description 20
- 230000001225 therapeutic effect Effects 0.000 description 20
- 102000035195 Peptidases Human genes 0.000 description 19
- 108091005804 Peptidases Proteins 0.000 description 19
- 238000004519 manufacturing process Methods 0.000 description 19
- 230000029087 digestion Effects 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 230000009456 molecular mechanism Effects 0.000 description 15
- 230000003990 molecular pathway Effects 0.000 description 15
- 108090000765 processed proteins & peptides Proteins 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 239000012634 fragment Substances 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 238000009472 formulation Methods 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 12
- 229940124597 therapeutic agent Drugs 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 239000004471 Glycine Substances 0.000 description 11
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 11
- 230000002797 proteolythic effect Effects 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 238000007792 addition Methods 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 230000003213 activating effect Effects 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 102000005962 receptors Human genes 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- 239000012636 effector Substances 0.000 description 8
- 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 8
- 239000002953 phosphate buffered saline Substances 0.000 description 8
- 235000019419 proteases Nutrition 0.000 description 8
- 238000003259 recombinant expression Methods 0.000 description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 7
- 235000004279 alanine Nutrition 0.000 description 7
- 229940127089 cytotoxic agent Drugs 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 229940127121 immunoconjugate Drugs 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 230000002285 radioactive effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 7
- 108700012359 toxins Proteins 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 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
- 239000002246 antineoplastic agent Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 239000003053 toxin Substances 0.000 description 6
- 231100000765 toxin Toxicity 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- -1 T225L Chemical compound 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 5
- 235000018417 cysteine Nutrition 0.000 description 5
- 239000002254 cytotoxic agent Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 238000000954 titration curve Methods 0.000 description 5
- 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 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 4
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Natural products OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000005875 antibody response Effects 0.000 description 4
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 4
- 238000005251 capillar electrophoresis Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 231100000599 cytotoxic agent Toxicity 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229960003180 glutathione Drugs 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000003094 microcapsule Substances 0.000 description 4
- 230000006337 proteolytic cleavage Effects 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108010087819 Fc receptors Proteins 0.000 description 3
- 102000009109 Fc receptors Human genes 0.000 description 3
- 101000851058 Homo sapiens Neutrophil elastase Proteins 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 3
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 229960000446 abciximab Drugs 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 102000052502 human ELANE Human genes 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108010068617 neonatal Fc receptor Proteins 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 2
- VPFUWHKTPYPNGT-UHFFFAOYSA-N 3-(3,4-dihydroxyphenyl)-1-(5-hydroxy-2,2-dimethylchromen-6-yl)propan-1-one Chemical compound OC1=C2C=CC(C)(C)OC2=CC=C1C(=O)CCC1=CC=C(O)C(O)=C1 VPFUWHKTPYPNGT-UHFFFAOYSA-N 0.000 description 2
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 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 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 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 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 241000282567 Macaca fascicularis Species 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 208000022873 Ocular disease Diseases 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108010039491 Ricin Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241000193996 Streptococcus pyogenes Species 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000000611 antibody drug conjugate Substances 0.000 description 2
- 229940049595 antibody-drug conjugate Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000012131 assay buffer Substances 0.000 description 2
- 239000008228 bacteriostatic water for injection Substances 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940044683 chemotherapy drug Drugs 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 239000000562 conjugate Substances 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000003405 delayed action preparation Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002022 differential scanning fluorescence spectroscopy Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000011194 good manufacturing practice Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000001823 molecular biology technique Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000001048 orange dye Substances 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- AQIXEPGDORPWBJ-UHFFFAOYSA-N pentan-3-ol Chemical compound CCC(O)CC AQIXEPGDORPWBJ-UHFFFAOYSA-N 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 229940068977 polysorbate 20 Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 229940107685 reopro Drugs 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 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
- 239000004474 valine Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- JKHVDAUOODACDU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCN1C(=O)C=CC1=O JKHVDAUOODACDU-UHFFFAOYSA-N 0.000 description 1
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- BQWBEDSJTMWJAE-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[(2-iodoacetyl)amino]benzoate Chemical compound C1=CC(NC(=O)CI)=CC=C1C(=O)ON1C(=O)CCC1=O BQWBEDSJTMWJAE-UHFFFAOYSA-N 0.000 description 1
- PMJWDPGOWBRILU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCC(C=C1)=CC=C1N1C(=O)C=CC1=O PMJWDPGOWBRILU-UHFFFAOYSA-N 0.000 description 1
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-UHFFFAOYSA-N 0.000 description 1
- WCMOHMXWOOBVMZ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCN1C(=O)C=CC1=O WCMOHMXWOOBVMZ-UHFFFAOYSA-N 0.000 description 1
- IHVODYOQUSEYJJ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]amino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)C(CC1)CCC1CN1C(=O)C=CC1=O IHVODYOQUSEYJJ-UHFFFAOYSA-N 0.000 description 1
- MFRNYXJJRJQHNW-DEMKXPNLSA-N (2s)-2-[[(2r,3r)-3-methoxy-3-[(2s)-1-[(3r,4s,5s)-3-methoxy-5-methyl-4-[methyl-[(2s)-3-methyl-2-[[(2s)-3-methyl-2-(methylamino)butanoyl]amino]butanoyl]amino]heptanoyl]pyrrolidin-2-yl]-2-methylpropanoyl]amino]-3-phenylpropanoic acid Chemical compound CN[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N(C)[C@@H]([C@@H](C)CC)[C@H](OC)CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MFRNYXJJRJQHNW-DEMKXPNLSA-N 0.000 description 1
- ALBODLTZUXKBGZ-JUUVMNCLSA-N (2s)-2-amino-3-phenylpropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound NCCCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC1=CC=CC=C1 ALBODLTZUXKBGZ-JUUVMNCLSA-N 0.000 description 1
- IEUUDEWWMRQUDS-UHFFFAOYSA-N (6-azaniumylidene-1,6-dimethoxyhexylidene)azanium;dichloride Chemical compound Cl.Cl.COC(=N)CCCCC(=N)OC IEUUDEWWMRQUDS-UHFFFAOYSA-N 0.000 description 1
- VILFTWLXLYIEMV-UHFFFAOYSA-N 1,5-difluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC([N+]([O-])=O)=C(F)C=C1F VILFTWLXLYIEMV-UHFFFAOYSA-N 0.000 description 1
- DIYPCWKHSODVAP-UHFFFAOYSA-N 1-[3-(2,5-dioxopyrrol-1-yl)benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 DIYPCWKHSODVAP-UHFFFAOYSA-N 0.000 description 1
- WLKSPGHQGFFKGE-UHFFFAOYSA-N 1-chloropropan-2-yl n-(3-chlorophenyl)carbamate Chemical compound ClCC(C)OC(=O)NC1=CC=CC(Cl)=C1 WLKSPGHQGFFKGE-UHFFFAOYSA-N 0.000 description 1
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- YBBNVCVOACOHIG-UHFFFAOYSA-N 2,2-diamino-1,4-bis(4-azidophenyl)-3-butylbutane-1,4-dione Chemical compound C=1C=C(N=[N+]=[N-])C=CC=1C(=O)C(N)(N)C(CCCC)C(=O)C1=CC=C(N=[N+]=[N-])C=C1 YBBNVCVOACOHIG-UHFFFAOYSA-N 0.000 description 1
- FZDFGHZZPBUTGP-UHFFFAOYSA-N 2-[[2-[bis(carboxymethyl)amino]-3-(4-isothiocyanatophenyl)propyl]-[2-[bis(carboxymethyl)amino]propyl]amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)C(C)CN(CC(O)=O)CC(N(CC(O)=O)CC(O)=O)CC1=CC=C(N=C=S)C=C1 FZDFGHZZPBUTGP-UHFFFAOYSA-N 0.000 description 1
- FBUTXZSKZCQABC-UHFFFAOYSA-N 2-amino-1-methyl-7h-purine-6-thione Chemical compound S=C1N(C)C(N)=NC2=C1NC=N2 FBUTXZSKZCQABC-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- NDMPLJNOPCLANR-UHFFFAOYSA-N 3,4-dihydroxy-15-(4-hydroxy-18-methoxycarbonyl-5,18-seco-ibogamin-18-yl)-16-methoxy-1-methyl-6,7-didehydro-aspidospermidine-3-carboxylic acid methyl ester Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 NDMPLJNOPCLANR-UHFFFAOYSA-N 0.000 description 1
- ZMRMMAOBSFSXLN-UHFFFAOYSA-N 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanehydrazide Chemical compound C1=CC(CCCC(=O)NN)=CC=C1N1C(=O)C=CC1=O ZMRMMAOBSFSXLN-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 108010066676 Abrin Proteins 0.000 description 1
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- QGZKDVFQNNGYKY-OUBTZVSYSA-N Ammonia-15N Chemical compound [15NH3] QGZKDVFQNNGYKY-OUBTZVSYSA-N 0.000 description 1
- 244000303258 Annona diversifolia Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- MECFLTFREHAZLH-ACZMJKKPSA-N Asn-Glu-Cys Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N MECFLTFREHAZLH-ACZMJKKPSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101000669426 Aspergillus restrictus Ribonuclease mitogillin Proteins 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101710158575 Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase Proteins 0.000 description 1
- OKTJSMMVPCPJKN-OUBTZVSYSA-N Carbon-13 Chemical compound [13C] OKTJSMMVPCPJKN-OUBTZVSYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-NJFSPNSNSA-N Carbon-14 Chemical compound [14C] OKTJSMMVPCPJKN-NJFSPNSNSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 102000011413 Chondroitinases and Chondroitin Lyases Human genes 0.000 description 1
- 108010023736 Chondroitinases and Chondroitin Lyases Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108700032819 Croton tiglium crotin II Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 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 1
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 206010061818 Disease progression 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
- 241000283086 Equidae Species 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 101710082714 Exotoxin A Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- LLQPHQFNMLZJMP-UHFFFAOYSA-N Fentrazamide Chemical compound N1=NN(C=2C(=CC=CC=2)Cl)C(=O)N1C(=O)N(CC)C1CCCCC1 LLQPHQFNMLZJMP-UHFFFAOYSA-N 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- 108700004714 Gelonium multiflorum GEL Proteins 0.000 description 1
- WQWMZOIPXWSZNE-WDSKDSINSA-N Gln-Asp-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O WQWMZOIPXWSZNE-WDSKDSINSA-N 0.000 description 1
- YYOBUPFZLKQUAX-FXQIFTODSA-N Glu-Asn-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YYOBUPFZLKQUAX-FXQIFTODSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101001041117 Homo sapiens Hyaluronidase PH-20 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 108010073807 IgG Receptors Proteins 0.000 description 1
- 102000009490 IgG Receptors Human genes 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical class C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 101710149643 Integrin alpha-IIb Proteins 0.000 description 1
- 238000012695 Interfacial polymerization Methods 0.000 description 1
- ZCYVEMRRCGMTRW-AHCXROLUSA-N Iodine-123 Chemical compound [123I] ZCYVEMRRCGMTRW-AHCXROLUSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 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
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 239000004907 Macro-emulsion Substances 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 244000302512 Momordica charantia Species 0.000 description 1
- 235000009811 Momordica charantia Nutrition 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- WTBIAPVQQBCLFP-UHFFFAOYSA-N N.N.N.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O Chemical compound N.N.N.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O WTBIAPVQQBCLFP-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- 101100413173 Phytolacca americana PAP2 gene Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 101710118538 Protease Proteins 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 241000205583 Streptococcus pyogenes MGAS1882 Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- KHPLUFDSWGDRHD-SLFFLAALSA-N Tyr-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O KHPLUFDSWGDRHD-SLFFLAALSA-N 0.000 description 1
- 244000000188 Vaccinium ovalifolium Species 0.000 description 1
- 240000001866 Vernicia fordii Species 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- IEDXPSOJFSVCKU-HOKPPMCLSA-N [4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[6-(2,5-dioxopyrrolidin-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate Chemical compound CC[C@H](C)[C@@H]([C@@H](CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C)[C@@H](O)c1ccccc1)OC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCc1ccc(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN2C(=O)CCC2=O)C(C)C)cc1)C(C)C IEDXPSOJFSVCKU-HOKPPMCLSA-N 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 108010001818 alpha-sarcin Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 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
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 238000011948 assay development Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 108010044540 auristatin Proteins 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- 229960001950 benzethonium chloride Drugs 0.000 description 1
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229940126587 biotherapeutics Drugs 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- HUTDDBSSHVOYJR-UHFFFAOYSA-H bis[(2-oxo-1,3,2$l^{5},4$l^{2}-dioxaphosphaplumbetan-2-yl)oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O HUTDDBSSHVOYJR-UHFFFAOYSA-H 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N butyl alcohol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229930195731 calicheamicin Natural products 0.000 description 1
- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000005354 coacervation Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012866 crystallographic experiment Methods 0.000 description 1
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- YBSJFWOBGCMAKL-UHFFFAOYSA-N dabigatran Chemical compound N=1C2=CC(C(=O)N(CCC(O)=O)C=3N=CC=CC=3)=CC=C2N(C)C=1CNC1=CC=C(C(N)=N)C=C1 YBSJFWOBGCMAKL-UHFFFAOYSA-N 0.000 description 1
- 229960003850 dabigatran Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229930191339 dianthin Natural products 0.000 description 1
- 108020001096 dihydrofolate reductase Proteins 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 125000005442 diisocyanate group Chemical group 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 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
- 150000002016 disaccharides Chemical class 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- ZWIBGKZDAWNIFC-UHFFFAOYSA-N disuccinimidyl suberate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)CCC1=O ZWIBGKZDAWNIFC-UHFFFAOYSA-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
- 229960003668 docetaxel Drugs 0.000 description 1
- AMRJKAQTDDKMCE-UHFFFAOYSA-N dolastatin Chemical compound CC(C)C(N(C)C)C(=O)NC(C(C)C)C(=O)N(C)C(C(C)C)C(OC)CC(=O)N1CCCC1C(OC)C(C)C(=O)NC(C=1SC=CN=1)CC1=CC=CC=C1 AMRJKAQTDDKMCE-UHFFFAOYSA-N 0.000 description 1
- 229930188854 dolastatin Natural products 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 108010028531 enomycin Proteins 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000002222 fluorine compounds Chemical class 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 238000013448 high performance strong cation exchange 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
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 229940044700 hylenex Drugs 0.000 description 1
- 229960002308 idarucizumab Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- APFVFJFRJDLVQX-AHCXROLUSA-N indium-111 Chemical compound [111In] APFVFJFRJDLVQX-AHCXROLUSA-N 0.000 description 1
- 229940055742 indium-111 Drugs 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229950005692 larotaxel Drugs 0.000 description 1
- SEFGUGYLLVNFIJ-QDRLFVHASA-N larotaxel dihydrate Chemical compound O.O.O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@@]23[C@H]1[C@@]1(CO[C@@H]1C[C@@H]2C3)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 SEFGUGYLLVNFIJ-QDRLFVHASA-N 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229940076783 lucentis Drugs 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Chemical class 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 108010010621 modeccin Proteins 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000001293 nucleolytic effect Effects 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- BWKDAMBGCPRVPI-ZQRPHVBESA-N ortataxel Chemical compound O([C@@H]1[C@]23OC(=O)O[C@H]2[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]2(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]21)OC(C)=O)C3(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)CC(C)C)C(=O)C1=CC=CC=C1 BWKDAMBGCPRVPI-ZQRPHVBESA-N 0.000 description 1
- 229950001094 ortataxel Drugs 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N p-hydroxybenzoic acid methyl ester Natural products COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000003094 perturbing effect Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- 108010076042 phenomycin Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229940096959 praxbind Drugs 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 231100000654 protein toxin Toxicity 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 229960003876 ranibizumab Drugs 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 102220075283 rs200435907 Human genes 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 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
- 239000012321 sodium triacetoxyborohydride Substances 0.000 description 1
- MKNJJMHQBYVHRS-UHFFFAOYSA-M sodium;1-[11-(2,5-dioxopyrrol-1-yl)undecanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCCCCCCN1C(=O)C=CC1=O MKNJJMHQBYVHRS-UHFFFAOYSA-M 0.000 description 1
- ULARYIUTHAWJMU-UHFFFAOYSA-M sodium;1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O ULARYIUTHAWJMU-UHFFFAOYSA-M 0.000 description 1
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 1
- MIDXXTLMKGZDPV-UHFFFAOYSA-M sodium;1-[6-(2,5-dioxopyrrol-1-yl)hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O MIDXXTLMKGZDPV-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000012414 sterilization procedure Methods 0.000 description 1
- 238000012799 strong cation exchange Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- MODVSQKJJIBWPZ-VLLPJHQWSA-N tesetaxel Chemical compound O([C@H]1[C@@H]2[C@]3(OC(C)=O)CO[C@@H]3CC[C@@]2(C)[C@H]2[C@@H](C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C(=CC=CN=4)F)C[C@]1(O)C3(C)C)O[C@H](O2)CN(C)C)C(=O)C1=CC=CC=C1 MODVSQKJJIBWPZ-VLLPJHQWSA-N 0.000 description 1
- 229950009016 tesetaxel Drugs 0.000 description 1
- 239000004308 thiabendazole Substances 0.000 description 1
- CNHYKKNIIGEXAY-UHFFFAOYSA-N thiolan-2-imine Chemical compound N=C1CCCS1 CNHYKKNIIGEXAY-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- RUELTTOHQODFPA-UHFFFAOYSA-N toluene 2,6-diisocyanate Chemical compound CC1=C(N=C=O)C=CC=C1N=C=O RUELTTOHQODFPA-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- LZAJKCZTKKKZNT-PMNGPLLRSA-N trichothecene Chemical compound C12([C@@]3(CC[C@H]2OC2C=C(CCC23C)C)C)CO1 LZAJKCZTKKKZNT-PMNGPLLRSA-N 0.000 description 1
- 229930013292 trichothecene Natural products 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004355 vindesine Drugs 0.000 description 1
- UGGWPQSBPIFKDZ-KOTLKJBCSA-N vindesine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(N)=O)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1N=C1[C]2C=CC=C1 UGGWPQSBPIFKDZ-KOTLKJBCSA-N 0.000 description 1
- XLYOFNOQVPJJNP-OUBTZVSYSA-N water-17o Chemical compound [17OH2] XLYOFNOQVPJJNP-OUBTZVSYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/53—Hinge
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/71—Decreased effector function due to an Fc-modification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/75—Agonist effect on antigen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present disclosure relates to antibody fragments (e.g., Fab and F(ab′) 2 ) having reduced or no reactivity towards pre-existing anti-hinge antibodies (AHA) and compositions comprising such antibody fragments, as well as methods of making and using such antibody fragments and compositions.
- antibody fragments e.g., Fab and F(ab′) 2
- AHA pre-existing anti-hinge antibodies
- Antibodies are composed of two Fab regions that are connected by a flexible hinge-region to the Fc. While the Fab mediates recognition and binding of the antigen, two important functions of the Fc are to mediate effector function by engagement with Fcy receptors (1) and to confer long serum half-life by binding to the salvage receptor, FcRn (2). In particular, the slow pharmacokinetics of IgG contribute to the success of antibodies as therapeutics as it enables less frequent dosing compared to other biotherapeutics. Consequently, the majority of approved therapeutic antibodies have full-length IgG format. Unlike IgG, the serum half-life of an isolated Fab fragment is short (3) and such property is required for indications when short plasma half-life is desired as with three FDA approved Fab molecules (4).
- Fab molecules directed against platelet surface receptor GPIIb/IIIa are commercially produced by proteolytic cleavage with papain (5), which is the original method of Fab production (6).
- proteolytic cleavage with papain (5) which is the original method of Fab production (6).
- recombinant expression of antibody fragments has become an attractive route to generate Fab molecules as exemplified by the second approved Fab therapeutic, anti-VEGF (ranibizumab, Lucentis®) (7) and the recently approved Fab against dabigatran (idarucizumab, Praxbind®) (33).
- Fab molecules are advantageous, for example, when transient systemic activity that does not persist past dosing is desired or when administration and activity are localized to a peripheral compartment such as the eye.
- AHA anti-hinge antibodies
- the present disclosure relates to antibody fragments (e.g., Fab and F(ab′) 2 ) having reduced or no reactivity towards pre-existing anti-hinge antibodies (AHA) and compositions comprising such antibody fragments, as well as methods of making and using such antibody fragments and compositions.
- antibody fragments e.g., Fab and F(ab′) 2
- AHA pre-existing anti-hinge antibodies
- the present disclosure is directed to an isolated antibody fragment and compositions comprising the same, wherein the antibody fragment has reduced or no reactivity towards pre-existing anti-hinge antibodies.
- an isolated antibody fragment of the present disclosure exhibits reduced and/or no binding to FcyRIIIa and/or C1q.
- the antibody fragment is a Fab, Fab′ or F(ab′) 2 .
- the present disclosure is directed to antibody fragments and compositions comprising the same, wherein the antibody fragment is a Fab.
- the present disclosure is directed to Fab molecules wherein the Fab terminates with residue D 221 .
- the Fab terminates with amino acids comprising an amino acid sequence selected from group consisting of CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK and CD.
- the Fab terminates with amino acids comprising an amino acid sequence selected from group consisting of KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY and K.
- the Fab comprises a heavy chain constant region that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and conservative modifications of.
- the present disclosure is directed to an antibody fragment and compositions comprising the same, wherein the antibody fragment is a F(ab′) 2 .
- the present disclosure is directed to F(ab′) 2 molecules, wherein the F(ab′) 2 comprises a C-terminal deletion of 1, 2, 3, 4, or 5 amino acids.
- the F(ab′) 2 comprises a deletion at position EU231.
- the F(ab′) 2 comprises a deletion at positions EU231-232.
- the F(ab′) 2 comprises a deletion at positions EU231-233.
- the F(ab′) 2 comprises a deletion at position EU231-234.
- the F(ab′) 2 comprises a deletion at position EU230-234.
- the present disclosure is directed to an isolated nucleic acid and compositions comprising the same, wherein the nucleic acid encodes an antibody fragment having reduced or no reactivity to AHA.
- the present disclosure is directed to a host cell comprising said nucleic acid.
- the present disclosure is directed to a method of producing an antibody fragment comprising culturing said host cell so that the antibody fragment is produced.
- the present disclosure is directed to a pharmaceutical formulation comprising an antibody fragment having reduced or no reactivity to AHA and a pharmaceutically acceptable carrier.
- the present disclosure is directed to an antibody fragment having reduced or no reactivity to AHA for use as a medicament. In certain embodiments, the present disclosure is directed to an antibody fragment having reduced or no reactivity to AHA for use in treating a disease. In certain embodiments, the present disclosure is directed to an antibody fragment having reduced or no reactivity to AHA for use in inhibiting or activating a molecular pathway and/or mechanism. In certain embodiments, the present disclosure is directed to the use of an antibody fragment having reduced or no reactivity to AHA in the manufacture of a medicament for the treatment of a disease. In certain embodiments, the present disclosure is directed to the use of an antibody fragment having reduced or no reactivity to AHA in the manufacture of a medicament for inhibiting or activating a molecular pathway and/or mechanism.
- the present disclosure is directed to methods of treating an individual having a disease comprising administering to the individual an effective amount of an antibody fragment having reduced or no reactivity to AHA. In certain embodiments, the present disclosure is directed to methods of inhibiting or activating a molecular pathway and/or mechanism in an individual comprising administering to the individual an effective amount of an antibody fragment having reduced or no reactivity to AHA to inhibit or activate a molecular pathway and/or mechanism
- FIGS. 1A-1D show the binding of pre-existing human antibodies to the Fab of human IgG1, IgG2 and IgG4.
- the upper hinge is a protruding unstructured region without structural and functional role.
- the residues of the upper hinge are displayed in magenta to indicate the T225L mutation ( 105 ) that is perturbing binding to pre-existing AHA. Numbering of residues is according to EU numbering nomenclature.
- 1A discloses SEQ ID NOS 14-15, respectively, in order of appearance.
- 1 B Pooled human serum was incubated with human IgG1 Fab with different upper hinge lengths and ends. Binding pre-existing antibodies detected by anti-Fc ELISA. Truncating the Fab C-terminus to D 221 (D) and the C-terminal variant T225L (DKTHL (SEQ ID NO: 20)) greatly reduced binding of pre-existing antibodies to almost background. Strong response was observed towards T223 as the C-terminal residue (DKT), coinciding with the cleavage site of human neutrophil elastase. The mean value of the individual data points is represented by the horizontal line.
- FIG. 1B discloses SEQ ID NOS 20-21 and 27, respectively, in order of appearance.
- FIG. 1C discloses SEQ ID NOS 21, 20, 21, 20, 21 and 20, respectively, in order of appearance.
- FIG. 1D discloses SEQ ID NO: 18.
- FIGS. 2A-2C show the cleavage of IgG1-2 chimera by IdeS.
- 2 A Model of the F(ab′) 2 region of antibody cAC10 modeled with MOE; light chain ( 201 ), heavy chain ( 202 ), interchain disulfide ( 203 ), and lower hinge ( 204 ).
- the P1 position of IdeS is G236. Numbering of residues is according to EU numbering nomenclature.
- FIG. 2A discloses SEQ ID NO: 30.
- FIG. 1 A Model of the F(ab′) 2 region of antibody cAC10 modeled with MOE; light chain ( 201 ), heavy chain ( 202 ), interchain disulfide ( 203 ), and lower hinge ( 204 ).
- 2B discloses SEQ ID NOS 31 and 59, respectively, in order of appearance.
- ( 2 C) Cleavage efficiency of human IgG1 and IgG1-2 chimera. 1 mg/ml of IgG1 and IgG1-2 were incubated for 24 hours at 37° C. with different IdeS amounts as indicated. Cleavage was analyzed by capillary electrophoresis. While IgG1 was efficiently cleaved into F(ab′) 2 at an IdeS:IgG ratio of 1:500, IgG1-2 requires 50-fold higher IdeS concentrations for complete cleavage.
- FIGS. 3A-3E show the cleavage of human IgG1 with variants in the P1 and P2 position by IdeS.
- 3 A Capillary electrophoresis of antibodies with variants at the P1 and P2 position were digested 24 hours at 37° C. with a 1:10 ratio of IdeS:IgG at 1 mg/ml. The P1 and P2 residues are designated in 1-letter code. Leucine and glycine (L235G236) are the natural amino acids at these positions. All antibody variants can be completely cleaved into F(ab′) 2 fragments.
- 3 B The cleavage efficiency of the variants was assessed by amount of F(ab′) 2 produced at different IdeS:IgG ratios.
- variant VG is cleaved comparably to the wild-type sequence (LG)
- other variants require increased amounts of IdeS for complete digestion.
- 3 C The cleavage efficiency of the variants was assessed by the amount of F(ab′) 2 produced at an IdeS:IgG ratio of 1:10.
- FIG. 3 D Schematic diagrams of the expression, purification and screening strategies for the human IgG1 variants.
- FIG. 3D discloses SEQ ID NOS 31-34, respectively, in order of appearance.
- FIG. 3 E Cleavage efficiency of the 76 human IgG1 variants with IdeS.
- FIG. 3E discloses SEQ ID NO: 31.
- FIGS. 4A-4B show the reactivity of P1 and P2 variants towards pre-existing AHA.
- IdeS is efficiently removed during purification and cannot be detected in the purified F(ab′) 2 variants by SDS-PAGE followed by coomassie staining (upper panel) or immunoblot analysis with anti-IdeS antibodies (lower panel).
- 4 B Pooled human serum was incubated with human IgG1 Fab with T225 C-terminus and F(ab′) 2 generated by IdeS cleavage of antibodies with variants in P1 and P2 positions. Binding pre-existing antibodies were detected by ELISA. The F(ab′) 2 showed a signal that was about 1.7 fold higher compared to the Fab. The hinge variants reduced reactivity to levels comparable to Fab but did not eliminate reactivity completely.
- FIGS. 5A-5F show the reactivity of truncated variants towards pre-existing AHA response.
- 5 A IdeS cleavage of antibodies with deletions in the IdeS P3, P4, and P5 sites. While deletion of the IdeS P3 residue (L234) in the lower hinge severely impacted cleavage efficiency, deletion of the P4 (E233) or P5 (P232) positions did not impact cleavage with IdeS compared to wild-type (WT).
- 5 B Deletion of the P4 and P5 positions was not sufficient to avoid binding of pre-existing AHA.
- FIG. 5F Schematic diagram depicting the deletions generated in the lower hinge region.
- FIG. 5F discloses SEQ ID NOS 35, 31, 36-40, 31, and 41-42, respectively, in order of appearance.
- FIGS. 6A-6B show the alignment of the amino acid residues ( 6 A) and EU numbering of the amino acid residues ( 6 B) within the upper, core and lower hinge regions of the IgG1, IgG2, IgG3 and IgG4 isotypes in human, cynomolgus monkey and rhesus monkey.
- FIG. 6A discloses SEQ ID NOS 43-54, respectively, in order of appearance.
- FIG. 6B discloses SEQ ID NOS 43, 46, 55 and 52, respectively, in order of appearance.
- FIG. 7 shows the expression levels of Fabs with upper hinge truncations or mutations in E. coli .
- Figure discloses SEQ ID NOS 14-15, respectively, in order of appearance.
- FIG. 8 shows the efficiency of the cleavage of the ⁇ P456 and ⁇ P4567 variant produced at an IdeS:IgG ratio of 1:500 or 1:10.
- Figure discloses SEQ ID NOS 56-58, respectively, in order of appearance.
- FIGS. 9A-9D show the reactivity of deletion variants with modified P1 and P2 residues with pre-existing AHA.
- 9 A The cleavage efficiency of the variants was assessed by amount of F(ab′) 2 produced at an IdeS:IgG ratio of 1:10.
- FIG. 9A discloses SEQ ID NO: 31.
- 9 B The cleavage efficiency of the variants was assessed by the amount of F(ab′) 2 produced at an IdeS:IgG ratio of 1:100.
- FIG. 9B discloses SEQ ID NO: 31.
- 9 C The cleavage efficiency of the variants was assessed by amount of F(ab′) 2 produced at an IdeS:IgG ratio of 1:500.
- FIG. 9C discloses SEQ ID NO: 31.
- 9 D Detection of the bound pre-existing AHA to the variants by anti-Fc ELISA.
- FIG. 9D discloses SEQ ID NO: 56.
- FIGS. 10A-10B show the reactivity of the ⁇ P456 and ⁇ P4567 variants with modified P1 and P2 residues to pre-existing AHA.
- 10 A The cleavage efficiency of the variants was assessed by amount of F(ab′) 2 produced at an IdeS:IgG ratio of 1:10 and 1:200.
- FIG. 10A discloses SEQ ID NOS 56-57, respectively, in order of appearance.
- 10 B Detection of pre-existing AHA bound to the variants by anti-Fc ELISA.
- FIG. 10B discloses SEQ ID NO: 56.
- FIG. 11 shows the titration curves of F(ab′) 2 and Fab molecules in the AHA ELISA.
- the dilutions corresponding to the OD 450 nm (1.15) at the middle of the F(ab′) 2 titration curves were 70 and 14 for F(ab′) 2 and Fab, respectively.
- F(ab′) 2 has five fold higher AHA reactivity than IgG1 Fab.
- F(ab′) 2 , F(ab′) 2 ⁇ P456, Fab T 225 , Fab T 225 L and Fab D 221 were coated on the wells.
- Serial dilutions of pooled human serum were added to the wells and control wells were uncoated. Similar results were obtained in 4 other experiments.
- the data shown here and in FIG. 1B and FIG. 5D were collected from the same experiment.
- Figure discloses SEQ ID NOS 21 and 20, respectively, in order of appearance.
- antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- an “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′) 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
- the antibody fragment is a Fab molecule.
- the antibody fragment is a F(ab′) 2 molecule.
- full length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
- “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures.
- native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (C H 1, C H 2, and C H 3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain.
- VH variable heavy domain
- VL variable region
- the light chain of an antibody can be assigned to one of two types, called kappa ( ⁇ ) and lambda (k), based on the amino acid sequence of its constant domain.
- Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
- Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
- Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIM (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
- Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
- Inhibiting receptor Fc ⁇ RIM contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
- ITAM immunoreceptor tyrosine-based activation motif
- ITIM immunoreceptor tyrosine-based inhibition motif
- FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).
- Other FcRs including those to be identified in the future, are encompassed by the term “FcR” herein.
- Fc receptor or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).
- Binding to human FcRn in vivo and serum half-life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered.
- WO 2000/042072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).
- “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody 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 receptor); and B cell activation.
- the “hinge region” is generally defined as stretching from 216-238 (EU numbering) or 226-251 (Kabat numbering) of human IgG1.
- the hinge can be further divided into three distinct regions, the upper, middle (e.g., core) and lower hinge. See, e.g., Brerski and Georgiou, Curr. Opin. Immunol. 40, 62-69 (2016), which is incorporated by reference herein in its entirety.
- the hinge region of a human IgG1 antibody is generally defined as follows:
- the upper hinge comprises amino acids having the sequence EPKSCDKTHT (SEQ ID NO: 22). In certain embodiments, the upper hinge comprises the amino acids at positions 216-225 (EU numbering) or 226-238 (Kabat numbering).
- the middle (e.g., core) hinge comprises amino acids having the sequence CPPC (SEQ ID NO: 23).
- the core hinge comprises the amino acids at positions 226-229 (EU numbering) or 239-242 (Kabat numbering).
- the lower hinge comprises amino acids having the sequence PAPELLGGP (SEQ ID NO: 24). In certain embodiments, the lower hinge comprises the amino acids at positions 230-238 (EU numbering) or 243-251 (Kabat numbering).
- the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
- the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
- an “isolated” antibody or antibody fragment is one which has been separated from a component of its natural environment.
- An antibody or an antibody fragment can be purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
- electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
- chromatographic e.g., ion exchange or reverse phase HPLC
- Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
- % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
- the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
- the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code.
- the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
- % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
- nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
- An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
- the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
- Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
- Progeny can be completely identical in nucleic acid content to a parent cell, or can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
- domesticated animals e.g., cows, sheep, cats, dogs, and horses
- primates e.g., humans and non-human primates such as monkeys
- rabbits e.g., mice and rats
- rodents e.g., mice and rats.
- the individual or subject is a human.
- treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- antibody fragments of the present disclosure are used to delay development of a disease or to slow the progression of a disease.
- an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
- pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
- a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
- Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal
- the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
- the present disclosure is based, in part, on methods of engineering antibody fragments to evade pre-existing anti-hinge antibodies (AHA).
- antibody fragments e.g., Fab and F(ab′) 2
- AHA anti-hinge antibodies
- antibody fragments of the present disclosure can provide superior safety in a therapeutic setting by minimizing immune response following drug treatments.
- the present disclosure provides antibody fragments (e.g., Fab, Fab′ and F(ab′) 2 ), and compositions comprising the same, that have reduced or no reactivity towards AHA.
- an antibody fragment disclosed herein exhibits AHA reactivity that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to a reference antibody fragment, e.g., an antibody fragment with a native hinge region.
- the reference antibody fragment is an IgG1 antibody fragment that has a native hinge region.
- the isolated antibody fragments of the present disclosure, and the compositions comprising the same exhibit reduced and/or no binding to Fc ⁇ RIIIa and/or C1q.
- an antibody fragment of the present disclosure exhibits binding to Fc ⁇ RIIIa and/or C1q that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to a reference antibody fragment, e.g., an antibody fragment with a native hinge region.
- the reference antibody fragment is an IgG1 antibody fragment that has a native hinge region.
- an antibody fragment employed in the context of the methods described herein comprises a native hinge region or a modified hinge region.
- an antibody fragment of the present disclosure can be a Fab fragment that comprises a native hinge region or modified hinge region.
- the antibody fragment of the present disclosure is a F(ab′) 2 that comprises a native hinge region or modified hinge region.
- a native hinge region is a hinge region normally associated with the C H 1 domain of an antibody molecule.
- the native hinge region of a presently disclosed antibody fragment can be of the IgG1, IgG2, IgG3 or IgG4 isotype.
- the Fab fragment can be of the IgG1, IgG2, IgG3 or IgG4 isotype.
- the antibody fragment, e.g., the Fab fragment is of the IgG2 isotype comprising a native hinge region.
- the antibody fragment, e.g., the Fab fragment is of the IgG4 isotype comprising a native hinge region.
- a modified hinge region is any hinge that differs in length and/or composition from the native hinge region.
- Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, pig, hamster, camel, llama or goat hinge regions.
- Other modified hinge regions can comprise a complete hinge region derived from an antibody of a different class or subclass from that of the C H 1 domain.
- a C H 1 domain of class ⁇ 1 can be attached to a hinge region of class ⁇ 4.
- the modified hinge region can comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region.
- the native hinge region is altered by substituting, deleting and/or adding one or more amino acid residues to generate a modified hinge region.
- the Fab fragment is of the IgG1 isotype comprising a modified hinge region.
- the Fab fragment is of the IgG2 isotype comprising a modified hinge region.
- the Fab fragment is of the IgG4 isotype comprising a modified hinge region.
- a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the upper hinge region.
- a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions EU216-225.
- a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the lower hinge region.
- a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions EU230-238.
- a modified hinge region can comprise the addition of one or more amino acids C-terminal to amino acid position EU238.
- a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the middle, e.g., core, hinge region.
- a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions EU226-229.
- the modification or alteration is a substitution of one or more, two or more, three or more, four or more, five or more or six or more amino acid residues.
- the substitution can be generated within the upper hinge region, middle hinge region and/or the lower hinge region.
- the amino acid residue at position 225 can be substituted.
- the amino acid residue at position 225 can be changed to any amino acid except for threonine (T).
- the amino acid at position 225 e.g., threonine
- an antibody fragment of the present disclosure is a Fab fragment comprising the substitution T225L.
- the upper hinge region of an IgG1 antibody fragment can be substituted with one or more amino acids residues present within the upper hinge region of an IgG2 and/or an IgG4 antibody because, for example, the upper hinge regions of IgG2 and IgG4 antibodies exhibit reduced or no reactivity towards AHA (see, e.g., FIG. 1 ).
- the upper hinge region of an IgG1 antibody fragment can be substituted with one or more amino acids residues present within the native hinge region of an IgG2 and/or an IgG4 antibody (see FIG. 6 ).
- a modified hinge region of an IgG1 antibody fragment retains a cysteine at amino acid position EU220 (e.g., as compared to the native hinge region of an IgG1 antibody). In certain embodiments, a modified hinge region of an IgG1 antibody fragment does not retain a cysteine at amino acid position EU220, e.g., in an IgG antibody fragment where the upper hinge region of the IgG1 antibody fragment is replaced with the upper hinge region (e.g., entire upper hinge region) of IgG4.
- the upper hinge region of an IgG1 antibody fragment can be substituted with one or more amino acids residues present within the upper hinge region of an IgG2, IgG3 and/or an IgG4 antibody, where the amino acid residue at position 131 of the IgG1 antibody is changed from a serine (S) to a cysteine (C), i.e., S131C.
- S serine
- C cysteine
- an antibody fragment of the present disclosure can comprise a substitution at amino acid positions EU235-236.
- the amino acid at position 236, e.g., glycine (G) can be changed to an alanine (A), e.g., G236A.
- an antibody fragment, e.g., a F(ab′) 2 can comprise a substitution at position 235, according to EU numbering.
- the amino acid at position 235 e.g., leucine (L)
- L can be changed to a valine (V), e.g., L235V, changed to an isoleucine (I) e.g., L235I, or changed to a methionine (M) e.g., L235M.
- V valine
- I isoleucine
- M methionine
- the modification or alteration is a deletion of one or more, two or more, three or more, four or more, five or more or six or more amino acid residues.
- the one or more deletions can be generated within the upper hinge region, middle hinge region and/or the lower hinge region.
- an antibody fragment of the present disclosure e.g., a Fab, F(ab′) 2 or Fab′, comprises a modified hinge region that has one or more deletions of one or more amino acids at positions EU230-238.
- the antibody fragment comprises a deletion at position EU231.
- the antibody fragment comprises a deletion at positions EU231 and EU232.
- the antibody fragment comprises deletions at positions EU231, EU232 and EU233. In certain embodiments, the antibody fragment comprises deletions at positions EU231, EU232, EU233 and EU234. In certain embodiments, the antibody fragment comprises deletions at positions EU230, EU231, EU232, EU233 and EU234.
- an antibody fragment of the present disclosure comprises a C-terminal deletion of one or more, two or more, three or more, four or more, five or more or six or more amino acids.
- an antibody fragment of the present comprises the deletion of one or more amino acids in the upper hinge region, e.g., to generate a C-terminal truncation.
- one or more amino acid at positions EU222-225 can be deleted to obtain a C-terminal truncation.
- an antibody fragment disclosed herein, e.g., a Fab fragment comprises a C-terminal truncation.
- the C-terminus of an antibody fragment disclosed herein terminates at amino acid residue D 221 (according to EU numbering).
- the C-terminus of an antibody fragment disclosed herein, e.g., a Fab fragment terminates at amino acid residue K222 (according to EU numbering).
- the C-terminus of the heavy chain of an antibody fragment terminates with amino acids having a sequence selected from CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK and CD.
- the C-terminus of the heavy chain of the Fab fragment terminates in the amino acid sequence CDKTHX (SEQ ID NO: 25), wherein X is any amino acid except T.
- a Fab fragment comprises a heavy chain constant region selected from “CDKTHT,” (SEQ ID NO: 14) “CDKTHL,” (SEQ ID NO: 15) “CDKTH,” (SEQ ID NO: 16) “CDKT,” (SEQ ID NO: 17) “CDK” or “CD,” as disclosed in Table 1.
- the presently disclosed subject matter provides antibody fragments, e.g., Fab fragment, that comprise a heavy chain constant region that comprises an amino acid sequence as set forth in SEQ ID NOs: 1, 2, 3, 4, 5 or 6.
- an antibody fragment of the present disclosure comprises a heavy chain constant region that comprises the amino acid sequence set forth in SEQ ID NO:5.
- an antibody fragment of the present disclosure comprises a heavy chain constant region that comprises the amino acid sequence set forth in SEQ ID NO:6.
- IgG2 or IgG4 Fab fragments can be used.
- an antibody fragment of the present disclosure can comprise a heavy chain constant region that comprises the amino acid sequence in SEQ ID NOs: 7 or 8.
- an IgG2 or IgG4 Fab fragment can comprise a C-terminal deletion of one or more, two or more, three or more, four or more or five or more amino acids.
- a Fab of the present disclosure is an IgG2 Fab fragment comprising a heavy chain constant region ending in the sequence VERK (SEQ ID NO: 26).
- the C-terminus of the heavy chain of an antibody fragment terminates with amino acids having a sequence selected from KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY and K.
- a Fab of the present disclosure is an IgG4 Fab fragment comprising a heavy chain constant region selected from “KYGPP,” (SEQ ID NO: 18) “KYGP,” (SEQ ID NO: 19) “KYG,” “KY” and “K,” as disclosed in Table 1.
- an antibody fragment of the present disclosure can comprise a heavy chain constant region that comprises the amino acid sequence set forth in SEQ ID NOs: 9, 10, 11, 12 or 13.
- the present disclosure further provides antibody fragments that comprise conservative modifications of the sequences disclosed herein.
- the present disclosure provides antibody fragments that comprise a heavy chain constant region that comprises an amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or conservative modifications thereof, and wherein the antibody fragment retains the desired properties of the antibody fragments disclosed herein.
- such antibody fragments have reduced or no reactivity towards AHA, as disclosed above.
- conservative sequence modification is intended to refer to amino acid modifications that do not significantly affect characteristics of the antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Exemplary conservative amino acid substitutions are shown in Table 2.
- a sequence disclosed herein can have up to about one, up to about two, up to about three, up to about four, up to about five, up to about six, up to about seven, up to about eight, up to about nine or up to about ten amino acid residues that are modified and/or substituted.
- Amino acids can be grouped according to common side-chain properties:
- modified hinge regions of the present disclosure can be entirely synthetic and can be designed to possess desired properties such as length, composition and flexibility.
- a modified hinge region of the present disclosure can be altered to increase or decrease the flexibility of the hinge region.
- modifications which can increase the flexibility of the hinge region include, but are not limited to, the substitution of one or more amino acids residues with one or more amino acid residues which increase the flexibility (e.g., glycine).
- modifications which can decrease the flexibility of the hinge region include, but are not limited to, the substitution of one or more amino acids residues with one or more amino acid residues which crease the rigidity of the polypeptide (e.g., proline).
- the antibody fragments are made by hinge engineering technology.
- the antibody fragment starting material for use in connection with the methods described herein can be obtained from any whole antibody (e.g., a whole monoclonal antibody), using any suitable enzymatic cleavage and/or digestion techniques.
- the antibody fragment can be obtained by cleavage with IdeS.
- Fab molecules are generated by proteolytic digestion or recombinant expression.
- Proteolytic digestion was the original method of Fab production (6).
- Generating Fab molecules via proteolytic digestion results in the C-terminal sequence of the Fab heavy chain defined by the protease cleavage site.
- a Fab molecule typically includes a portion of the upper hinge of the antibody. This upper hinge region of the antibody serves as the linker between Fab and Fc region but has no structural or functional role in a Fab molecule. It can be considered as an unstructured appendix (see FIG. 1A ) as it is often not fully resolved in crystal structures of Fab molecules.
- One therapeutic Fab molecule directed against platelet surface receptor GPITh/IIIa is commercially produced by proteolytic cleavage.
- the recombinant expression of antibody fragments is an attractive route to generate Fab molecules (7).
- the recombinant expression of Fab molecules provides flexibility in defining the length of the included upper hinge region.
- the Fab fragments are produced by recombinant expression.
- the high affinity of an antibody is often enabled by bivalent target engagement, facilitating avidity.
- the target engagement of a Fab is monovalent. This often leads to lower target affinity compared to the full-length IgG.
- By joining two Fab fragments to create a F(ab′) 2 avidity can be restored while preserving key properties of the Fab, such as short serum half-life.
- targeting multiple disease mediators by bispecific antibodies has become increasingly important for therapeutic antibody development (8).
- F(ab′) 2 molecules can provide a natural scaffold to produce small bispecific antibody fragments.
- F(ab′) 2 In contrast to the production of Fab molecules, the recombinant expression of F(ab′) 2 is not naturally possible because expressed Fab′ molecules require non-native homo- or heterodimerization domains as a fusion (9, 10). Hence, there are two main approaches to generate F(ab′) 2 molecules: (i) chemical conjugation and (ii) proteolytic digestion. For chemical conjugation, recombinantly generated Fab′ molecules are coupled by homo- or heterobifunctional crosslinkers (3, 9, 11, 12). Analogous to the proteolytic digestion approach to produce Fab molecules, a number of known proteases can cleave the intact antibody in the lower hinge region to produce F(ab′) 2 molecules (13).
- Such proteolytic digestion results in very stable F(ab′) 2 molecules where the two Fab molecules are connected by the two disulfide-bonds of the core-hinge.
- Pepsin is most widely used (14) but a highly specific IgG degrading enzyme of Streptococcus pyogenes , IdeS, has been described more recently (15, 16).
- IdeS a highly specific IgG degrading enzyme of Streptococcus pyogenes
- the use of IdeS enables generation of highly homogenous product by eliminating the C-terminal heterogeneity observed from pepsin digest (3).
- the F(ab′) 2 , fragments are produced by IdeS cleavage.
- Antibody fragments can be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567.
- an isolated nucleic acid encoding an antibody fragment described herein or composition comprising such nucleic acid is provided.
- one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided.
- a host cell comprising such nucleic acid is also provided.
- the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell).
- a method of making a Fab molecule comprises culturing a host cell comprising a nucleic acid encoding the Fab, as provided above, under conditions suitable for expression of the Fab, and optionally recovering the Fab from the host cell (or host cell culture medium).
- a nucleic acid encoding a Fab is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
- Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the Fab).
- Suitable host cells for cloning or expression of Fab-encoding vectors include prokaryotic or eukaryotic cells described herein.
- Fabs can be produced in bacteria.
- For expression of antibody fragments, such as Fabs, in bacteria see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli .)
- the Fab can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for Fab-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized.” See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
- Suitable host cells for the expression of glycosylated proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frupperda cells.
- Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
- Vertebrate cells can also be used as hosts.
- mammalian cell lines that are adapted to grow in suspension can be useful.
- Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
- monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
- Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR′′ CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
- compositions of an antibody fragments are prepared by mixing such antibody fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
- lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958.
- aqueous antibody formulations can include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.
- an antibody fragment of the present disclosure can be of a purity greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8% or greater than about 99.9%.
- Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
- sHASEGP soluble neutral-active hyaluronidase glycoproteins
- rHuPH20 HYLENEX®, Baxter International, Inc.
- Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
- a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
- the formulation herein can also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
- active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
- a composition of the present disclosure can be administered by a variety of methods known in the art.
- the route and/or mode of administration vary depending upon the desired results.
- the active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
- the pharmaceutical compositions are manufactured under Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.
- GMP Good Manufacturing Practice
- the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
- the active compound i.e., antibody fragment
- the active compound can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.
- Active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
- sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- the formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
- the disclosed pharmaceutical compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
- the antibodies of the present invention when administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, from about 0.01% to about 99.5% (or about 0.1 to about 90%) of an antibody fragment in combination with a pharmaceutically acceptable carrier.
- an antibody fragment for use as a medicament is provided.
- an antibody fragment for use in treating a particular disease indication is provided.
- an antibody fragment of the present disclosure can be used treat an ocular disease and/or disorder.
- an antibody fragment of the present disclosure can be used treat a disease and/or a disorder that would benefit from the application of an antibody fragment that exhibits a short systemic half-life.
- an antibody fragment for use in a method of treatment is provided.
- the present disclosure provides an antibody fragment for use in a method of treating an individual having a specific disease comprising administering to the individual an effective amount of the antibody fragment or compositions comprising the same. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In certain embodiments, the present disclosure provides an antibody fragment for use in inhibiting a particular molecular pathway and/or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in a method of inhibiting a particular molecular pathway and/or mechanism in an individual that comprises administering to the individual an effective of the antibody fragment to inhibit the particular molecular pathway and/or mechanism.
- the present disclosure provides an antibody fragment for use in activating a particular molecular pathway and/or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in a method of activating a particular molecular pathway and/or mechanism in an individual that comprises administering to the individual an effective of the antibody fragment to inhibit the particular molecular pathway and/or mechanism.
- An “individual” according to any of the above embodiments can be a human.
- the present disclosure provides for the use of an antibody fragment in the manufacture or preparation of a medicament.
- the medicament is for treatment of a particular disease.
- the medicament is for use in a method of treating a particular disease comprising administering to an individual having the disease an effective amount of the medicament.
- the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
- the medicament is for inhibiting or activating a particular molecular pathway and/or mechanism.
- the medicament is for use in a method of inhibiting or activating a particular molecular pathway and/or mechanism in an individual comprising administering to the individual an amount effective of the medicament to inhibit a particular molecular pathway and/or mechanism.
- An “individual” according to any of the above embodiments can be a human.
- the present disclosure provides a method for treating a particular disease.
- the method comprises administering to an individual having such disease an effective amount of an antibody fragment.
- the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.
- An “individual” according to any of the above embodiments can be a human.
- the present disclosure provides a method for inhibiting a particular molecular pathway and/or mechanism in an individual.
- the method comprises administering to the individual an effective amount of an antibody fragment to inhibit a particular molecular pathway and/or mechanism.
- an “individual” is a human.
- the present disclosure provides pharmaceutical formulations comprising any of the antibody fragments provided herein, e.g., for use in any of the above therapeutic methods.
- a pharmaceutical formulation comprises any of the antibody fragments provided herein and a pharmaceutically acceptable carrier.
- a pharmaceutical formulation comprises any of the antibody fragments provided herein and at least one additional therapeutic agent, e.g., as described below.
- Antibody fragments of the present disclosure can be used either alone or in combination with other agents in a therapy.
- an antibody fragment of the present disclosure can be co-administered with at least one additional therapeutic agent.
- Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the present disclosure can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents.
- administration of the antibody fragment and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.
- the antibody fragments described herein can also be used in combination with radiation therapy.
- An antibody fragment can be administered by any suitable means, including parenteral, intrapulmonary, intraocular, and intranasal, and, if desired for local treatment, intralesional administration.
- Parenteral infusions include intramuscular, intravenous, intraarterial, intraocular, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
- Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
- Antibody fragments are formulated, dosed and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
- the antibody fragment need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of such other agents depends on the amount of antibody fragment present in the formulation, the type of disease or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
- an antibody fragment of the present disclosure when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of antibody fragment, the severity and course of the disease, whether the antibody fragment is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody fragment, and the discretion of the attending physician.
- the antibody fragment is suitably administered to the patient at one time or over a series of treatments.
- about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody fragment can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
- One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
- One exemplary dosage of the antibody fragment would be in the range from about 0.05 mg/kg to about 10 mg/kg.
- one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient.
- Such doses can be administered intermittently, e.g. every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the antibody fragment).
- An initial higher loading dose, followed by one or more lower doses can be administered. The progress of this therapy is easily monitored by conventional techniques and assays.
- immunoconjugates which include an antibody fragment, disclosed herein, conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
- cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
- cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
- an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody fragment is conjugated to one or more drugs, including but not limited to, a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos.
- ADC antibody-drug conjugate
- drugs including but not limited to, a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMA
- an immunoconjugate includes an antibody fragment as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin
- an immunoconjugate includes an antibody fragment, as described herein, conjugated to a radioactive atom to form a radioconjugate.
- a variety of radioactive isotopes are available for the production of radioconjugates.
- Non-limiting examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
- a radioconjugate can include a radioactive atom for scintigraphic studies, for example tc99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
- NMR nuclear magnetic resonance
- Conjugates of an antibody fragment and cytotoxic agent can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
- a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987).
- Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethyl ene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026.
- the linker can be a “cleavable linker” facilitating release of a cytotoxic drug in the cell.
- an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.
- Non-limiting examples of linkers are disclosed above.
- the immunuoconjugates disclosed herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-STAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).
- cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC
- an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
- Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
- the containers can be formed from a variety of materials such as glass or plastic.
- the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and can have a sterile access port (for example the container can 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 antibody fragment of the present disclosure.
- the label or package insert indicates that the composition is used for treating the condition of choice.
- the article of manufacture can comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody fragment of the present disclosure; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
- the article of manufacture in this embodiment of the present disclosure can further comprise a package insert indicating that the compositions can be used to treat a particular condition.
- the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
- BWFI bacteriostatic water for injection
- phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
- any of the above articles of manufacture can include an immunoconjugate in place of or in addition to an antibody fragment as described herein.
- Fab and F(ab′) 2 antibody fragments serve as alternative formats to full-length antibodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function.
- proteases associated with invasive diseases are known to cleave antibodies in the hinge-region and result in anti-hinge antibodies (AHA) towards the neoepitopes.
- AHA anti-hinge antibodies
- Pre-existing AHA in serum can act as surrogate Fc and reintroduce the properties of the Fc lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments.
- T225 L mutation also referred to herein as the “T225L variant”
- T225L variant the natural C-terminal D 221
- IdeS was expressed as N-terminal glutathione S-transferase (GST) fusion protein.
- GST glutathione S-transferase
- the mature sequence of IdeS from Streptococcus pyogenes MGAS1882 (Uniprot ID H8HDRO) was codon optimized for E. coli expression and synthesized by GeneArtTM and cloned by standard molecular biology techniques into E. coli expression vector (23).
- IdeS was expressed using conditions described in Simmons et al. (23) and purified using glutathione sepharose (GSH) column.
- Eluate fractions in 50mM Tris-HCl, pH 8.0, 20 mM glutathione from the GSH column were concentrated and loaded onto S200 column and eluted with 200 mM K 2 HPO 4 , pH 6.2, 250mM KCl.
- Antibody and Fab purification After expression, the cells were pelleted by gravity. The supernatants were transferred to a 50 ml Falcon tube (Corning, Corning, N.Y., USA). 400 ⁇ l of 50% MabSelect SuReTM protein affinity slurry or Gamma BindTM Plus slurry (GE Healthcare, Pittsburgh, Pa., USA) was added to the supernatants for IgG and Fab purification respectively. The mixture was incubated overnight at room temperature on an Innova 2000 platform shaker (New Brunswick Scientific, Enfield, Conn., USA). Supernatants were removed and the resin transferred to a 96-well 2 ml filter plate with a 25 ⁇ m size membrane (Thompson Instrument, Oceanside, Calif., USA).
- the resin was washed three times with 1 ml of 1 ⁇ PBS pH 7.4 by centrifugation at 1,120 ⁇ g for 5 minutes using a SorvallTM HT6 Centrifuge (Thermo Scientific, Waltham, Mass., USA).
- the resin was further washed with 0.2 ⁇ PBS pH 5.0 before elution.
- the IgG was eluted using 50 mM phosphoric acid pH 2.9 and the eluate neutralized with 20 ⁇ PBS pH 11.0 by centrifugation at 1,000 ⁇ g for 5 minutes.
- the Fab fragments were eluted using 10 mM sodium citrate pH 2.9 and neutralized with 0.3 M Tris pH 9.0.
- the eluted IgG and Fab was filtered through 0.2 ⁇ m 96-well filter plate (Orochem, Naperville, Ill., USA) by centrifugation at 1,000 ⁇ g for 5 minutes using an Sorvall HT6 Centrifuge (Thermo Scientific, Waltham, Mass., USA).
- IdeS digestion of IgG hinge variants IgG at 1 mg/ml was incubated with stated IdeS:IgG ratio (w/w) at 37° C. for 24 hours.
- IdeS:IgG ratio w/w
- F(ab′) 2 purification after IgG cleavage by IdeS The IdeS cleaved sample was diluted with 25 mM Sodium Acetate, pH 4.4 (Buffer A) and loaded onto a 1 mL SP Sepharose High Performance strong cation exchange column (GE Healthcare, Pittsburgh, Pa., USA) at 150 cm/hr (0.7 cm diameter, 10 cm bed height) equilibrated in Buffer A. The column was washed to baseline with Buffer A and F(ab′) 2 eluted with a linear salt gradient from 0 to 0.5 M NaCl over 30 column volumes.
- Buffer A SP Sepharose High Performance strong cation exchange column
- the eluate was neutralized by addition of 3 M Tris pH 9.0 to adjust the pH to 7.0 and filtered through 0.22 ⁇ m STERIFLIP® (EMD Millipore, Billerica, Mass., USA).
- the SP eluted F(ab′) 2 was further purified on a MonoS 5/50 GL strong cation exchange column (GE Healthcare, Pittsburgh, Pa., USA) after diluting with Buffer A to lower the conductivity ⁇ 5 mS/cm.
- the column was washed to baseline ( ⁇ 0.05 mAU) with Buffer A and the F(ab′) 2 eluted using a salt gradient from 0 to 0.6 M NaC1 over 40 column volumes.
- the eluted F(ab′) 2 solution was neutralized with 3 M Tris pH 9.0 to adjust the pH to 7.0 and filtered through 0.22 ⁇ m STERIFLIP® (EMD Millipore, Billerica, Mass., USA).
- Mass spectrometry of Fab and F(ab′) 2 fragments Mass spectrometric data was acquired using an Agilent 6224 TOF LC-MS system (Agilent Technology, Santa Clara, Calif., USA). F(ab′) 2 was reduced with 100 mM dithiothreitol at 37° C. for 20 minutes. The polypeptide chains were separated with a PLRP-S reversed phase column (Agilent Technologies, Santa Clara, Calif., USA). Intact masses of the reduced light and heavy chains were obtained by Maximun Entropy Deconvolution using MassHunter software (Qualitative Analysis B.03.01).
- ELISA enzyme-linked immunosorbent assay
- the reaction was stopped with 1 M phosphoric acid and absorbance was read at 450 nm. The absorbance readings at 1:30 dilution were used for the figures to allow presentation of all samples. Comparable results were observed at a 1:10 serum dilution.
- the titration curve of F(ab′) 2 was fitted with a 4 parameter curve fitting program (KaleidaGraph, Synerg Software, Reading, Pa.). MidOD (the average OD of the top and bottom OD readings) of the F(ab′) 2 titration curve was determined. The dilutions of Fab DKTHT (SEQ ID NO: 21) and F(ab′) 2 corresponding to this midOD were calculated and used to calculate the relative AHA reactivity.
- SDS-PAGE and immunoblots For SDS-PAGE, 5 ⁇ g of purified F(ab′) 2 variants and GST-IdeS were mixed with SDS-sample buffer, heated for 5 min at 95° C. and spun for 1 min at 16,000 relative centrifugal force. The samples were loaded onto a NuPAGE 4-12% BisTris/MES gels (Invitrogen). For immunoblotting, 5 ng of protein samples were used for SDS-PAGE. Gels were transferred by IBLOT® (Invitrogen) onto nitrocellulose membrane, immunoblotted with anti-IdeS (Genovis, USA; catalog no. A3-AF1-010, lot no.
- A3AF1-7C17H as primary antibody and IRDye800CW conjugated donkey anti-goat antibody (Li-COR®, USA; catalog no. 926-32214, lot no. B80821-03) as the secondary antibody, and imaged with a LI-COR® Odyssey® Imager (LI-COR®, USA).
- LI-COR® LI-COR® Imaging System
- LI-COR® Two-color protein molecular weight marker was used for immunoblots and pre-stained SEEBLUE® Plus2 (Invitrogen, USA) was used for Coomassie stained gels.
- Protein stability measurements by differential scanning fluorimetry Protein stability was determined in a Biorad CFX96 TOUCHTM Real-Time System (Biorad, USA) with a final dilution of 1:200 of the SYPRO® Orange dye stock (Molecular ProbesTM, USA). 1 ⁇ l of the SYPRO® Orange dye stock was added to 24 ⁇ l of the purified antibody at 100 ⁇ g/ml. Fluorescence of the final 25 ⁇ l sample in PBS was recorded from 20-100° C. (0.2° C. increments, 10 seconds hold per step).
- Fab C-terminus determines response to pre-existing AHA: Originally, reactivity of autoantibodies in human serum towards the upper hinge of Fab molecules was studied with a papain cleaved antibody, abciximab (5). Papain cleavage leaves a C-terminal H 224 on the Fab. Later, a more comprehensive study was carried out using biotinylated peptide analogs to dissect the contribution of individual C-terminal residues of the upper hinge (20). In this study, only minimal AHA reactivity towards the upper hinge residues K 222 through H 224 was observed. No signal was observed towards peptides with T 225 as the C-terminal residue.
- T 225 at the C-terminus having the sequence DKTHT (SEQ ID NO: 21), also referred to herein as “CDKTHT” (SEQ ID NO: 14)
- DKTHT sequence DKTHT
- CDKTHT sequence D
- D 221 at the C-terminus having the sequence D, also referred to herein as “CD”
- binding of AHA was reduced almost to background.
- terminating the Fab at D 221 provides a solution to minimize recognition by pre-existing AHA while maintaining a natural antibody sequence.
- the C-terminal Fab residue has a profound impact on AHA binding. Whether binding by pre-existing antibodies could also be obviated by a single amino acid change at the C-terminus and provide an alternative route to the D 221 to minimize reactivity towards AHA was investigated.
- a T 225 L variant was introduced to place a non-natural residue at the Fab C-terminus, and its binding of AHA was tested. The T 225 L variant has been described previously (7).
- the mutation perturbed binding of the pre-existing AHA ( FIG. 1B ), further highlighting the importance of the C-terminus for binding.
- IgG2 and IgG4 are commonly used for therapeutic antibodies, the use of IgG2 and IgG4 Fabs has not been leveraged for therapeutic development so far.
- IgG2 and IgG4 Fabs with the complete upper hinge region ( FIG. 1D ; C-terminus K 218 and P 225 , respectively) were tested.
- the IgG2 and IgG4 Fabs were not recognized by pre-existing AHA.
- the upper hinge of the IgG4 was truncated.
- the length of the IgG4 isotype upper hinge is shorter when compared to IgG1 (see FIG. 6A-B ); however, since the cysteine involved in the heavy-light interchain disulfide is located in the center of the C H 1 primary structure, it was able to include residues K 218 and Y 219 in the truncation experiments. These truncated upper hinge Fabs displayed a signal similar to the intact IgG1 upper hinge ( FIG. 1B ).
- T30M Fab determined by differential scanning fluorimetry Human isotype C-terminus Tm (° C.) IgG1 ..DKTHL (SEQ ID NO: 20) 81.6 ..DKTHT (SEQ ID NO: 21) 81.8 ..DKTH (SEQ ID NO: 27) 81.7 ..DKT 81.8 ..DK 81.7 ..D 81.9 IgG2 ..K 75.5 IgG4 ..KYGPP (SEQ ID NO: 18) 75.9 ..KYG 75.7 ..KY 75.8 ..K 75.6
- IgG1 with the lower hinge of IgG2 cannot be cleaved efficiently: Pre-existing AHA towards the lower hinge region of F(ab′) 2 have been extensively described in the literature (13, 27). Analogous to the AHA to the upper hinge of Fab molecules, these AHA can act as surrogate Fc or introduce assay artifacts. Thus, development of a F(ab′) 2 format that impedes AHA binding is desirable. While AHA in serum are found towards F(ab′) 2 of IgG1 isotype, it has not been possible to establish existence of autoantibodies to the lower hinge of IgG2 isotype.
- IdeS protease an IgG specific endoprotease from Streptococcus pyrogenes that cleaves after G236. In addition to the cleavage site in the antibody hinge region, it recognizes a second site in the Fc that contributes to its high specificity towards IgG (15, 16). The inefficient cleavage of IgG2 antibodies could be caused by the exosite outside of the cleavage site.
- the lower hinge residues of IgG2 were grafted onto IgG1 to create an IgG1-2 chimera ( FIG. 2B ).
- the cleavage efficiency at different IdeS:IgG ratios was tested ( FIG. 2C ). While IgG1 wild-type was efficiently cleaved into F(ab′) 2 at a IdeS:IgG ratio of 1:500, at least 50-fold higher protease concentration was necessary to achieve similar cleavage of the IgG1-2 chimera. It was concluded that the sequence differences in the lower hinge at least partially contribute to the poor cleavage efficiency of IgG2 antibodies. Thus, the IgG1-2 chimera may not be a practical strategy to generate F(ab′) 2 molecules that do not bind to pre-existing AHA.
- the antibodies were purified and digested with IdeS at an IdeS:IgG ratio of 1:10 to identify variants that can be cleaved by IdeS.
- IdeS:IgG ratio 1:10 to identify variants that can be cleaved by IdeS.
- Such high protease to antibody ratio was chosen to assess proteolysis without taking cleavage efficiency into account.
- Seven variants were identified that were cleaved by IdeS ( FIG. 3A ). While the P2 position tolerated all four residues tested, only two amino acids with very small side chains, the natural glycine and alanine, were accepted in the P1 position.
- This subset was further investigated for cleavage efficiency at three different IdeS:IgG ratios: 1:500, 1:200 and 1:100 ( FIG. 3B ) and 1:10 ( FIG. 3C ).
- the variant L 235 V in the P2 position demonstrated minimal change in cleavage efficiency compared to the wild-type sequence. All other variants were characterized by a dominant single cleavage at only one side of the hinge, leaving the other side of the antibody intact. While L 2351 and L 235 M variants with glycine in the P1 position could be completely cleaved at IdeS:IgG ratio of 1:200, all P2 variants with alanine in P1 needed significantly higher protease amounts. Hence, efficient cleavage by IdeS requires glycine in the P1 position.
- C-terminal F(ab′) 2 variants do not prevent recognition by pre-existing AHA: IdeS can confound results of the pre-existing AHA assay with a false positive result. This can be explained by binding of serum antibodies by IdeS and their subsequent recognition by the anti-Fc detection antibody. Therefore, it was ensured that F(ab′) 2 molecules used in the assay did not contain IdeS from the preceding proteolysis reaction. The removal of IdeS in the purified F(ab′) 2 proteins was confirmed by SDS-PAGE followed by Coomassie staining and anti-IdeS immunoblot ( FIG. 4A ).
- the purified P1 and P2 F(ab′) 2 variants were then tested for their recognition to pre-existing AHA ( FIG. 4B ). While there was a reduction of signal compared to the wild-type, none of the antibodies with C-terminal modifications eliminated the reactivity with serum autoantibodies. Changes to the P2 position only had a modest impact. The G 236 A change in the P1 position reduced the signal to levels comparable to a Fab. Without being limited to a particular theory, it is thus not possible to design a lower hinge with an amino acid variant that retains cleavage by IdeS and concurrently eliminates pre-existing antibody response against the F(ab′) 2 .
- Antibodies with deletions of P4, P5 and a combination of both were tested for binding to AHA ( FIG. 5B ). For these variants, a lower signal was observed compared to the wild-type hinge sequence. To further reduce hinge recognition by pre-existing AHA, the deletion was extended to include the P6 (EU231) and P7 (EU230) residues (see FIG. 5F ).
- the observed AHA binding signal of the ⁇ P456 variant F(ab′) 2 is comparable to the two Fab C-terminal variants, Fab D 221 and Fab T 225 L.
- the dilutions corresponding to the OD (1.15) at the middle of the F(ab′) 2 titration curves were 70 and 14 for F(ab′) 2 and Fab, respectively.
- the five-fold higher AHA activity seen with F(ab′) 2 compared to Fab T 225 can be explained by the potential bivalent binding of AHA to the F(ab′) 2 , which indicates that F(ab′) 2 molecules need avoid pre-existing AHA.
- the variant L 235 V or G 236 A in combination with a deletion at P5 resulted in a reduced AHA signal compared to the deletion of P5 alone.
- the ⁇ P456 variant that included L 235 V, L 235 I, or L 235 M at P2 position with alanine or glycine in P1 exhibited similar reductions in AHA signal compared to ⁇ P456 alone ( FIG. 10B ); however, the deletion of ⁇ P456 alone exhibited better cleavage efficiency compared to the variants in combination with ⁇ P456 ( FIG. 10A ).
- Antibody fragments such as Fab and F(ab′) 2 are attractive therapeutic formats when a short systemic half-life and an effector-silent molecule are concurrently desired. Certain fragments are also natural products of proteases associated with invasive diseases such as tumor cells and bacteria and are generated in an effort to evade immune surveillance. As a consequence, the C-terminal neoepitope of the Fab and F(ab′) 2 fragment is recognized by the immune system and result in AHA that can provide surrogate Fc.
- IgG1 isotype seems to be the prime target for these proteases. Indeed, several proteases have been described to cleave in the upper hinge of human IgG1, including plasmin, human neutrophil elastase and LysC. This study shows that pre-existing AHA exist towards all cleavage sites of the upper hinge of human IgG1 with the exception of D 221 .
- the absence of AHA toward D 221 may be a reflection of the inability of human proteases to cleave after this residue or the inability to raise antibodies toward this neoepitope.
- the highest reactivity was observed towards the C-terminal T 223 Fab.
- this C-terminus coincides with the cleavage point of human neutrophil elastase, a protease that is secreted by neutrophils and macrophages during inflammation to destroy bacteria and host tissue (29).
- Pre-existing AHA can rapidly recruit effector-function to a molecule that is designed to be effector-less.
- Using a Fab of IgG2 or IgG4 isotype can provide one strategy to supply a molecule without pre-existing antibody response.
- introducing a non-natural residue at the heavy chain C-terminus, like the T 225 L mutation, or truncating the upper hinge to D 221 is a strategy for the IgG1 isotype. While the T 225 L mutation eliminates the response towards pre-existing AHA, it implies that in principle, it can elicit an immune response as well. This is further supported by a recent study with an anti-TNFR1 domain antibody (34).
- the Fab-D 221 can also be considered for the recombinant expression of Fab for crystallographic studies, since the upper hinge is not commonly resolved in crystal structures due to the unstructured nature. This example demonstrates that equal stability and expression can be achieved with a Fab-D 221 construct. Eliminating the unstructured areas may further improve crystallization outcomes.
- F(ab′) 2 molecules so far is by proteolytic digest and proteases with high-specificity such as IdeS are preferred.
- pre-existing AHA also exist towards the lower hinge of F(ab′) 2 molecules.
- the AHA titer towards IdeS-cleaved antibody is higher compared to a Fab. Without being limited to a particular theory, this may be due to the bivalent nature of the F(ab′) 2 that provides an avidity based binding component in the assay or a natural higher abundance of F(ab′) 2 that lead to increased titers.
- Several strategies were employed to remove the AHA reactivity while maintaining cleavage efficiency by IdeS.
- the first strategy was to mutate the C-terminal residue of the F(ab′) 2 to remove binding activity of AHA.
- the selected set of mutations in the P2-site had only modest impact on AHA binding, further confirming the importance of the C-terminal residue for reactivity with AHA.
- the coinciding differences in AHA binding with the IdeS cleavage efficiency of the P1 and P2 variants perhaps explains the reason why the IgG2 isotype with valine in P2 and alanine in P1 position is less susceptible to anti-hinge antibody response.
- the requirement for glycine in position P1 for efficient cleavage is accompanied by the high conservation of this residue within different isotypes and across species.
- Pre-existing antibodies have also been demonstrated to decrease effector-function (32) in vitro and can also confound immunogenicity assays during drug development. While the majority of anti-therapeutic antibodies (ATAs) towards a humanized antibody are targeting the idiotype, rheumatoid factor, a low affinity antibody towards the Fc region has been described.
- One way to eliminate artifacts by rheumatoid factor in the immunogenicity assay is to use an antibody fragment devoid of the Fc region. However, it is import to use fragments that are not recognized by other pre-existing antibodies.
- Fab molecules by choosing the appropriate antibody fragment, it is possible to evade recognition by pre-existing AHA.
- Fab molecules several options exist: (1) using an IgG2 or IgG4 isotype, (2) a mutation of the C-terminal residue (T225L), or (3) terminating the Fab with residue D 221 .
- Options are more limited for F(ab′) 2 molecules due to the need for proteolytic digestion.
- a deletion was identified in the lower hinge of IgG1, however, that maintains high cleavage efficiency and specificity and removes reactivity with pre-existing AHA. Using these formats can enable further minimization of safety concerns with antibody fragments in a therapeutic setting and remove interference in assay development.
- F(ab) 2 A1 3 456 has Reduced AHA Mediated Fc ⁇ RIIIa and C1q Binding
- AHA antibodies can act as surrogate Fc and restore ADCC/CDC function that was lost by IdeS generated F(ab′) 2 (20, 22).
- IdeS generated F(ab′) 2 (20, 22).
- Fab and F(ab′) 2 variants were further reflected by reduced recruitment of Fc ⁇ receptors and C1q.
- bridging experiments were employed.
- human serum was added to Fab or F(ab′) 2 coated wells and incubated for 2 hours as described above.
- soluble Fc ⁇ RIIIa(V158)-His-GST consisting of the extracellular domain fused with Gly-His6-glutathione-S-transferase (“Gly-His6” disclosed as SEQ ID NO: 28) at the carboxy-terminus
- Gly-His6 Gly-His6
- Bound Fc ⁇ RIIIa(V158)-His-GST was detected using horseradish peroxidase-conjugated mouse anti-His antibody (Penta-His (SEQ ID NO: 29), Qiagen, Germantown, Md.) followed by TMB as the substrate.
- human serum was added to Fab or F(ab′) 2 coated wells and incubated for 2 hours as described above. After the plates were washed, purified human C1q (Quidel, San Diego, Calif.) was added. Bound C1q was detected with goat anti-C1q antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) followed by rabbit anti-goat IgG -HRP (Jackson ImmunoResearch, West Grove, Pa.) and TMB as the substrate.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The instant disclosure provides antibody fragments (e.g., Fab and F(ab′)2) having reduced or no reactivity towards pre-existing anti-hinge antibodies (AHA) and compositions comprising such antibody fragments, as well as methods of making and using such antibody fragments and compositions.
Description
- This application is a continuation of U.S. patent application Ser. No. 15/967,154, filed Apr. 30, 2018, which is a continuation of International Patent Application No. PCT/US2016/059137, filed Oct. 27, 2016, which claims priority to U.S. Provisional Patent Application Ser. No. 62/248,792, filed Oct. 30, 2015, and U.S. Provisional Patent Application Ser. No. 62/346,905, filed Jun. 7, 2016, the contents of each of which are incorporated by reference in their entirety, and to each of which priority is claimed.
- The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 17, 2020, is named 00B206_0908_SL.txt and is 26,245 bytes in size.
- The present disclosure relates to antibody fragments (e.g., Fab and F(ab′)2) having reduced or no reactivity towards pre-existing anti-hinge antibodies (AHA) and compositions comprising such antibody fragments, as well as methods of making and using such antibody fragments and compositions.
- Antibodies are composed of two Fab regions that are connected by a flexible hinge-region to the Fc. While the Fab mediates recognition and binding of the antigen, two important functions of the Fc are to mediate effector function by engagement with Fcy receptors (1) and to confer long serum half-life by binding to the salvage receptor, FcRn (2). In particular, the slow pharmacokinetics of IgG contribute to the success of antibodies as therapeutics as it enables less frequent dosing compared to other biotherapeutics. Consequently, the majority of approved therapeutic antibodies have full-length IgG format. Unlike IgG, the serum half-life of an isolated Fab fragment is short (3) and such property is required for indications when short plasma half-life is desired as with three FDA approved Fab molecules (4). One therapeutic Fab molecule directed against platelet surface receptor GPIIb/IIIa (abciximab, REOPRO®) is commercially produced by proteolytic cleavage with papain (5), which is the original method of Fab production (6). With the advances in molecular cloning, recombinant expression of antibody fragments has become an attractive route to generate Fab molecules as exemplified by the second approved Fab therapeutic, anti-VEGF (ranibizumab, Lucentis®) (7) and the recently approved Fab against dabigatran (idarucizumab, Praxbind®) (33). Fab molecules are advantageous, for example, when transient systemic activity that does not persist past dosing is desired or when administration and activity are localized to a peripheral compartment such as the eye.
- Many proteases against the antibody hinge region have been implicated as the mechanism by which pathogens and tumor cells attempt to evade the host immune response (13). Resulting C-terminal neoepitopes, however, are eventually recognized by the immune system and anti-hinge antibodies (AHA) are generated. Such autoantibodies to the upper-hinge region of the Fab and the lower-hinge region of the F(ab′)2 have been shown in several studies (17-21). These pre-existing AHA titers vary from donor to donor (20) and it may represent past and current exposure to such neoepitopes. In certain instances, AHA can act as surrogate Fc and restore effector function of proteolytically inactivated antibodies (22). As one rationale for using a Fab or F(ab′)2 molecule as the therapeutic format is to eliminate effector function, it would be undesirable to have effector function reinstated by pre-existing AHA and risk any potential safety concerns. Accordingly, there is a need in the art for novel Fab and F(ab′)2 molecules that have reduced or no reactivity with pre-existing AHA in human serum to, inter alia, potentially provide a superior safety profile in a therapeutic setting by minimizing immune responses following drug treatment.
- The present disclosure relates to antibody fragments (e.g., Fab and F(ab′)2) having reduced or no reactivity towards pre-existing anti-hinge antibodies (AHA) and compositions comprising such antibody fragments, as well as methods of making and using such antibody fragments and compositions.
- In certain embodiments, the present disclosure is directed to an isolated antibody fragment and compositions comprising the same, wherein the antibody fragment has reduced or no reactivity towards pre-existing anti-hinge antibodies. In certain embodiments, an isolated antibody fragment of the present disclosure exhibits reduced and/or no binding to FcyRIIIa and/or C1q. In certain embodiments, the antibody fragment is a Fab, Fab′ or F(ab′)2.
- In certain embodiments, the present disclosure is directed to antibody fragments and compositions comprising the same, wherein the antibody fragment is a Fab. In certain embodiments, the present disclosure is directed to Fab molecules wherein the Fab terminates with residue D221. In certain embodiments, the Fab terminates with amino acids comprising an amino acid sequence selected from group consisting of CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK and CD. In certain embodiments, the Fab terminates with amino acids comprising an amino acid sequence selected from group consisting of KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY and K. In certain embodiments, the Fab comprises a heavy chain constant region that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and conservative modifications of.
- In certain embodiments, the present disclosure is directed to an antibody fragment and compositions comprising the same, wherein the antibody fragment is a F(ab′)2. In certain embodiments, the present disclosure is directed to F(ab′)2 molecules, wherein the F(ab′)2 comprises a C-terminal deletion of 1, 2, 3, 4, or 5 amino acids. In certain embodiments of the present disclosure, the F(ab′)2 comprises a deletion at position EU231. In certain embodiments of the present disclosure, the F(ab′)2 comprises a deletion at positions EU231-232. In certain embodiments of the present disclosure, the F(ab′)2 comprises a deletion at positions EU231-233. In certain embodiments of the present disclosure, the F(ab′)2 comprises a deletion at position EU231-234. In certain embodiments, the F(ab′)2 comprises a deletion at position EU230-234.
- In certain embodiments, the present disclosure is directed to an isolated nucleic acid and compositions comprising the same, wherein the nucleic acid encodes an antibody fragment having reduced or no reactivity to AHA. In certain embodiments, the present disclosure is directed to a host cell comprising said nucleic acid. In certain embodiments, the present disclosure is directed to a method of producing an antibody fragment comprising culturing said host cell so that the antibody fragment is produced. In certain embodiments, the present disclosure is directed to a pharmaceutical formulation comprising an antibody fragment having reduced or no reactivity to AHA and a pharmaceutically acceptable carrier.
- In certain embodiments, the present disclosure is directed to an antibody fragment having reduced or no reactivity to AHA for use as a medicament. In certain embodiments, the present disclosure is directed to an antibody fragment having reduced or no reactivity to AHA for use in treating a disease. In certain embodiments, the present disclosure is directed to an antibody fragment having reduced or no reactivity to AHA for use in inhibiting or activating a molecular pathway and/or mechanism. In certain embodiments, the present disclosure is directed to the use of an antibody fragment having reduced or no reactivity to AHA in the manufacture of a medicament for the treatment of a disease. In certain embodiments, the present disclosure is directed to the use of an antibody fragment having reduced or no reactivity to AHA in the manufacture of a medicament for inhibiting or activating a molecular pathway and/or mechanism.
- In certain embodiments, the present disclosure is directed to methods of treating an individual having a disease comprising administering to the individual an effective amount of an antibody fragment having reduced or no reactivity to AHA. In certain embodiments, the present disclosure is directed to methods of inhibiting or activating a molecular pathway and/or mechanism in an individual comprising administering to the individual an effective amount of an antibody fragment having reduced or no reactivity to AHA to inhibit or activate a molecular pathway and/or mechanism
-
FIGS. 1A-1D show the binding of pre-existing human antibodies to the Fab of human IgG1, IgG2 and IgG4. (1A) X-ray crystal structure of the Fab region (PDB: 1HZH) including the upper hinge; light chain (101), heavy chain (102), interchain disulfide (103), and upper hinge (104). In the isolated Fab molecule the upper hinge is a protruding unstructured region without structural and functional role. The residues of the upper hinge are displayed in magenta to indicate the T225L mutation (105) that is perturbing binding to pre-existing AHA. Numbering of residues is according to EU numbering nomenclature.FIG. 1A discloses SEQ ID NOS 14-15, respectively, in order of appearance. (1B) Pooled human serum was incubated with human IgG1 Fab with different upper hinge lengths and ends. Binding pre-existing antibodies detected by anti-Fc ELISA. Truncating the Fab C-terminus to D221 (D) and the C-terminal variant T225L (DKTHL (SEQ ID NO: 20)) greatly reduced binding of pre-existing antibodies to almost background. Strong response was observed towards T223 as the C-terminal residue (DKT), coinciding with the cleavage site of human neutrophil elastase. The mean value of the individual data points is represented by the horizontal line.FIG. 1B discloses SEQ ID NOS 20-21 and 27, respectively, in order of appearance. (1C) Three different Fabs were incubated with pooled human serum and binding of pre-existing antibodies detected by ELISA. Significant signal was observed for different Fabs with DKTHT (SEQ ID NO: 21) C-terminus. Reduced binding of pre-existing antibodies to the D221 and T225L C-terminus was detected across different Fabs. Fab-1 includes the antibody variable domain used in (B) and all other AHA binding experiments throughout Example 1.FIG. 1C disclosesSEQ ID NOS FIG. 1D discloses SEQ ID NO: 18. -
FIGS. 2A-2C show the cleavage of IgG1-2 chimera by IdeS. (2A) Model of the F(ab′)2 region of antibody cAC10 modeled with MOE; light chain (201), heavy chain (202), interchain disulfide (203), and lower hinge (204). The P1 position of IdeS is G236. Numbering of residues is according to EU numbering nomenclature.FIG. 2A discloses SEQ ID NO: 30. (2B) Alignment of the lower hinge of IgG1 and the IgG1-2 chimera. Residues in cyan are IgG2 isotype residues introduced into the lower hinge of IgG2.FIG. 2B discloses SEQ ID NOS 31 and 59, respectively, in order of appearance. (2C) Cleavage efficiency of human IgG1 and IgG1-2 chimera. 1 mg/ml of IgG1 and IgG1-2 were incubated for 24 hours at 37° C. with different IdeS amounts as indicated. Cleavage was analyzed by capillary electrophoresis. While IgG1 was efficiently cleaved into F(ab′)2 at an IdeS:IgG ratio of 1:500, IgG1-2 requires 50-fold higher IdeS concentrations for complete cleavage. -
FIGS. 3A-3E show the cleavage of human IgG1 with variants in the P1 and P2 position by IdeS. (3A) Capillary electrophoresis of antibodies with variants at the P1 and P2 position were digested 24 hours at 37° C. with a 1:10 ratio of IdeS:IgG at 1 mg/ml. The P1 and P2 residues are designated in 1-letter code. Leucine and glycine (L235G236) are the natural amino acids at these positions. All antibody variants can be completely cleaved into F(ab′)2 fragments. (3B) The cleavage efficiency of the variants was assessed by amount of F(ab′)2 produced at different IdeS:IgG ratios. While the variant VG is cleaved comparably to the wild-type sequence (LG), other variants require increased amounts of IdeS for complete digestion. (3C) The cleavage efficiency of the variants was assessed by the amount of F(ab′)2 produced at an IdeS:IgG ratio of 1:10. - (3D) Schematic diagrams of the expression, purification and screening strategies for the human IgG1 variants.
FIG. 3D discloses SEQ ID NOS 31-34, respectively, in order of appearance. (3E) Cleavage efficiency of the 76 human IgG1 variants with IdeS.FIG. 3E discloses SEQ ID NO: 31. -
FIGS. 4A-4B show the reactivity of P1 and P2 variants towards pre-existing AHA. (4A) IdeS is efficiently removed during purification and cannot be detected in the purified F(ab′)2 variants by SDS-PAGE followed by coomassie staining (upper panel) or immunoblot analysis with anti-IdeS antibodies (lower panel). (4B) Pooled human serum was incubated with human IgG1 Fab with T225 C-terminus and F(ab′)2 generated by IdeS cleavage of antibodies with variants in P1 and P2 positions. Binding pre-existing antibodies were detected by ELISA. The F(ab′)2 showed a signal that was about 1.7 fold higher compared to the Fab. The hinge variants reduced reactivity to levels comparable to Fab but did not eliminate reactivity completely. -
FIGS. 5A-5F show the reactivity of truncated variants towards pre-existing AHA response. (5A) IdeS cleavage of antibodies with deletions in the IdeS P3, P4, and P5 sites. While deletion of the IdeS P3 residue (L234) in the lower hinge severely impacted cleavage efficiency, deletion of the P4 (E233) or P5 (P232) positions did not impact cleavage with IdeS compared to wild-type (WT). (5B) Deletion of the P4 and P5 positions was not sufficient to avoid binding of pre-existing AHA. (5C) IdeS cleavage of antibodies with deletions of the IdeS P4 through P6 (AP456) and P4 through P7 (ΔP4567) sites. While cleavage efficiency of the ΔP4567 variant was slightly reduced compared to the wild-type lower hinge sequence (WT), ΔP456 displayed cleavage efficiency comparable to the wild-type at IdeS:IgG ratio of 1:200. (5D) Pooled human serum was incubated with F(ab′)2 produced by IdeS digestion and binding antibodies detected by ELISA. Lower hinge deletions ΔP456 and ΔP4567 were not recognized by pre-existing AHA. (5E) IdeS cleaved the ΔP456 hinge variant with high specificity. After digestion of wild-type (WT) and ΔP456 hinge IgG, reduced F(ab′)2 was analyzed by mass spectrometry. Only a single heavy chain species corresponding to the expected molecular mass was observed. (5F) Schematic diagram depicting the deletions generated in the lower hinge region.FIG. 5F discloses SEQ ID NOS 35, 31, 36-40, 31, and 41-42, respectively, in order of appearance. -
FIGS. 6A-6B show the alignment of the amino acid residues (6A) and EU numbering of the amino acid residues (6B) within the upper, core and lower hinge regions of the IgG1, IgG2, IgG3 and IgG4 isotypes in human, cynomolgus monkey and rhesus monkey.FIG. 6A discloses SEQ ID NOS 43-54, respectively, in order of appearance.FIG. 6B discloses SEQ ID NOS 43, 46, 55 and 52, respectively, in order of appearance. -
FIG. 7 shows the expression levels of Fabs with upper hinge truncations or mutations in E. coli. Figure discloses SEQ ID NOS 14-15, respectively, in order of appearance. -
FIG. 8 shows the efficiency of the cleavage of the ΔP456 and ΔP4567 variant produced at an IdeS:IgG ratio of 1:500 or 1:10. Figure discloses SEQ ID NOS 56-58, respectively, in order of appearance. -
FIGS. 9A-9D show the reactivity of deletion variants with modified P1 and P2 residues with pre-existing AHA. (9A) The cleavage efficiency of the variants was assessed by amount of F(ab′)2 produced at an IdeS:IgG ratio of 1:10.FIG. 9A discloses SEQ ID NO: 31. (9B) The cleavage efficiency of the variants was assessed by the amount of F(ab′)2 produced at an IdeS:IgG ratio of 1:100.FIG. 9B discloses SEQ ID NO: 31. (9C) The cleavage efficiency of the variants was assessed by amount of F(ab′)2 produced at an IdeS:IgG ratio of 1:500.FIG. 9C discloses SEQ ID NO: 31. (9D) Detection of the bound pre-existing AHA to the variants by anti-Fc ELISA.FIG. 9D discloses SEQ ID NO: 56. -
FIGS. 10A-10B show the reactivity of the ΔP456 and ΔP4567 variants with modified P1 and P2 residues to pre-existing AHA. (10A) The cleavage efficiency of the variants was assessed by amount of F(ab′)2 produced at an IdeS:IgG ratio of 1:10 and 1:200.FIG. 10A discloses SEQ ID NOS 56-57, respectively, in order of appearance. (10B) Detection of pre-existing AHA bound to the variants by anti-Fc ELISA.FIG. 10B discloses SEQ ID NO: 56. -
FIG. 11 shows the titration curves of F(ab′)2 and Fab molecules in the AHA ELISA. The dilutions corresponding to theOD 450 nm (1.15) at the middle of the F(ab′)2 titration curves were 70 and 14 for F(ab′)2 and Fab, respectively. Thus, F(ab′)2 has five fold higher AHA reactivity than IgG1 Fab. F(ab′)2, F(ab′)2 ΔP456, Fab T225, Fab T225L and Fab D221 were coated on the wells. Serial dilutions of pooled human serum were added to the wells and control wells were uncoated. Similar results were obtained in 4 other experiments. The data shown here and inFIG. 1B andFIG. 5D were collected from the same experiment. Figure disclosesSEQ ID NOS 21 and 20, respectively, in order of appearance. - The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody fragment is a Fab molecule. In certain embodiments, the antibody fragment is a F(ab′)2 molecule.
- The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
- “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (
C H1,C H2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (k), based on the amino acid sequence of its constant domain. - The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
- “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIM (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIM contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.
- The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).
- Binding to human FcRn in vivo and serum half-life of human FcRn high affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered. WO 2000/042072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).
- “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody 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 receptor); and B cell activation.
- The “hinge region” is generally defined as stretching from 216-238 (EU numbering) or 226-251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions, the upper, middle (e.g., core) and lower hinge. See, e.g., Brerski and Georgiou, Curr. Opin. Immunol. 40, 62-69 (2016), which is incorporated by reference herein in its entirety. In certain embodiments, the hinge region of a human IgG1 antibody is generally defined as follows:
- The upper hinge comprises amino acids having the sequence EPKSCDKTHT (SEQ ID NO: 22). In certain embodiments, the upper hinge comprises the amino acids at positions 216-225 (EU numbering) or 226-238 (Kabat numbering).
- The middle (e.g., core) hinge comprises amino acids having the sequence CPPC (SEQ ID NO: 23). In certain embodiments, the core hinge comprises the amino acids at positions 226-229 (EU numbering) or 239-242 (Kabat numbering).
- The lower hinge comprises amino acids having the sequence PAPELLGGP (SEQ ID NO: 24). In certain embodiments, the lower hinge comprises the amino acids at positions 230-238 (EU numbering) or 243-251 (Kabat numbering).
- The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ϵ, γ and μ, respectively.
- An “isolated” antibody or antibody fragment is one which has been separated from a component of its natural environment. An antibody or an antibody fragment can be purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
- “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
- In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
-
100 times the fraction X/Y - where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
- An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
- The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
- The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny can be completely identical in nucleic acid content to a parent cell, or can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
- As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In certain embodiments, antibody fragments of the present disclosure are used to delay development of a disease or to slow the progression of a disease.
- An “effective amount” of an agent, e.g., an antibody fragment disclosed herein, or a pharmaceutical formulation comprising an agent, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
- The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
- A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.
- As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
- In certain embodiments, the present disclosure is based, in part, on methods of engineering antibody fragments to evade pre-existing anti-hinge antibodies (AHA). In certain embodiments, antibody fragments (e.g., Fab and F(ab′)2) that have reduced or no reactivity towards AHA and methods of making these antibody fragments are provided. In certain embodiments, antibody fragments of the present disclosure can provide superior safety in a therapeutic setting by minimizing immune response following drug treatments.
- In certain embodiments, the present disclosure provides antibody fragments (e.g., Fab, Fab′ and F(ab′)2), and compositions comprising the same, that have reduced or no reactivity towards AHA. For example, but not by way of limitation, an antibody fragment disclosed herein exhibits AHA reactivity that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to a reference antibody fragment, e.g., an antibody fragment with a native hinge region. In certain embodiments, the reference antibody fragment is an IgG1 antibody fragment that has a native hinge region.
- In certain embodiments, the isolated antibody fragments of the present disclosure, and the compositions comprising the same, exhibit reduced and/or no binding to FcγRIIIa and/or C1q. For example, and not by way of limitation, an antibody fragment of the present disclosure exhibits binding to FcγRIIIa and/or C1q that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% relative to a reference antibody fragment, e.g., an antibody fragment with a native hinge region. In certain embodiments, the reference antibody fragment is an IgG1 antibody fragment that has a native hinge region.
- In certain embodiments, an antibody fragment employed in the context of the methods described herein comprises a native hinge region or a modified hinge region. For example, and not by way of limitation, an antibody fragment of the present disclosure can be a Fab fragment that comprises a native hinge region or modified hinge region. In certain embodiments, the antibody fragment of the present disclosure is a F(ab′)2 that comprises a native hinge region or modified hinge region.
- A native hinge region is a hinge region normally associated with the
C H1 domain of an antibody molecule. In certain embodiments, the native hinge region of a presently disclosed antibody fragment can be of the IgG1, IgG2, IgG3 or IgG4 isotype. For example, and not by way of limitation, the Fab fragment can be of the IgG1, IgG2, IgG3 or IgG4 isotype. In certain embodiments, the antibody fragment, e.g., the Fab fragment, is of the IgG2 isotype comprising a native hinge region. In certain embodiments, the antibody fragment, e.g., the Fab fragment, is of the IgG4 isotype comprising a native hinge region. - A modified hinge region is any hinge that differs in length and/or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions can comprise a complete hinge region derived from an antibody of a different class or subclass from that of the
C H1 domain. Thus, for instance, aC H1 domain of class γ1 can be attached to a hinge region of class γ4. Alternatively, the modified hinge region can comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region. - In certain embodiments, the native hinge region is altered by substituting, deleting and/or adding one or more amino acid residues to generate a modified hinge region. In certain embodiments, the Fab fragment is of the IgG1 isotype comprising a modified hinge region. In certain embodiments, the Fab fragment is of the IgG2 isotype comprising a modified hinge region. In certain embodiments, the Fab fragment is of the IgG4 isotype comprising a modified hinge region.
- In certain embodiments, a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the upper hinge region. For example, and not by way of limitation, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions EU216-225. Alternatively or additionally, a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the lower hinge region. In certain embodiments, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions EU230-238. Alternatively or additionally, a modified hinge region can comprise the addition of one or more amino acids C-terminal to amino acid position EU238. In certain embodiments, a modified hinge region comprises the substitution, deletion and/or addition of one or more amino acids within the middle, e.g., core, hinge region. For example, and not by way of limitation, a modified hinge region of the disclosed subject matter can have one or more substitutions, deletions and/or additions at amino acid positions EU226-229.
- In certain embodiments, the modification or alteration is a substitution of one or more, two or more, three or more, four or more, five or more or six or more amino acid residues. In certain embodiments, the substitution can be generated within the upper hinge region, middle hinge region and/or the lower hinge region. In certain embodiments, the amino acid residue at
position 225 can be substituted. For example, and not by way of limitation, the amino acid residue atposition 225 can be changed to any amino acid except for threonine (T). In certain embodiments, the amino acid atposition 225, e.g., threonine, can be changed to a leucine (L), e.g., T225L, according to EU numbering. In certain embodiments, an antibody fragment of the present disclosure is a Fab fragment comprising the substitution T225L. - In certain embodiments, the upper hinge region of an IgG1 antibody fragment can be substituted with one or more amino acids residues present within the upper hinge region of an IgG2 and/or an IgG4 antibody because, for example, the upper hinge regions of IgG2 and IgG4 antibodies exhibit reduced or no reactivity towards AHA (see, e.g.,
FIG. 1 ). For example, and not by way of limitation, the upper hinge region of an IgG1 antibody fragment can be substituted with one or more amino acids residues present within the native hinge region of an IgG2 and/or an IgG4 antibody (seeFIG. 6 ). In certain embodiments, a modified hinge region of an IgG1 antibody fragment retains a cysteine at amino acid position EU220 (e.g., as compared to the native hinge region of an IgG1 antibody). In certain embodiments, a modified hinge region of an IgG1 antibody fragment does not retain a cysteine at amino acid position EU220, e.g., in an IgG antibody fragment where the upper hinge region of the IgG1 antibody fragment is replaced with the upper hinge region (e.g., entire upper hinge region) of IgG4. In certain embodiments, the upper hinge region of an IgG1 antibody fragment can be substituted with one or more amino acids residues present within the upper hinge region of an IgG2, IgG3 and/or an IgG4 antibody, where the amino acid residue at position 131 of the IgG1 antibody is changed from a serine (S) to a cysteine (C), i.e., S131C. - In certain embodiments, an antibody fragment of the present disclosure, e.g., a Fab, F(ab′)2 or Fab′, can comprise a substitution at amino acid positions EU235-236. For example, and not by way of limitation, the amino acid at
position 236, e.g., glycine (G), can be changed to an alanine (A), e.g., G236A. In certain embodiments, an antibody fragment, e.g., a F(ab′)2, can comprise a substitution at position 235, according to EU numbering. In certain embodiments, the amino acid at position 235, e.g., leucine (L), can be changed to a valine (V), e.g., L235V, changed to an isoleucine (I) e.g., L235I, or changed to a methionine (M) e.g., L235M. - In certain embodiments, the modification or alteration is a deletion of one or more, two or more, three or more, four or more, five or more or six or more amino acid residues. In certain embodiments, the one or more deletions can be generated within the upper hinge region, middle hinge region and/or the lower hinge region. In certain embodiments, an antibody fragment of the present disclosure, e.g., a Fab, F(ab′)2 or Fab′, comprises a modified hinge region that has one or more deletions of one or more amino acids at positions EU230-238. In certain embodiments, the antibody fragment comprises a deletion at position EU231. In certain embodiments, the antibody fragment comprises a deletion at positions EU231 and EU232. In certain embodiments, the antibody fragment comprises deletions at positions EU231, EU232 and EU233. In certain embodiments, the antibody fragment comprises deletions at positions EU231, EU232, EU233 and EU234. In certain embodiments, the antibody fragment comprises deletions at positions EU230, EU231, EU232, EU233 and EU234.
- In certain embodiments, an antibody fragment of the present disclosure comprises a C-terminal deletion of one or more, two or more, three or more, four or more, five or more or six or more amino acids. In certain embodiments, an antibody fragment of the present comprises the deletion of one or more amino acids in the upper hinge region, e.g., to generate a C-terminal truncation. In certain embodiments, one or more amino acid at positions EU222-225 can be deleted to obtain a C-terminal truncation. In certain embodiments, an antibody fragment disclosed herein, e.g., a Fab fragment, comprises a C-terminal truncation. For example, and not by way of limitation, the C-terminus of an antibody fragment disclosed herein, e.g., a Fab fragment, terminates at amino acid residue D221 (according to EU numbering). In certain embodiments, the C-terminus of an antibody fragment disclosed herein, e.g., a Fab fragment, terminates at amino acid residue K222 (according to EU numbering).
- In certain embodiments, the C-terminus of the heavy chain of an antibody fragment, e.g., a Fab fragment, disclosed herein, terminates with amino acids having a sequence selected from CDKTHT (SEQ ID NO: 14), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK and CD. In certain embodiments, the C-terminus of the heavy chain of the Fab fragment terminates in the amino acid sequence CDKTHX (SEQ ID NO: 25), wherein X is any amino acid except T. In certain embodiments, a Fab fragment comprises a heavy chain constant region selected from “CDKTHT,” (SEQ ID NO: 14) “CDKTHL,” (SEQ ID NO: 15) “CDKTH,” (SEQ ID NO: 16) “CDKT,” (SEQ ID NO: 17) “CDK” or “CD,” as disclosed in Table 1. In certain embodiments, the presently disclosed subject matter provides antibody fragments, e.g., Fab fragment, that comprise a heavy chain constant region that comprises an amino acid sequence as set forth in SEQ ID NOs: 1, 2, 3, 4, 5 or 6. In certain embodiments, an antibody fragment of the present disclosure comprises a heavy chain constant region that comprises the amino acid sequence set forth in SEQ ID NO:5. In certain embodiments, an antibody fragment of the present disclosure comprises a heavy chain constant region that comprises the amino acid sequence set forth in SEQ ID NO:6.
- In certain embodiments, as an alternative to truncation and/or mutation at the C-terminus, to avoid pre-existing AHA responses, IgG2 or IgG4 Fab fragments can be used. For example, and not by way of limitation, an antibody fragment of the present disclosure can comprise a heavy chain constant region that comprises the amino acid sequence in SEQ ID NOs: 7 or 8. In certain embodiments, an IgG2 or IgG4 Fab fragment can comprise a C-terminal deletion of one or more, two or more, three or more, four or more or five or more amino acids. In certain embodiments, a Fab of the present disclosure is an IgG2 Fab fragment comprising a heavy chain constant region ending in the sequence VERK (SEQ ID NO: 26). In certain embodiments, the C-terminus of the heavy chain of an antibody fragment, e.g., an IgG4 Fab fragment, disclosed herein, terminates with amino acids having a sequence selected from KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG, KY and K. In certain embodiments, a Fab of the present disclosure is an IgG4 Fab fragment comprising a heavy chain constant region selected from “KYGPP,” (SEQ ID NO: 18) “KYGP,” (SEQ ID NO: 19) “KYG,” “KY” and “K,” as disclosed in Table 1. For example, and not by way of limitation, an antibody fragment of the present disclosure can comprise a heavy chain constant region that comprises the amino acid sequence set forth in SEQ ID NOs: 9, 10, 11, 12 or 13.
-
TABLE 1 Fab Heavy Chain Sequences Fab heavy chain ASTKG PSVFPLAPSS KSTSGGTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA “CDKTHT” (SEQ ID VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NO: 14) NHKPSNTKVD KKVEPKSCDK THT (SEQ ID NO: 1) Fab heavy chain ASTKG PSVFPLAPSS KSTSGGTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA “CDKTHL” (SEQ ID VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NO: 15) NHKPSNTKVD KKVEPKSCDK THL (SEQ ID NO: 2) Fab heavy chain ASTKG PSVFPLAPSS KSTSGGTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA “CDKTH” (SEQ ID VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NO: 16) NHKPSNTKVD KKVEPKSCDK TH (SEQ ID NO: 3) Fab heavy chain ASTKG PSVFPLAPSS KSTSGGTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA “CDKT” (SEQ ID VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NO: 17) NHKPSNTKVD KKVEPKSCDK T (SEQ ID NO: 4) Fab heavy chain ASTKG PSVFPLAPSS KSTSGGTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA “CDK”) VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK (SEQ ID NO: 5) Fab heavy chain ASTKG PSVFPLAPSS KSTSGGTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA “CD” VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCD (SEQ ID NO: 6) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG2 VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERK (SEQ ID NO: 7) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG4 VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP (SEQ ID NO: 8) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG4 (“KYG”) VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYG (SEQ ID NO: 9) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG4 (“KYGP”) VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV (SEQ ID NO: 19) DHKPSNTKVD KRVESKYGP (SEQ ID NO: 10) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG4 (“KYGPP”) VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV (SEQ ID NO: 18) DHKPSNTKVD KRVESKYGPP (SEQ ID NO: 11) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG4 (“KY”) VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKY (SEQ ID NO: 12) Fab heavy chain ASTKG PSVFPLAPCS RSTSESTAAL constant region GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA IgG4 (“K”) VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESK (SEQ ID NO: 13) - The present disclosure further provides antibody fragments that comprise conservative modifications of the sequences disclosed herein. For example, and not by way of limitation, the present disclosure provides antibody fragments that comprise a heavy chain constant region that comprises an amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or conservative modifications thereof, and wherein the antibody fragment retains the desired properties of the antibody fragments disclosed herein. For example, and not by way of limitation, such antibody fragments have reduced or no reactivity towards AHA, as disclosed above.
- As used herein, the term “conservative sequence modification” is intended to refer to amino acid modifications that do not significantly affect characteristics of the antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Exemplary conservative amino acid substitutions are shown in Table 2.
- In certain embodiments, a sequence disclosed herein can have up to about one, up to about two, up to about three, up to about four, up to about five, up to about six, up to about seven, up to about eight, up to about nine or up to about ten amino acid residues that are modified and/or substituted.
- Amino acids can be grouped according to common side-chain properties:
-
- i. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
- ii. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
- iii. acidic: Asp, Glu;
- iv. basic: His, Lys, Arg;
- v. residues that influence chain orientation: Gly, Pro;
- vi. aromatic: Trp, Tyr, Phe.
In certain embodiments, non-conservative substitutions can entail exchanging a member of one of these classes for another class.
-
TABLE 2 Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine - Other modified hinge regions of the present disclosure can be entirely synthetic and can be designed to possess desired properties such as length, composition and flexibility. For example, and not by way of limitation, a modified hinge region of the present disclosure can be altered to increase or decrease the flexibility of the hinge region. For example, and not by way of limitation, modifications which can increase the flexibility of the hinge region include, but are not limited to, the substitution of one or more amino acids residues with one or more amino acid residues which increase the flexibility (e.g., glycine). In certain embodiments, modifications which can decrease the flexibility of the hinge region include, but are not limited to, the substitution of one or more amino acids residues with one or more amino acid residues which crease the rigidity of the polypeptide (e.g., proline).
- In certain embodiments, the antibody fragments are made by hinge engineering technology.
- In certain embodiments, the antibody fragment starting material for use in connection with the methods described herein can be obtained from any whole antibody (e.g., a whole monoclonal antibody), using any suitable enzymatic cleavage and/or digestion techniques. In certain embodiments, the antibody fragment can be obtained by cleavage with IdeS.
- In certain embodiments, Fab molecules are generated by proteolytic digestion or recombinant expression. Proteolytic digestion was the original method of Fab production (6). Generating Fab molecules via proteolytic digestion results in the C-terminal sequence of the Fab heavy chain defined by the protease cleavage site. In turn, a Fab molecule typically includes a portion of the upper hinge of the antibody. This upper hinge region of the antibody serves as the linker between Fab and Fc region but has no structural or functional role in a Fab molecule. It can be considered as an unstructured appendix (see
FIG. 1A ) as it is often not fully resolved in crystal structures of Fab molecules. One therapeutic Fab molecule directed against platelet surface receptor GPITh/IIIa (abciximab, REOPRO®) is commercially produced by proteolytic cleavage. - With the advances in molecular cloning, recombinant expression of antibody fragments is an attractive route to generate Fab molecules (7). In contrast to proteolytic digestion as a production route, the recombinant expression of Fab molecules provides flexibility in defining the length of the included upper hinge region. In certain embodiments, the Fab fragments are produced by recombinant expression.
- The high affinity of an antibody is often enabled by bivalent target engagement, facilitating avidity. In contrast, the target engagement of a Fab is monovalent. This often leads to lower target affinity compared to the full-length IgG. By joining two Fab fragments to create a F(ab′)2, avidity can be restored while preserving key properties of the Fab, such as short serum half-life. In addition, targeting multiple disease mediators by bispecific antibodies has become increasingly important for therapeutic antibody development (8). F(ab′)2 molecules can provide a natural scaffold to produce small bispecific antibody fragments. In contrast to the production of Fab molecules, the recombinant expression of F(ab′)2 is not naturally possible because expressed Fab′ molecules require non-native homo- or heterodimerization domains as a fusion (9, 10). Hence, there are two main approaches to generate F(ab′)2 molecules: (i) chemical conjugation and (ii) proteolytic digestion. For chemical conjugation, recombinantly generated Fab′ molecules are coupled by homo- or heterobifunctional crosslinkers (3, 9, 11, 12). Analogous to the proteolytic digestion approach to produce Fab molecules, a number of known proteases can cleave the intact antibody in the lower hinge region to produce F(ab′)2 molecules (13). Such proteolytic digestion results in very stable F(ab′)2 molecules where the two Fab molecules are connected by the two disulfide-bonds of the core-hinge. Pepsin is most widely used (14) but a highly specific IgG degrading enzyme of Streptococcus pyogenes, IdeS, has been described more recently (15, 16). The use of IdeS enables generation of highly homogenous product by eliminating the C-terminal heterogeneity observed from pepsin digest (3). In certain embodiments, the F(ab′)2, fragments are produced by IdeS cleavage.
- Antibody fragments can be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In certain embodiments, an isolated nucleic acid encoding an antibody fragment described herein or composition comprising such nucleic acid is provided. In addition, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. A host cell comprising such nucleic acid is also provided. In certain embodiments, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In certain embodiments, a method of making a Fab molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the Fab, as provided above, under conditions suitable for expression of the Fab, and optionally recovering the Fab from the host cell (or host cell culture medium).
- For recombinant production of a Fab, a nucleic acid encoding a Fab, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the Fab).
- Suitable host cells for cloning or expression of Fab-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, Fabs can be produced in bacteria. For expression of antibody fragments, such as Fabs, in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the Fab can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
- In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for Fab-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized.” See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
- Suitable host cells for the expression of glycosylated proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frupperda cells.
- Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).
- Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982);
MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR″ CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for Fab production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). - Pharmaceutical formulations of an antibody fragments, e.g., Fab and F(ab′)2, as described herein, are prepared by mixing such antibody fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. For example, but not by way of limitation, lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. In certain embodiments, aqueous antibody formulations can include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.
- In certain embodiments, an antibody fragment of the present disclosure can be of a purity greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8% or greater than about 99.9%.
- Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
- The formulation herein can also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
- A composition of the present disclosure can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. The active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical compositions are manufactured under Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.
- The carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody fragment, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.
- Active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
- Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
- The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
- The disclosed pharmaceutical compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms can be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
- In certain embodiments, when the antibodies of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, from about 0.01% to about 99.5% (or about 0.1 to about 90%) of an antibody fragment in combination with a pharmaceutically acceptable carrier.
- Any of the antibody fragments provided herein can be used in therapeutic methods. In certain embodiments, an antibody fragment for use as a medicament is provided. In certain embodiments, an antibody fragment for use in treating a particular disease indication is provided. In certain embodiments, an antibody fragment of the present disclosure can be used treat an ocular disease and/or disorder. In certain embodiments, an antibody fragment of the present disclosure can be used treat a disease and/or a disorder that would benefit from the application of an antibody fragment that exhibits a short systemic half-life. In certain embodiments, an antibody fragment for use in a method of treatment is provided.
- In certain embodiments, the present disclosure provides an antibody fragment for use in a method of treating an individual having a specific disease comprising administering to the individual an effective amount of the antibody fragment or compositions comprising the same. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In certain embodiments, the present disclosure provides an antibody fragment for use in inhibiting a particular molecular pathway and/or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in a method of inhibiting a particular molecular pathway and/or mechanism in an individual that comprises administering to the individual an effective of the antibody fragment to inhibit the particular molecular pathway and/or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in activating a particular molecular pathway and/or mechanism. In certain embodiments, the present disclosure provides an antibody fragment for use in a method of activating a particular molecular pathway and/or mechanism in an individual that comprises administering to the individual an effective of the antibody fragment to inhibit the particular molecular pathway and/or mechanism. An “individual” according to any of the above embodiments can be a human.
- In certain embodiments, the present disclosure provides for the use of an antibody fragment in the manufacture or preparation of a medicament. In certain embodiments, the medicament is for treatment of a particular disease. In certain embodiments, the medicament is for use in a method of treating a particular disease comprising administering to an individual having the disease an effective amount of the medicament. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In certain embodiments, the medicament is for inhibiting or activating a particular molecular pathway and/or mechanism. In certain embodiments, the medicament is for use in a method of inhibiting or activating a particular molecular pathway and/or mechanism in an individual comprising administering to the individual an amount effective of the medicament to inhibit a particular molecular pathway and/or mechanism. An “individual” according to any of the above embodiments can be a human.
- In certain embodiments, the present disclosure provides a method for treating a particular disease. In certain embodiments, the method comprises administering to an individual having such disease an effective amount of an antibody fragment. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An “individual” according to any of the above embodiments can be a human.
- In certain embodiments, the present disclosure provides a method for inhibiting a particular molecular pathway and/or mechanism in an individual. In certain embodiments, the method comprises administering to the individual an effective amount of an antibody fragment to inhibit a particular molecular pathway and/or mechanism. In certain embodiments, an “individual” is a human.
- In certain embodiments, the present disclosure provides pharmaceutical formulations comprising any of the antibody fragments provided herein, e.g., for use in any of the above therapeutic methods. In certain embodiments, a pharmaceutical formulation comprises any of the antibody fragments provided herein and a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical formulation comprises any of the antibody fragments provided herein and at least one additional therapeutic agent, e.g., as described below.
- Antibody fragments of the present disclosure can be used either alone or in combination with other agents in a therapy. For instance, an antibody fragment of the present disclosure can be co-administered with at least one additional therapeutic agent.
- Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the present disclosure can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In certain embodiments, administration of the antibody fragment and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. The antibody fragments described herein can also be used in combination with radiation therapy.
- An antibody fragment (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, intraocular, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraocular, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
- Antibody fragments are formulated, dosed and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody fragment need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of such other agents depends on the amount of antibody fragment present in the formulation, the type of disease or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
- For the prevention or treatment of disease, the appropriate dosage of an antibody fragment of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody fragment, the severity and course of the disease, whether the antibody fragment is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody fragment, and the discretion of the attending physician. The antibody fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody fragment can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody fragment would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the antibody fragment). An initial higher loading dose, followed by one or more lower doses can be administered. The progress of this therapy is easily monitored by conventional techniques and assays.
- It is understood that any of the above formulations or therapeutic methods can be carried out using an immunoconjugate in place of or in addition to an antibody fragment of the present disclosure.
- The presently disclosed subject matter also provides immunoconjugates, which include an antibody fragment, disclosed herein, conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. For example, an antibody fragment of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.
- In certain embodiments, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody fragment is conjugated to one or more drugs, including but not limited to, a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and
European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065. - In certain embodiments, an immunoconjugate includes an antibody fragment as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin
- A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In certain embodiments, an immunoconjugate includes an antibody fragment, as described herein, conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Non-limiting examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it can include a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
- Conjugates of an antibody fragment and cytotoxic agent can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethyl ene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026. The linker can be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used. Non-limiting examples of linkers are disclosed above. - The immunuoconjugates disclosed herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-STAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).
- In certain embodiments of the present disclosure, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and can have a sterile access port (for example the container can 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 antibody fragment of the present disclosure. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture can comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody fragment of the present disclosure; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the present disclosure can further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
- It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
- It is understood that any of the above articles of manufacture can include an immunoconjugate in place of or in addition to an antibody fragment as described herein.
- The following are examples of the methods and compositions of the present disclosure. It is understood that various other embodiments can be practiced, given the general description provided above.
- Fab and F(ab′)2 antibody fragments serve as alternative formats to full-length antibodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region and result in anti-hinge antibodies (AHA) towards the neoepitopes. Pre-existing AHA in serum can act as surrogate Fc and reintroduce the properties of the Fc lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In this study, a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease was identified. The resulting neoepitope at the F(ab′)2 C-terminus did not have detectable pre-existing AHA, providing a practical route to produce F(ab′)2 in vitro by proteolytic digestion when pre-existing AHA response is undesired. In this study, the upper hinge region of the antibody was also studied, which provided a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies were observed towards the Fab of IgG2 and IgG4 isotype, a significant response was observed towards most residues of the upper hinge of human IgG1. A T225L mutation (also referred to herein as the “T225L variant”) and the natural C-terminal D221 were identified as solutions with minimal serum reactivity. This study enabled the production of Fab and F(ab′)2 fragments for therapeutic and immune assays that have minimal reactivity towards pre-existing AHA.
-
- Materials and Methodsa
- Plasmid construction and antibody expression: Antibodies were cloned by standard molecular biology techniques into E. coli expression vectors (9, 23) or mammalian expression vectors (24) as previously described. E. coli expression was carried out as described in Simmons et al. (23). IgG and Fab was expressed in 30 mL transient transfection cultures of CHO (25) or HEK293T (26) cells as previously described.
- Cloning, expression, and purification of IdeS: IdeS was expressed as N-terminal glutathione S-transferase (GST) fusion protein. The mature sequence of IdeS from Streptococcus pyogenes MGAS1882 (Uniprot ID H8HDRO) was codon optimized for E. coli expression and synthesized by GeneArt™ and cloned by standard molecular biology techniques into E. coli expression vector (23). IdeS was expressed using conditions described in Simmons et al. (23) and purified using glutathione sepharose (GSH) column. Eluate fractions in 50mM Tris-HCl, pH 8.0, 20 mM glutathione from the GSH column were concentrated and loaded onto S200 column and eluted with 200 mM K2HPO4, pH 6.2, 250mM KCl.
- Antibody and Fab purification: After expression, the cells were pelleted by gravity. The supernatants were transferred to a 50 ml Falcon tube (Corning, Corning, N.Y., USA). 400 μl of 50% MabSelect SuRe™ protein affinity slurry or Gamma Bind™ Plus slurry (GE Healthcare, Pittsburgh, Pa., USA) was added to the supernatants for IgG and Fab purification respectively. The mixture was incubated overnight at room temperature on an Innova 2000 platform shaker (New Brunswick Scientific, Enfield, Conn., USA). Supernatants were removed and the resin transferred to a 96-well 2 ml filter plate with a 25 μm size membrane (Thompson Instrument, Oceanside, Calif., USA). The resin was washed three times with 1 ml of 1× PBS pH 7.4 by centrifugation at 1,120× g for 5 minutes using a Sorvall™ HT6 Centrifuge (Thermo Scientific, Waltham, Mass., USA). For Fab purification the resin was further washed with 0.2× PBS pH 5.0 before elution. The IgG was eluted using 50 mM phosphoric acid pH 2.9 and the eluate neutralized with 20× PBS pH 11.0 by centrifugation at 1,000× g for 5 minutes. The Fab fragments were eluted using 10 mM sodium citrate pH 2.9 and neutralized with 0.3 M Tris pH 9.0. The eluted IgG and Fab was filtered through 0.2 μm 96-well filter plate (Orochem, Naperville, Ill., USA) by centrifugation at 1,000× g for 5 minutes using an Sorvall HT6 Centrifuge (Thermo Scientific, Waltham, Mass., USA).
- IdeS digestion of IgG hinge variants: IgG at 1 mg/ml was incubated with stated IdeS:IgG ratio (w/w) at 37° C. for 24 hours. For scaled up digestion to generate highly pure F(ab′)2 material in large quantities for AHA assays, up to 1:10 IdeS:IgG ratio was used to drive complete digestion.
- F(ab′)2 purification after IgG cleavage by IdeS: The IdeS cleaved sample was diluted with 25 mM Sodium Acetate, pH 4.4 (Buffer A) and loaded onto a 1 mL SP Sepharose High Performance strong cation exchange column (GE Healthcare, Pittsburgh, Pa., USA) at 150 cm/hr (0.7 cm diameter, 10 cm bed height) equilibrated in Buffer A. The column was washed to baseline with Buffer A and F(ab′)2 eluted with a linear salt gradient from 0 to 0.5 M NaCl over 30 column volumes. The eluate was neutralized by addition of 3 M Tris pH 9.0 to adjust the pH to 7.0 and filtered through 0.22 μm STERIFLIP® (EMD Millipore, Billerica, Mass., USA). The SP eluted F(ab′)2 was further purified on a
MonoS 5/50 GL strong cation exchange column (GE Healthcare, Pittsburgh, Pa., USA) after diluting with Buffer A to lower the conductivity <5 mS/cm. The column was washed to baseline (<0.05 mAU) with Buffer A and the F(ab′)2 eluted using a salt gradient from 0 to 0.6 M NaC1 over 40 column volumes. The eluted F(ab′)2 solution was neutralized with 3 M Tris pH 9.0 to adjust the pH to 7.0 and filtered through 0.22 μm STERIFLIP® (EMD Millipore, Billerica, Mass., USA). - Mass spectrometry of Fab and F(ab′)2 fragments: Mass spectrometric data was acquired using an Agilent 6224 TOF LC-MS system (Agilent Technology, Santa Clara, Calif., USA). F(ab′)2 was reduced with 100 mM dithiothreitol at 37° C. for 20 minutes. The polypeptide chains were separated with a PLRP-S reversed phase column (Agilent Technologies, Santa Clara, Calif., USA). Intact masses of the reduced light and heavy chains were obtained by Maximun Entropy Deconvolution using MassHunter software (Qualitative Analysis B.03.01).
- Analysis of protein by capillary electrophoresis: All samples were prepared by mixing 5 μl of sample volume with 7 μl of HT Protein Express Sample buffer and incubated for 5 minutes at 70° C. 32 μl of water was added to the samples and centrifuged at 1,000× g for 5 minutes. The chip was prepared according to manufacturer's instructions provided in the LabChip GXII User Guide and samples were analyzed on a Caliper GX II microfluidic system (PerkinElmer® Biotechnology, Waltham, Mass., USA). Samples were analyzed on a Caliper GXII microfluidic system (PerkinElmer® Biotechnology, Waltham, Mass., USA). All reagents were obtained from PerkinElmer®.
- Pre-existing anti-hinge antibody enzyme-linked immunosorbent assay (ELISA): MAXISORP® plates (384-well, Nunc, Thermo Fisher Scientific, Rochester, NY, USA) were coated with 1 μg/m1 F(ab′)2 or Fab in 50 mM carbonate, pH 9.6 at 4° C. overnight. Plates were washed with 0.05
% polysorbate 20 in PBS, pH 7.4 and then blocked with 0.5% BSA, 15 ppm proclin in PBS, pH 7.4. Pooled human serum from 25 female and 25 male individuals (BioreclamationlVT, Westbury, N.Y., USA) were serially diluted in assay buffer (0.5% BSA, 0.05% polysorbate 20, 15 ppm PROCLIN™, in PBS, pH 7.4) and added to the plates. After a 2 hour incubation, bound pre-existing anti-hinge antibody was detected using horseradish peroxidase (HRP) goat anti-F(ab′)2 conjugated anti-human IgG Fc (Jackson ImmunoResearch, West Grove, Pa.) in assay buffer, followed by 3,3′,5,5′-tetramethyl benzidine (TMB, Moss Inc., Pasadena, Md., USA) as the substrate. The reaction was stopped with 1 M phosphoric acid and absorbance was read at 450 nm. The absorbance readings at 1:30 dilution were used for the figures to allow presentation of all samples. Comparable results were observed at a 1:10 serum dilution. To calculate the relative AHA reactivity, the titration curve of F(ab′)2 was fitted with a 4 parameter curve fitting program (KaleidaGraph, Synerg Software, Reading, Pa.). MidOD (the average OD of the top and bottom OD readings) of the F(ab′)2 titration curve was determined. The dilutions of Fab DKTHT (SEQ ID NO: 21) and F(ab′)2 corresponding to this midOD were calculated and used to calculate the relative AHA reactivity. - SDS-PAGE and immunoblots: For SDS-PAGE, 5μg of purified F(ab′)2 variants and GST-IdeS were mixed with SDS-sample buffer, heated for 5 min at 95° C. and spun for 1 min at 16,000 relative centrifugal force. The samples were loaded onto a NuPAGE 4-12% BisTris/MES gels (Invitrogen). For immunoblotting, 5 ng of protein samples were used for SDS-PAGE. Gels were transferred by IBLOT® (Invitrogen) onto nitrocellulose membrane, immunoblotted with anti-IdeS (Genovis, USA; catalog no. A3-AF1-010, lot no. A3AF1-7C17H) as primary antibody and IRDye800CW conjugated donkey anti-goat antibody (Li-COR®, USA; catalog no. 926-32214, lot no. B80821-03) as the secondary antibody, and imaged with a LI-COR® Odyssey® Imager (LI-COR®, USA). Odyssey® Two-color protein molecular weight marker (LI-COR®, USA) was used for immunoblots and pre-stained SEEBLUE® Plus2 (Invitrogen, USA) was used for Coomassie stained gels.
- Protein stability measurements by differential scanning fluorimetry: Protein stability was determined in a Biorad CFX96 TOUCH™ Real-Time System (Biorad, USA) with a final dilution of 1:200 of the SYPRO® Orange dye stock (Molecular Probes™, USA). 1μl of the SYPRO® Orange dye stock was added to 24 μl of the purified antibody at 100 μg/ml. Fluorescence of the final 25 μl sample in PBS was recorded from 20-100° C. (0.2° C. increments, 10 seconds hold per step).
-
- Results
- Fab C-terminus determines response to pre-existing AHA: Originally, reactivity of autoantibodies in human serum towards the upper hinge of Fab molecules was studied with a papain cleaved antibody, abciximab (5). Papain cleavage leaves a C-terminal H224 on the Fab. Later, a more comprehensive study was carried out using biotinylated peptide analogs to dissect the contribution of individual C-terminal residues of the upper hinge (20). In this study, only minimal AHA reactivity towards the upper hinge residues K222 through H224 was observed. No signal was observed towards peptides with T225 as the C-terminal residue. The use of synthetic peptides can confound results because the Fab-tail spanning the upper hinge residues D221 to T225 (
FIG. 1A ) is presented outside of the context of the intact molecule. Thus, the Fab-tail contribution for binding to pre-existing AHA in the setting of an intact Fab was studied. - The recombinant expression of Fab molecules in E. coli and mammalian cells allows to readily produce molecules with defined C-terminal ends without the need of proteolytic cleavage. To ensure integrity of the C-terminus, correct mass of the purified Fab was confirmed by intact mass-spectrometry. The Fab molecules were coated on microtiter plates, and after incubation with pooled human donor serum, binding of pre-existing AHA was quantified by anti-Fc detection. In agreement with the previous study (20), T223 at the C-terminus (having the sequence DKT, also referred to herein as “CDKT” (SEQ ID NO: 17)) showed the highest reactivity of all upper hinge variants towards pre-existing AHA (
FIG. 1B ). Significant difference to the previous studies is observed with T225 at the C-terminus (having the sequence DKTHT (SEQ ID NO: 21), also referred to herein as “CDKTHT” (SEQ ID NO: 14)). This variant did not bind AHA as peptide (20); however, substantial AHA reactivity was observed when tested as Fab. With D221 at the C-terminus (having the sequence D, also referred to herein as “CD”), binding of AHA was reduced almost to background. Thus, terminating the Fab at D221 provides a solution to minimize recognition by pre-existing AHA while maintaining a natural antibody sequence. - As demonstrated by these experiments, the C-terminal Fab residue has a profound impact on AHA binding. Whether binding by pre-existing antibodies could also be obviated by a single amino acid change at the C-terminus and provide an alternative route to the D221 to minimize reactivity towards AHA was investigated. A T225L variant was introduced to place a non-natural residue at the Fab C-terminus, and its binding of AHA was tested. The T225L variant has been described previously (7). The mutation perturbed binding of the pre-existing AHA (
FIG. 1B ), further highlighting the importance of the C-terminus for binding. To exclude the possibility that the reduced AHA in T225L was due to reduced coating efficiency, an antigen capture format was used to capture the Fab molecules, and a similar fold of AHA signal reduction was also observed. To determine whether this observation can be generalized, three different Fabs were incubated with pooled human serum and binding of pre-existing antibodies was detected by ELISA. Significant signal was observed for the three different Fabs having a DKTHT (SEQ ID NO: 21) C-terminus while reduced binding of pre-existing antibodies to the D221 and T225L C-terminus was indeed detected. (FIG. 1C ). - Next, Fab molecules of IgG2 and IgG4 isotypes were studied. While IgG1, IgG2 and IgG4 are commonly used for therapeutic antibodies, the use of IgG2 and IgG4 Fabs has not been leveraged for therapeutic development so far. Thus, IgG2 and IgG4 Fabs with the complete upper hinge region (
FIG. 1D ; C-terminus K218 and P225, respectively) were tested. In contrast to IgG1 Fab, the IgG2 and IgG4 Fabs were not recognized by pre-existing AHA. Next, the upper hinge of the IgG4 was truncated. The length of the IgG4 isotype upper hinge is shorter when compared to IgG1 (seeFIG. 6A-B ); however, since the cysteine involved in the heavy-light interchain disulfide is located in the center of theC H1 primary structure, it was able to include residues K218 and Y219 in the truncation experiments. These truncated upper hinge Fabs displayed a signal similar to the intact IgG1 upper hinge (FIG. 1B ). - All Fab molecules within the same isotype yielded similar expression levels in E. coli and CHO (
FIG. 7 ). No change in thermal stability was observed within the same isotype (Table 3). Thermal stability of IgG2 and IgG4 Fabs was decreased by about 6° C. compared to IgG1 isotype. - In conclusion, multiple Fab formats with minimal reactivity towards pre-existing AHA exist: IgG1-D221, IgG1-T225L, IgG2 and IgG4.
-
TABLE 3 Thermal Stability of T30M Fab determined by differential scanning fluorimetry Human isotype C-terminus Tm (° C.) IgG1 ..DKTHL (SEQ ID NO: 20) 81.6 ..DKTHT (SEQ ID NO: 21) 81.8 ..DKTH (SEQ ID NO: 27) 81.7 ..DKT 81.8 ..DK 81.7 ..D 81.9 IgG2 ..K 75.5 IgG4 ..KYGPP (SEQ ID NO: 18) 75.9 ..KYG 75.7 ..KY 75.8 ..K 75.6 - IgG1 with the lower hinge of IgG2 cannot be cleaved efficiently: Pre-existing AHA towards the lower hinge region of F(ab′)2 have been extensively described in the literature (13, 27). Analogous to the AHA to the upper hinge of Fab molecules, these AHA can act as surrogate Fc or introduce assay artifacts. Thus, development of a F(ab′)2 format that impedes AHA binding is desirable. While AHA in serum are found towards F(ab′)2 of IgG1 isotype, it has not been possible to establish existence of autoantibodies to the lower hinge of IgG2 isotype. Interestingly, the lack of such autoantibodies coincides with the inability of physiologically relevant human proteases to efficiently cleave IgG2 into F(ab′)2 fragments (28). However, inefficient cleavage of IgG2 has been observed using IdeS protease (28). IdeS is an IgG specific endoprotease from Streptococcus pyrogenes that cleaves after G236. In addition to the cleavage site in the antibody hinge region, it recognizes a second site in the Fc that contributes to its high specificity towards IgG (15, 16). The inefficient cleavage of IgG2 antibodies could be caused by the exosite outside of the cleavage site. Thus, the lower hinge residues of IgG2 were grafted onto IgG1 to create an IgG1-2 chimera (
FIG. 2B ). The cleavage efficiency at different IdeS:IgG ratios was tested (FIG. 2C ). While IgG1 wild-type was efficiently cleaved into F(ab′)2 at a IdeS:IgG ratio of 1:500, at least 50-fold higher protease concentration was necessary to achieve similar cleavage of the IgG1-2 chimera. It was concluded that the sequence differences in the lower hinge at least partially contribute to the poor cleavage efficiency of IgG2 antibodies. Thus, the IgG1-2 chimera may not be a practical strategy to generate F(ab′)2 molecules that do not bind to pre-existing AHA. - Characterizing the P1 and P2 positions for efficient IdeS cleavage: It was demonstrated that binding of pre-existing AHA can be prevented by a single C-terminal T225L mutation with the Fab experiments described. A similar strategy was employed for the F(ab′)2. As a first step, residues in the P1 (EU236) and P2 (EU235) site that allow cleavage by IdeS protease were identified. A set of 76 Fab variants that included L235, L235V, L2351, or L235M at the P2 position for IdeS were generated and combined with any amino acid except cysteine in P1 (
FIG. 3D and E). The antibodies were purified and digested with IdeS at an IdeS:IgG ratio of 1:10 to identify variants that can be cleaved by IdeS. Such high protease to antibody ratio was chosen to assess proteolysis without taking cleavage efficiency into account. Seven variants were identified that were cleaved by IdeS (FIG. 3A ). While the P2 position tolerated all four residues tested, only two amino acids with very small side chains, the natural glycine and alanine, were accepted in the P1 position. This subset was further investigated for cleavage efficiency at three different IdeS:IgG ratios: 1:500, 1:200 and 1:100 (FIG. 3B ) and 1:10 (FIG. 3C ). The variant L235V in the P2 position demonstrated minimal change in cleavage efficiency compared to the wild-type sequence. All other variants were characterized by a dominant single cleavage at only one side of the hinge, leaving the other side of the antibody intact. While L2351 and L235M variants with glycine in the P1 position could be completely cleaved at IdeS:IgG ratio of 1:200, all P2 variants with alanine in P1 needed significantly higher protease amounts. Hence, efficient cleavage by IdeS requires glycine in the P1 position. - C-terminal F(ab′)2 variants do not prevent recognition by pre-existing AHA: IdeS can confound results of the pre-existing AHA assay with a false positive result. This can be explained by binding of serum antibodies by IdeS and their subsequent recognition by the anti-Fc detection antibody. Therefore, it was ensured that F(ab′)2 molecules used in the assay did not contain IdeS from the preceding proteolysis reaction. The removal of IdeS in the purified F(ab′)2 proteins was confirmed by SDS-PAGE followed by Coomassie staining and anti-IdeS immunoblot (
FIG. 4A ). The purified P1 and P2 F(ab′)2 variants were then tested for their recognition to pre-existing AHA (FIG. 4B ). While there was a reduction of signal compared to the wild-type, none of the antibodies with C-terminal modifications eliminated the reactivity with serum autoantibodies. Changes to the P2 position only had a modest impact. The G236A change in the P1 position reduced the signal to levels comparable to a Fab. Without being limited to a particular theory, it is thus not possible to design a lower hinge with an amino acid variant that retains cleavage by IdeS and concurrently eliminates pre-existing antibody response against the F(ab′)2. - Deletions in the lower hinge prevents recognition by pre-existing AHA while retaining IdeS cleavage: Another strategy was employed to remove the epitope of the AHA by truncating the lower hinge. Leaving the P1 (EU236) and P2 (EU235) residues untouched for their significance in cleavage efficiency, single and double residue deletions in the P3 (EU234), P4 (EU233) and P5 (EU232) sites of IdeS were generated to identify residues with a minimal impact in cleavage efficiency (see
FIG. 5F ). Significantly poor cleavage efficiency was observed with deletion of the P3 position, thus this position was not considered for further studies (FIG. 5A ). Antibodies with deletions of P4, P5 and a combination of both (ΔP45; also referred to herein as a deletion of amino acid residues at positions EU232-233) were tested for binding to AHA (FIG. 5B ). For these variants, a lower signal was observed compared to the wild-type hinge sequence. To further reduce hinge recognition by pre-existing AHA, the deletion was extended to include the P6 (EU231) and P7 (EU230) residues (seeFIG. 5F ). While the deletion of P4 through P7 sites (AP4567; also referred to herein as a deletion of amino acid residues at positions EU230-233) resulted in modestly reduced cleavage efficiency, the deletion of P4 through P6 sites (AP456; also referred to herein as a deletion of amino acid residues at positions EU231-233) had cleavage efficiency comparable to wild-type at IdeS:IgG ratio of 1:200 (FIG. 5C andFIG. 8 ). Both variants did not result in pre-existing AHA recognition (FIG. 5D ). To ensure high cleavage specificity of IdeS is maintained, ESI-TOF mass spectrometry was used to analyze the IdeS cleaved F(ab′)2. Only a single mass corresponding to the expected cleavage site at G236 was observed for the F(ab′)2 from the wild-type as well as the ΔP456 variant F(ab′)2 (FIG. 5E ). - As shown in
FIG. 11 , the observed AHA binding signal of the ΔP456 variant F(ab′)2 is comparable to the two Fab C-terminal variants, Fab D221 and Fab T225L. The dilutions corresponding to the OD (1.15) at the middle of the F(ab′)2 titration curves were 70 and 14 for F(ab′)2 and Fab, respectively. Without being limited to a particular theory, the five-fold higher AHA activity seen with F(ab′)2 compared to Fab T225 can be explained by the potential bivalent binding of AHA to the F(ab′)2, which indicates that F(ab′)2 molecules need avoid pre-existing AHA. To exclude the possibility that the reduced AHA reactivity seen in F(ab′)2 ΔP456 and Fab T225L was due to reduced coating efficiency, an antigen capture format was used to detect AHA. The OD readings (n=4) were 2.2±0.1, 0.34±0.03, 1.3±0.1, 0.36±0.02 and 0.32±0.01 on antigen coated wells receiving F(ab′)2, ΔP456 F(ab′)2, Fab, Fab T225L and buffer at 1:30 serum dilution, respectively. Thus, these results confirmed that ΔP456 F(ab′)2 and Fab T225L had reduced AHA reactivity compared to their corresponding wild type molecules. - These data show that the ΔP456 variant provides a solution to avoid pre-existing AHA response towards the lower hinge of F(ab′)2 while maintaining the possibility to produce the F(ab′)2 antibody fragment by the well-established route of proteolytic digest.
- Deletions in the lower hinge in C-terminal F(ab′)2 variants: The C-terminal variants having deletions in the lower hinge region were analyzed to determine whether the recognition by pre-existing AHA were altered. A set of 6 variants that included L235, L235V, L235I, or L235M at the P2 position for IdeS with alanine or glycine at P1 were generated with various hinge region deletions (
FIG. 9 ). This subset was further investigated for cleavage efficiency at three different IdeS:IgG ratios: 1:10 (FIG. 9A ), 1:100 (FIG. 9B ) and 1:500 (FIG. 9C ). As shown inFIG. 9D , the variant L235V or G236A in combination with a deletion at P5, resulted in a reduced AHA signal compared to the deletion of P5 alone. As shown inFIG. 10 , the ΔP456 variant that included L235V, L235I, or L235M at P2 position with alanine or glycine in P1 exhibited similar reductions in AHA signal compared to ΔP456 alone (FIG. 10B ); however, the deletion of ΔP456 alone exhibited better cleavage efficiency compared to the variants in combination with ΔP456 (FIG. 10A ). -
- Discussion
- Antibody fragments such as Fab and F(ab′)2 are attractive therapeutic formats when a short systemic half-life and an effector-silent molecule are concurrently desired. Certain fragments are also natural products of proteases associated with invasive diseases such as tumor cells and bacteria and are generated in an effort to evade immune surveillance. As a consequence, the C-terminal neoepitope of the Fab and F(ab′)2 fragment is recognized by the immune system and result in AHA that can provide surrogate Fc.
- In this study, the reactivity of pre-existing AHA towards the individual C-terminal residues spanning the upper hinge region of human IgG1, IgG2 and IgG4 isotype was dissected. While it was previously reported that pre-existing AHA did not exist towards neoepitopes in the lower hinge of IdeS-cleaved human IgG2 antibodies, reactivity towards the upper hinge of human isotypes was incompletely investigated so far. In this study, pre-existing AHA towards the upper hinge of IgG2 and IgG4 isotype was not detected. This in turn may suggest that these isotypes are not the target of proteases of invasive diseases. This can be explained by the effector-attenuated nature of these isotypes and the fact that removing the Fc region of these isotypes does not provide an advantage for tumors and bacteria. In contrast, IgG1 isotype seems to be the prime target for these proteases. Indeed, several proteases have been described to cleave in the upper hinge of human IgG1, including plasmin, human neutrophil elastase and LysC. This study shows that pre-existing AHA exist towards all cleavage sites of the upper hinge of human IgG1 with the exception of D221. Without being limited to a particular theory, the absence of AHA toward D221 may be a reflection of the inability of human proteases to cleave after this residue or the inability to raise antibodies toward this neoepitope. The highest reactivity was observed towards the C-terminal T223 Fab. Interestingly, this C-terminus coincides with the cleavage point of human neutrophil elastase, a protease that is secreted by neutrophils and macrophages during inflammation to destroy bacteria and host tissue (29).
- Pre-existing AHA can rapidly recruit effector-function to a molecule that is designed to be effector-less. Using a Fab of IgG2 or IgG4 isotype can provide one strategy to supply a molecule without pre-existing antibody response. Alternatively, introducing a non-natural residue at the heavy chain C-terminus, like the T225L mutation, or truncating the upper hinge to D221 is a strategy for the IgG1 isotype. While the T225L mutation eliminates the response towards pre-existing AHA, it implies that in principle, it can elicit an immune response as well. This is further supported by a recent study with an anti-TNFR1 domain antibody (34). The addition of a C-terminal alanine was sufficient to reduce binding of pre-existing human anti-VH antibodies during screening in vitro; however, one subject was found to develop high levels of antibodies specific toward the modified C-terminus in a Phase I clinical trial. In addition, potential exopeptidase activity on a longer tail can eventually result in a neoepitope that is recognized by pre-existing AHA. Removing the unstructured upper hinge altogether like the Fab-D221 further minimizes the risk of such secondary responses. This is supported by the crystal structure of an anti-hinge antibody that has been crystalized in complex with a peptide spanning the IdeS-cleaved lower hinge of human IgG1 (30). The peptide is bound to the antibody in an extended conformation, suggesting that truncating the upper or lower hinge can successfully remove the neoepitope and suppress an immune response for antibodies against the hinge region.
- These findings can also have implications on the design of studies in cynomolgus monkeys. While the lower hinge region is highly conserved between cynomolgus and human IgG, considerable differences exist in the upper hinge region (31) that will prevent cross-species reactivity. This may have an impact on toxicological studies in cynomolgus as liabilities from pre-existing AHA towards a human Fab cannot be addressed by these studies.
- Beyond therapeutic use, the Fab-D221 can also be considered for the recombinant expression of Fab for crystallographic studies, since the upper hinge is not commonly resolved in crystal structures due to the unstructured nature. This example demonstrates that equal stability and expression can be achieved with a Fab-D221 construct. Eliminating the unstructured areas may further improve crystallization outcomes.
- The most efficient route to generate F(ab′)2 molecules so far is by proteolytic digest and proteases with high-specificity such as IdeS are preferred. As discussed earlier, pre-existing AHA also exist towards the lower hinge of F(ab′)2 molecules. The AHA titer towards IdeS-cleaved antibody is higher compared to a Fab. Without being limited to a particular theory, this may be due to the bivalent nature of the F(ab′)2 that provides an avidity based binding component in the assay or a natural higher abundance of F(ab′)2 that lead to increased titers. Several strategies were employed to remove the AHA reactivity while maintaining cleavage efficiency by IdeS.
- Since the C-terminal residue has an important role in epitope recognition (22), the first strategy was to mutate the C-terminal residue of the F(ab′)2 to remove binding activity of AHA. However, this was not possible due to the strict requirement of glycine in the P1-position for efficient cleavage by IdeS. The selected set of mutations in the P2-site had only modest impact on AHA binding, further confirming the importance of the C-terminal residue for reactivity with AHA. The coinciding differences in AHA binding with the IdeS cleavage efficiency of the P1 and P2 variants perhaps explains the reason why the IgG2 isotype with valine in P2 and alanine in P1 position is less susceptible to anti-hinge antibody response. The requirement for glycine in position P1 for efficient cleavage is accompanied by the high conservation of this residue within different isotypes and across species.
- By deleting three residues in the lower hinge (ΔP456), it was able to maintain the cleavage efficiency of IdeS while removing pre-existing AHA recognition. It demonstrates that positions upstream of the P3 site have minor relevance for efficient cleavage. In addition to removing the reactivity towards pre-existing AHA, truncating the lower hinge can dampen an immune response towards this epitope. Based on structural studies, an AHA binds the lower hinge in an extended conformation and the five C-terminal residues interact with the antibody complementarity determining regions (CDR) (30). By removing three residues from the lower hinge, only 4 residues remained after IdeS cleavage. This short sequence may not be sufficient for a robust immune response and may reduce the likelihood of developing de novo antibodies toward the engineered hinge.
- Pre-existing antibodies have also been demonstrated to decrease effector-function (32) in vitro and can also confound immunogenicity assays during drug development. While the majority of anti-therapeutic antibodies (ATAs) towards a humanized antibody are targeting the idiotype, rheumatoid factor, a low affinity antibody towards the Fc region has been described. One way to eliminate artifacts by rheumatoid factor in the immunogenicity assay is to use an antibody fragment devoid of the Fc region. However, it is import to use fragments that are not recognized by other pre-existing antibodies. These findings provide multiple Fab formats and a F(ab′)2 format that can fulfill these criteria.
- In summary, by choosing the appropriate antibody fragment, it is possible to evade recognition by pre-existing AHA. For Fab molecules, several options exist: (1) using an IgG2 or IgG4 isotype, (2) a mutation of the C-terminal residue (T225L), or (3) terminating the Fab with residue D221. Options are more limited for F(ab′)2 molecules due to the need for proteolytic digestion. A deletion was identified in the lower hinge of IgG1, however, that maintains high cleavage efficiency and specificity and removes reactivity with pre-existing AHA. Using these formats can enable further minimization of safety concerns with antibody fragments in a therapeutic setting and remove interference in assay development.
- It has been previously described that purified AHA antibodies can act as surrogate Fc and restore ADCC/CDC function that was lost by IdeS generated F(ab′)2 (20, 22). To study if the reduced binding of AHA by the disclosed engineered Fab and F(ab′)2 variants is further reflected by reduced recruitment of Fcγreceptors and C1q, bridging experiments were employed. To assess binding of AHA to FcγRIIIa, human serum was added to Fab or F(ab′)2 coated wells and incubated for 2 hours as described above. After the plates were washed, soluble FcγRIIIa(V158)-His-GST (consisting of the extracellular domain fused with Gly-His6-glutathione-S-transferase (“Gly-His6” disclosed as SEQ ID NO: 28) at the carboxy-terminus) was added at 0.5 μg/ml. Bound FcγRIIIa(V158)-His-GST was detected using horseradish peroxidase-conjugated mouse anti-His antibody (Penta-His (SEQ ID NO: 29), Qiagen, Germantown, Md.) followed by TMB as the substrate. To assess binding of AHA to human C1q, human serum was added to Fab or F(ab′)2 coated wells and incubated for 2 hours as described above. After the plates were washed, purified human C1q (Quidel, San Diego, Calif.) was added. Bound C1q was detected with goat anti-C1q antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) followed by rabbit anti-goat IgG -HRP (Jackson ImmunoResearch, West Grove, Pa.) and TMB as the substrate.
- Significant binding of FcγRIIIa and C1q for F(ab′)2 was observed while little signal was detected for the ΔP456 F(ab′)2 variant, indicating that the engineering of the F(ab′)2 variant significantly reduced the risk of ADCC/CDC activation. The OD readings (n=3) were 0.45±0.05, 0.10±0.02 and 0.09±0.02 for FcγRIIIa binding and 0.98±0.09, 0.158±0.004 and 0.107±0.009 for C1q binding on F(ab′)2, ΔP456 variant, and uncoated wells at 1:10 diluted serum, respectively.
- 1. Desjarlais, J. R., and Lazar, G. A. (2011) Modulation of antibody effector function. Experimental Cell Research. 317, 1278-1285.
- 2. Olafsen, T. (2012) Fc engineering: serum half-life modulation through FcRn binding. Methods Mol Biol. 907, 537-556.
- 3. Gadkar, K., Pastuskovas, C. V., Le Couter, J. E., Elliott, J. M., Zhang, J., Lee, C. V., Sanowar, S., Fuh, G., Kim, H. S., Lombana, T. N., Spiess, C., Nakamura, M., Hass, P., Shatz, W., Meng, Y. G., and Scheer, J. M. (2015) Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics. Invest. Ophthalmol. Vis. Sci. 56, 5390-5400.
- 4. Walsh, G. (2014) Biopharmaceutical benchmarks 2014. Nat Biotechnol. 32, 992-1000.
- 5. Curtis, B. R., Swyers, J., Divgi, A., McFarland, J. G., and Aster, R. H. (2002) Thrombocytopenia after second exposure to abciximab is caused by antibodies that recognize abciximab-coated platelets. Blood. 99, 2054-2059.
- 6. Porter, R. R. (1950) The formation of a specific inhibitor by hydrolysis of rabbit antiovalbumin.
Biochem 1 46, 479-484. - 7. Chen, Y., Wiesmann, C., Fuh, G., Li, B., Christinger, H. W., McKay, P., de Vos, A. M., and Lowman, H. B. (1999) Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured fab in complex with antigen. J Mol Biol. 293, 865-881.
- 8. Spiess, C., Zhai, Q., and Carter, P. J. (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. 67, 95-106.
- 9. Carter, P., Kelley, R. F., Rodrigues, M. L., and Snedecor, B. (1992) High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Nature. 10.1038/nbt0292-163.
- 10. Kostelny, S. A., Cole, M. S., and Tso, J. Y. (1992) Formation of a bispecific antibody by the use of leucine zippers. J Immunol. 148, 1547-1553. 11. Glennie, M. J., McBride, H. M., Worth, A. T., and Stevenson, G. T.
- (1987) Preparation and performance of bispecific F(ab′ gamma)2 antibody containing thioether-linked Fab″ gamma fragments. Jlmmunol. 139, 2367-2375.
- 12. Ellerman, D., and Scheer, J. M. (2011) Generation of bispecific antibodies by chemical conjugation (Kontermann, R. E. ed), pp. 47-63, Springer-Verlag Berlin Heidelberg, 10.1007/978-3-642-20910-9_3.
- 13. Brerski, R. J., and Jordan, R. E. (2010) Cleavage of IgGs by proteases associated with invasive diseases: an evasion tactic against host immunity? MAbs. 2, 212-220.
- 14. Nisonoff, A., Wissler, F. C., and Lipman, L. N. (1960) Properties of the major component of a peptic digest of rabbit antibody. Science. 132, 1770-1771.
- 15. Pawel-Rammingen, von, U., Johansson, B. P., and Bjorck, L. (2002) IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J. 21, 1607-1615.
- 16. Vincents, B., Pawel-Rammingen, von, U., Bjorck, L., and Abrahamson, M. (2004) Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry. 43, 15540-15549.
- 17. Osterland, C. K., Harboe, M., and Kunkel, H. G. (1963) Anti-gamma-globulin factors in human sera revealed by enzymatic splitting of anti-Rh antibodies. Vox Sang. 8, 133-152.
- 18. Mellbye, O. J., and Natvig, J. B. (1971) Evidence for immune complexes containing antibody to the pepsin site of IgG in rheumatoid synovial fluids. Clin Exp Immunol. 8, 889-899.
- 19. Terness, P., Kohl, I., Hübener, G., Battistutta, R., Moroder, L., Welschof, M., Dufter, C., Finger, M., Hain, C., and Jung, M. (1995) The natural human IgG anti-F(ab′)2 antibody recognizes a conformational IgG1 hinge epitope. J Immunol. 154, 6446-6452.
- 20. Brerski, R. J., Luongo, J. L., Petrone, D., Ryan, M. H., Zhong, D., Tam, S. H., Schmidt, A. P., Kruszynski, M., Whitaker, B. P., Knight, D. M., and Jordan, R. E. (2008) Human anti-IgG1 hinge autoantibodies reconstitute the effector functions of proteolytically inactivated IgGs. J Immunol. 181, 3183-3192.
- 21. Rispens, T., de Vrieze, H., de Groot, E., Wouters, D., Stapel, S., Wolbink, G. J., and Aarden, L. A. (2012) Antibodies to constant domains of therapeutic monoclonal antibodies: anti-hinge antibodies in immunogenicity testing. J Immunol Methods. 375, 93-99.
- 22. Brerski, R. J., Kinder, M., Grugan, K. D., Soring, K. L., Carton, J., Greenplate, A. R., Petley, T., Capaldi, D., Brosnan, K., Emmell, E., Watson, S., and Jordan, R. E. (2014) A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo. MAbs.
- 23. Simmons, L. C., Reilly, D., Klimowski, L., Raju, T. S., Meng, G., Sims, P., Hong, K., Shields, R. L., Damico, L. A., Rancatore, P., and Yansura, D. G. (2002) Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies. J Immunol Methods. 263, 133-147
- 24. Eaton, D. L., Wood, W. I., Eaton, D., Hass, P. E., Hollingshead, P., Wion, K., Mather, J., Lawn, R. M., Vehar, G. A., and Gorman, C. (1986) Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry. 25, 8343-8347.
- 25. Wong, A. W., Baginski, T. K., and Reilly, D. E. (2010) Enhancement of DNA uptake in FUT8-deleted CHO cells for transient production of afucosylated antibodies. Biotechnol Bioeng. 106, 751-763.
- 26. Bos, A. B., Duque, J. N., Bhakta, S., Farahi, F., Chirdon, L. A., Junutula, J. R., Harms, P. D., and Wong, A. W. (2014) Development of a semi-automated high throughput transient transfection system. Journal of Biotechnology. 180, 10-16.
- 27. Van Schie, K. A., Wolbink, G.-J., and Rispens, T. (2015) Cross-reactive and pre-existing antibodies to therapeutic antibodies-Effects on treatment and immunogenicity. MAbs. 7, 662-671.
- 28. Brerski, R. J., Oberholtzer, A., Strake, B., and Jordan, R. E. (2011) The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge. MAbs. 3, 558-567.
- 29. Belaaouaj, A., Kim, K. S., and Shapiro, S. D. (2000) Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science. 289, 1185-1188
- 30. Malia, T. J., Teplyakov, A., Brerski, R. J., Luo, J., Kinder, M., Sweet, R. W., Almagro, J. C., Jordan, R. E., and Gilliland, G. L. (2014) Structure and specificity of an antibody targeting a proteolytically cleaved IgG hinge. Proteins. 10.1002/prot.24545.
- 31. Jacobsen, F. W., Padaki, R., Morris, A. E., Aldrich, T. L., Armitage, R. J., Allen, M. J., Lavallee, J. C., and Arora, T. (2011) Molecular and Functional Characterization of Cynomolgus Monkey IgG Subclasses. J Immunol. 186, 341-349.
- 32. Jones, J. D., Shyu, I., Newkirk, M. M., and Rigby, W. F. C. (2013) A rheumatoid factor paradox: inhibition of rituximab effector function. Arthritis Res. Ther. 15, R20.
- 33. Pollack, C. V., Reilly, P. A., Eikelboom, J., Glund, S., Verhamme, P., Bernstein, R. A., Dubiel, R., Huisman, M. V., Hylek, E. M., Kamphuisen, P. W., Kreuzer, J., Levy, J. H., Sellke, F. W., Stangier, J., Steiner, T., Wang, B., Kam, C.-W., and Weitz, J. I. (2015) Idarucizumab for Dabigatran Reversal. N. Engl. J. Med. 373, 511-520.
- 34. Cordy, J. C., Morley, P. J., Wright, T. J., Birchler, M. A., Lewis, A. P., Emmins, R., Chen, Y. Z., Powley, W. M., Bareille, P. J., Wilson, R., Tonkyn, J., Bayliffe, A. I., and Lazaar, A. L. (2015) Specificity of human anti-variable heavy (VH) chain autoantibodies and impact on the design and clinical testing of a VH domain antibody antagonist of TNF-
α receptor 1. Clin Exp Immunol. 10.1111/cei.12680. - Although the foregoing compositions and methods have been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the instant disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
Claims (22)
1. A composition comprising an isolated Fab antibody fragment, wherein the Fab antibody fragment has reduced or no reactivity towards pre-existing anti-hinge antibodies.
2. The composition of claim 1 , wherein the Fab antibody fragment is an IgG1 Fab.
3. The composition of claim 2 , wherein the IgG1 Fab terminates with residue D221.
4. The composition of claim 2 , wherein the IgG1 Fab terminates with residue K222.
5. The composition of claim 2 , wherein the IgG1 Fab comprises a T225X mutation, wherein X is any amino acid except T.
6. The composition of claim 2 , wherein the IgG1 Fab terminates with amino acids comprising an amino acid sequence selected from the group consisting of CDKTHT (SEQ ID NO: 4), CDKTHL (SEQ ID NO: 15), CDKTH (SEQ ID NO: 16), CDKT (SEQ ID NO: 17), CDK and CD.
7. The composition of claim 6 , wherein the IgG1 Fab terminates with amino acids comprising the amino acid sequence CDKTHT (SEQ ID NO: 4)
8. The composition of claim 6 , wherein the IgG1 Fab terminates with amino acids comprising the amino acid sequence CDKTHL (SEQ ID NO: 15).
9. The composition of claim 6 , wherein the IgG1 Fab terminates with amino acids comprising the amino acid sequence CDKTH (SEQ ID NO: 16).
10. The composition of claim 6 , wherein the IgG1 Fab terminates with amino acids comprising the amino acid sequence CDKT (SEQ ID NO: 17).
11. The composition of claim 6 , wherein the IgG1 Fab terminates with amino acids comprising the amino acid sequence CDK.
12. The composition of claim 6 , wherein the IgG1 Fab terminates with amino acids comprising the amino acid sequence CD.
13. The composition of claim 1 , wherein the Fab antibody fragment is an IgG4 Fab.
14. The composition of claim 13 , wherein the IgG4 Fab terminates with amino acids comprising an amino acid sequence selected from group consisting of KYGPP (SEQ ID NO: 18), KYGP (SEQ ID NO: 19), KYG and KY.
15. The composition of claim 14 , wherein the IgG4 Fab terminates with amino acids comprising the amino acid sequence KYGPP (SEQ ID NO: 18).
16. The composition of claim 14 , wherein the IgG4 Fab terminates with amino acids comprising the amino acid sequence KYGP (SEQ ID NO: 19).
17. The composition of claim 14 , wherein the IgG4 Fab terminates with amino acids comprising the amino acid sequence KYG.
18. The composition of claim 14 , wherein the IgG4 Fab terminates with amino acids comprising the amino acid sequence KY.
19. The composition of claim 13 , wherein the IgG4 Fab terminates with residue K218.
20. The composition of claim 1 , wherein the Fab antibody fragment comprises a heavy chain constant region that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and conservative modifications of.
21. The composition of claim 1 , wherein the Fab antibody fragment exhibits reduced binding to FcγRIIIa, C1q or a combination thereof.
22. A pharmaceutical formulation comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/852,136 US20200308304A1 (en) | 2015-10-30 | 2020-04-17 | Hinge modified antibody fragments and methods of making |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562248792P | 2015-10-30 | 2015-10-30 | |
US201662346905P | 2016-06-07 | 2016-06-07 | |
PCT/US2016/059137 WO2017075229A1 (en) | 2015-10-30 | 2016-10-27 | Hinge modified antibody fragments and methods of making |
US15/967,154 US10662254B2 (en) | 2015-10-30 | 2018-04-30 | Hinge modified antibody fragments and methods of making |
US16/852,136 US20200308304A1 (en) | 2015-10-30 | 2020-04-17 | Hinge modified antibody fragments and methods of making |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/967,154 Continuation US10662254B2 (en) | 2015-10-30 | 2018-04-30 | Hinge modified antibody fragments and methods of making |
Publications (1)
Publication Number | Publication Date |
---|---|
US20200308304A1 true US20200308304A1 (en) | 2020-10-01 |
Family
ID=57288500
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/967,154 Active US10662254B2 (en) | 2015-10-30 | 2018-04-30 | Hinge modified antibody fragments and methods of making |
US16/852,136 Pending US20200308304A1 (en) | 2015-10-30 | 2020-04-17 | Hinge modified antibody fragments and methods of making |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/967,154 Active US10662254B2 (en) | 2015-10-30 | 2018-04-30 | Hinge modified antibody fragments and methods of making |
Country Status (9)
Country | Link |
---|---|
US (2) | US10662254B2 (en) |
EP (2) | EP3368579B1 (en) |
JP (3) | JP6920292B2 (en) |
CN (2) | CN109071674B (en) |
ES (1) | ES2904553T3 (en) |
HR (1) | HRP20220064T1 (en) |
PL (1) | PL3368579T3 (en) |
SI (1) | SI3368579T1 (en) |
WO (1) | WO2017075229A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11254734B2 (en) * | 2016-11-14 | 2022-02-22 | Morphosys Ag | Fab molecules with a rodent hinge region and a non-rodent CH1 region |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109071674B (en) * | 2015-10-30 | 2022-08-05 | 豪夫迈·罗氏有限公司 | Hinge-modified antibody fragments and methods of making the same |
CN116981695A (en) * | 2022-05-06 | 2023-10-31 | 上海药明合联生物技术有限公司 | Antibodies comprising engineered hinge regions and uses thereof |
CN115873833A (en) * | 2022-11-08 | 2023-03-31 | 上海泰昶生物技术有限公司 | Engineering strain and process for producing immunoglobulin G degrading enzyme |
Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005092925A2 (en) * | 2004-03-24 | 2005-10-06 | Xencor, Inc. | Immunoglobulin variants outside the fc region |
WO2006015371A2 (en) * | 2004-08-05 | 2006-02-09 | Genentech, Inc. | Humanized anti-cmet antagonists |
JP2006166905A (en) * | 2004-11-17 | 2006-06-29 | Oita Univ | HUMAN Fab ANTIBODY GENE CLUSTER FOR EFFECTIVELY NEUTRALIZING RABIES VIRUS |
WO2008156269A1 (en) * | 2007-06-18 | 2008-12-24 | Isu Abxis Co., Ltd. | Recombinant antibodies capable of binding to a cancer specific marker and uses thereof |
US20090138208A1 (en) * | 2002-10-31 | 2009-05-28 | Ge Healthcare Bio-Sciences Ab | Immunoglobulin g binding pocket |
WO2010122148A1 (en) * | 2009-04-24 | 2010-10-28 | Boehringer Ingelheim International Gmbh | An improved antibody domain and antibody fragments and antibodies based thereon |
US8273352B2 (en) * | 2008-04-28 | 2012-09-25 | Genentech, Inc. | Humanized anti-factor D antibodies and uses thereof |
WO2013026835A1 (en) * | 2011-08-23 | 2013-02-28 | Roche Glycart Ag | Fc-free antibodies comprising two fab fragments and methods of use |
US20130331297A1 (en) * | 2010-07-16 | 2013-12-12 | Avantgen, Inc. | Novel peptides and uses thereof |
WO2014164709A2 (en) * | 2013-03-11 | 2014-10-09 | Genzyme Corporation | Engineered anti-tgf-beta antibodies and antigen-binding fragments |
US9796776B2 (en) * | 2014-02-27 | 2017-10-24 | Allergan, Inc. | Complement factor Bb antibodies |
US10662254B2 (en) * | 2015-10-30 | 2020-05-26 | Genentech, Inc. | Hinge modified antibody fragments and methods of making |
Family Cites Families (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US5606040A (en) | 1987-10-30 | 1997-02-25 | American Cyanamid Company | Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group |
US5770701A (en) | 1987-10-30 | 1998-06-23 | American Cyanamid Company | Process for preparing targeted forms of methyltrithio antitumor agents |
CA2026147C (en) | 1989-10-25 | 2006-02-07 | Ravi J. Chari | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
EP0861893A3 (en) | 1991-09-19 | 1999-11-10 | Genentech, Inc. | High level expression of immunoglobulin polypeptides |
DK0669836T3 (en) | 1992-11-13 | 1996-10-14 | Idec Pharma Corp | Therapeutic use of chimeric and radiolabeled antibodies and human B lymphocyte restricted differentiation antigen for the treatment of B cell lymphoma |
US5635483A (en) | 1992-12-03 | 1997-06-03 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Tumor inhibiting tetrapeptide bearing modified phenethyl amides |
US5780588A (en) | 1993-01-26 | 1998-07-14 | Arizona Board Of Regents | Elucidation and synthesis of selected pentapeptides |
US5773001A (en) | 1994-06-03 | 1998-06-30 | American Cyanamid Company | Conjugates of methyltrithio antitumor agents and intermediates for their synthesis |
US5789199A (en) | 1994-11-03 | 1998-08-04 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5840523A (en) | 1995-03-01 | 1998-11-24 | Genetech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
US5712374A (en) | 1995-06-07 | 1998-01-27 | American Cyanamid Company | Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates |
US5714586A (en) | 1995-06-07 | 1998-02-03 | American Cyanamid Company | Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
US6171586B1 (en) | 1997-06-13 | 2001-01-09 | Genentech, Inc. | Antibody formulation |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
KR101077001B1 (en) | 1999-01-15 | 2011-10-26 | 제넨테크, 인크. | Polypeptide Variants with Altered Effector Function |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
AU782626B2 (en) | 1999-10-04 | 2005-08-18 | Medicago Inc. | Method for regulating transcription of foreign genes |
DE60031793T2 (en) | 1999-12-29 | 2007-08-23 | Immunogen Inc., Cambridge | DOXORUBICIN AND DAUNORUBICIN-CONTAINING CYTOTOXIC AGENTS AND THEIR THERAPEUTIC USE |
WO2003102157A2 (en) * | 2002-06-03 | 2003-12-11 | Genentech, Inc. | Synthetic antibody phage libraries |
US7217797B2 (en) | 2002-10-15 | 2007-05-15 | Pdl Biopharma, Inc. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
US7871607B2 (en) | 2003-03-05 | 2011-01-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
KR101520209B1 (en) | 2003-11-06 | 2015-05-13 | 시애틀 지네틱스, 인크. | Monomethylvaline compounds capable of conjugation to ligands |
JO3000B1 (en) | 2004-10-20 | 2016-09-05 | Genentech Inc | Antibody Formulations. |
EP2144931A2 (en) * | 2007-04-04 | 2010-01-20 | The Government Of The U.S.A, As Represented By The Secretary, Dept. Of Health And Human Services | Monoclonal antibodies against dengue and other viruses with deletion in fc region |
CN102271708A (en) * | 2008-11-13 | 2011-12-07 | 新兴产品开发西雅图有限公司 | CD37 immunotherapeutic combination therapies and uses thereof |
GB0920127D0 (en) * | 2009-11-17 | 2009-12-30 | Ucb Pharma Sa | Antibodies |
ES2813432T3 (en) * | 2011-08-17 | 2021-03-23 | Glaxo Group Ltd | Modified proteins and peptides |
TW201444872A (en) * | 2013-03-06 | 2014-12-01 | Merrimack Pharmaceuticals Inc | Anti-c-Met tandem Fc bispecific antibodies |
-
2016
- 2016-10-27 CN CN201680077081.2A patent/CN109071674B/en active Active
- 2016-10-27 PL PL16795192T patent/PL3368579T3/en unknown
- 2016-10-27 EP EP16795192.0A patent/EP3368579B1/en active Active
- 2016-10-27 JP JP2018521860A patent/JP6920292B2/en active Active
- 2016-10-27 HR HRP20220064TT patent/HRP20220064T1/en unknown
- 2016-10-27 EP EP21207346.4A patent/EP4036120A1/en active Pending
- 2016-10-27 CN CN202210935154.4A patent/CN115925962A/en active Pending
- 2016-10-27 WO PCT/US2016/059137 patent/WO2017075229A1/en active Application Filing
- 2016-10-27 SI SI201631449T patent/SI3368579T1/en unknown
- 2016-10-27 ES ES16795192T patent/ES2904553T3/en active Active
-
2018
- 2018-04-30 US US15/967,154 patent/US10662254B2/en active Active
-
2020
- 2020-04-17 US US16/852,136 patent/US20200308304A1/en active Pending
-
2021
- 2021-07-21 JP JP2021120669A patent/JP7413322B2/en active Active
-
2023
- 2023-12-27 JP JP2023221318A patent/JP2024045138A/en active Pending
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090138208A1 (en) * | 2002-10-31 | 2009-05-28 | Ge Healthcare Bio-Sciences Ab | Immunoglobulin g binding pocket |
WO2005092925A2 (en) * | 2004-03-24 | 2005-10-06 | Xencor, Inc. | Immunoglobulin variants outside the fc region |
WO2006015371A2 (en) * | 2004-08-05 | 2006-02-09 | Genentech, Inc. | Humanized anti-cmet antagonists |
JP2006166905A (en) * | 2004-11-17 | 2006-06-29 | Oita Univ | HUMAN Fab ANTIBODY GENE CLUSTER FOR EFFECTIVELY NEUTRALIZING RABIES VIRUS |
WO2008156269A1 (en) * | 2007-06-18 | 2008-12-24 | Isu Abxis Co., Ltd. | Recombinant antibodies capable of binding to a cancer specific marker and uses thereof |
US8273352B2 (en) * | 2008-04-28 | 2012-09-25 | Genentech, Inc. | Humanized anti-factor D antibodies and uses thereof |
WO2010122148A1 (en) * | 2009-04-24 | 2010-10-28 | Boehringer Ingelheim International Gmbh | An improved antibody domain and antibody fragments and antibodies based thereon |
US20130331297A1 (en) * | 2010-07-16 | 2013-12-12 | Avantgen, Inc. | Novel peptides and uses thereof |
WO2013026835A1 (en) * | 2011-08-23 | 2013-02-28 | Roche Glycart Ag | Fc-free antibodies comprising two fab fragments and methods of use |
WO2014164709A2 (en) * | 2013-03-11 | 2014-10-09 | Genzyme Corporation | Engineered anti-tgf-beta antibodies and antigen-binding fragments |
US9796776B2 (en) * | 2014-02-27 | 2017-10-24 | Allergan, Inc. | Complement factor Bb antibodies |
US10662254B2 (en) * | 2015-10-30 | 2020-05-26 | Genentech, Inc. | Hinge modified antibody fragments and methods of making |
Non-Patent Citations (3)
Title |
---|
Edwards et al., J Mol Biol. 334(1): 103-118 (Year: 2003) * |
Lloyd et al., Protein Engineering, Design & Selection 22:159-168 (Year: 2009) * |
Van Schie et al, mAbs 7(4): 662-671 (Year: 2015) * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11254734B2 (en) * | 2016-11-14 | 2022-02-22 | Morphosys Ag | Fab molecules with a rodent hinge region and a non-rodent CH1 region |
Also Published As
Publication number | Publication date |
---|---|
JP6920292B2 (en) | 2021-08-18 |
JP7413322B2 (en) | 2024-01-15 |
EP3368579B1 (en) | 2021-11-24 |
CN115925962A (en) | 2023-04-07 |
ES2904553T3 (en) | 2022-04-05 |
JP2021181452A (en) | 2021-11-25 |
CN109071674B (en) | 2022-08-05 |
JP2019504820A (en) | 2019-02-21 |
JP2024045138A (en) | 2024-04-02 |
HRP20220064T1 (en) | 2022-04-15 |
CN109071674A (en) | 2018-12-21 |
SI3368579T1 (en) | 2022-04-29 |
EP3368579A1 (en) | 2018-09-05 |
US10662254B2 (en) | 2020-05-26 |
WO2017075229A1 (en) | 2017-05-04 |
EP4036120A1 (en) | 2022-08-03 |
US20180305462A1 (en) | 2018-10-25 |
PL3368579T3 (en) | 2022-03-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20200308304A1 (en) | Hinge modified antibody fragments and methods of making | |
JP6707090B2 (en) | Fc region variants with altered FcRn binding and methods of use | |
US11286300B2 (en) | Humanized anti-human CD19 antibodies and methods of use | |
US10344085B2 (en) | Anti-IL-1beta antibodies | |
US10087246B2 (en) | Anti-PDGF-B antibodies and methods of use | |
US20190389971A1 (en) | The contorsbody - a single chain target binder | |
US11542308B2 (en) | FLT3 ligand fusion proteins and methods of use | |
US11820816B2 (en) | Anti-VEGF antibodies and methods of use | |
WO2022220275A1 (en) | ANTI-C1s ANTIBODY |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCV | Information on status: appeal procedure |
Free format text: NOTICE OF APPEAL FILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |