US20220135686A1 - Antibody that binds to human pd-l1 - Google Patents
Antibody that binds to human pd-l1 Download PDFInfo
- Publication number
- US20220135686A1 US20220135686A1 US17/574,960 US202217574960A US2022135686A1 US 20220135686 A1 US20220135686 A1 US 20220135686A1 US 202217574960 A US202217574960 A US 202217574960A US 2022135686 A1 US2022135686 A1 US 2022135686A1
- Authority
- US
- United States
- Prior art keywords
- antibody
- cancer
- human
- antigen
- binds
- 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
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 claims abstract description 50
- 102000048776 human CD274 Human genes 0.000 claims abstract description 50
- 102000008096 B7-H1 Antigen Human genes 0.000 claims abstract 5
- 108010074708 B7-H1 Antigen Proteins 0.000 claims abstract 5
- 239000000427 antigen Substances 0.000 claims description 67
- 102000036639 antigens Human genes 0.000 claims description 66
- 108091007433 antigens Proteins 0.000 claims description 66
- 230000027455 binding Effects 0.000 claims description 64
- 238000009739 binding Methods 0.000 claims description 64
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 55
- 239000012634 fragment Substances 0.000 claims description 52
- 238000000034 method Methods 0.000 claims description 39
- 206010028980 Neoplasm Diseases 0.000 claims description 36
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 29
- 239000013604 expression vector Substances 0.000 claims description 28
- 239000002773 nucleotide Substances 0.000 claims description 23
- 125000003729 nucleotide group Chemical group 0.000 claims description 23
- 241001529936 Murinae Species 0.000 claims description 22
- 201000011510 cancer Diseases 0.000 claims description 17
- 239000008194 pharmaceutical composition Substances 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 15
- 230000002018 overexpression Effects 0.000 claims description 10
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 8
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 8
- 201000005202 lung cancer Diseases 0.000 claims description 8
- 208000020816 lung neoplasm Diseases 0.000 claims description 8
- 201000002528 pancreatic cancer Diseases 0.000 claims description 8
- 206010006187 Breast cancer Diseases 0.000 claims description 7
- 208000026310 Breast neoplasm Diseases 0.000 claims description 7
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 7
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 7
- 208000032612 Glial tumor Diseases 0.000 claims description 7
- 206010018338 Glioma Diseases 0.000 claims description 7
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 7
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 7
- 206010033128 Ovarian cancer Diseases 0.000 claims description 7
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 7
- 206010038389 Renal cancer Diseases 0.000 claims description 7
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 claims description 7
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 7
- 201000010881 cervical cancer Diseases 0.000 claims description 7
- 208000029742 colonic neoplasm Diseases 0.000 claims description 7
- 201000004101 esophageal cancer Diseases 0.000 claims description 7
- 201000010982 kidney cancer Diseases 0.000 claims description 7
- 201000007270 liver cancer Diseases 0.000 claims description 7
- 208000014018 liver neoplasm Diseases 0.000 claims description 7
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 7
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 7
- 206010025323 Lymphomas Diseases 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 6
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 6
- 208000005017 glioblastoma Diseases 0.000 claims description 6
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 claims description 6
- 208000032839 leukemia Diseases 0.000 claims description 6
- 201000001441 melanoma Diseases 0.000 claims description 6
- 201000002510 thyroid cancer Diseases 0.000 claims description 6
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 4
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 4
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 230000009452 underexpressoin Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 abstract description 13
- 239000000126 substance Substances 0.000 abstract description 5
- 230000004048 modification Effects 0.000 abstract description 4
- 238000012986 modification Methods 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 33
- 210000004027 cell Anatomy 0.000 description 26
- 238000002965 ELISA Methods 0.000 description 17
- 241000699670 Mus sp. Species 0.000 description 13
- 238000010494 dissociation reaction Methods 0.000 description 13
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 13
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 description 12
- 230000005593 dissociations Effects 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 11
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 210000001744 T-lymphocyte Anatomy 0.000 description 8
- 229940098773 bovine serum albumin Drugs 0.000 description 8
- 210000004408 hybridoma Anatomy 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 241000700159 Rattus Species 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 102100020873 Interleukin-2 Human genes 0.000 description 5
- 108010002350 Interleukin-2 Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 229960003852 atezolizumab Drugs 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 210000000987 immune system Anatomy 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 239000012089 stop solution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- 241000283707 Capra Species 0.000 description 4
- 102100037850 Interferon gamma Human genes 0.000 description 4
- 108010074328 Interferon-gamma Proteins 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 238000005571 anion exchange chromatography Methods 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000005277 cation exchange chromatography Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000007405 data analysis Methods 0.000 description 4
- 210000004443 dendritic cell Anatomy 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000003211 malignant effect Effects 0.000 description 4
- 230000001613 neoplastic effect Effects 0.000 description 4
- 229960003301 nivolumab Drugs 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 229940066453 tecentriq Drugs 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 229920002873 Polyethylenimine Polymers 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 101000839781 Homo sapiens Immunoglobulin heavy variable 4-59 Proteins 0.000 description 2
- 101001090250 Homo sapiens Immunoglobulin kappa variable 6-21 Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 102100028405 Immunoglobulin heavy variable 4-59 Human genes 0.000 description 2
- 102100034806 Immunoglobulin kappa variable 6-21 Human genes 0.000 description 2
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 229960002303 citric acid monohydrate Drugs 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 210000005220 cytoplasmic tail Anatomy 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 238000012453 sprague-dawley rat model Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 1
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 description 1
- BDOYKFSQFYNPKF-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O BDOYKFSQFYNPKF-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 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
- 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
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 101000895912 Elizabethkingia meningoseptica Endo-beta-N-acetylglucosaminidase F2 Proteins 0.000 description 1
- 239000012739 FreeStyle 293 Expression medium Substances 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical group OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 1
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 238000011190 asparagine deamidation Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 229930195712 glutamate Chemical group 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 102000048362 human PDCD1 Human genes 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 201000010225 mixed cell type cancer Diseases 0.000 description 1
- 208000029638 mixed neoplasm Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 201000002740 oral squamous cell carcinoma Diseases 0.000 description 1
- 229940043515 other immunoglobulins in atc Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000012557 regeneration buffer Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229940087562 sodium acetate trihydrate Drugs 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 206010044412 transitional cell carcinoma Diseases 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- 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
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- 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
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
-
- 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
- C07K2317/70—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
-
- 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
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to the field of antibodies. More particularly, the present invention discloses a tetravalent bispecific antibody against PD-1 and PD-L1.
- Human programmed cell death receptor-1 (PD-1) is a type I membrane protein of 288 amino acids and is one of the known major immune checkpoints (Blank et al, 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed in activated T lymphocytes, and it binds to the ligands PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2) to inhibit the activity of T lymphocytes and related cellular immune responses in vivo.
- PD-L1 programmed cell death-Ligand 1
- PD-L2 programmeed cell death-Ligand 2
- PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, tissue cells of lung, liver, heart and the like. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in vivo, but also the main mechanism and cause of PD-L1 expression-positive tumor cells to evade immune surveillance. By blocking the negative regulation of cancer cells on the PD-1/PD-L1 signaling pathway and activating the immune system, T cell-related tumor-specific cellular immune responses can be promoted, thereby opening a new door for tumor treatment—tumor immunotherapy.
- PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Studies have shown that PD-1 negatively regulates antigen receptor signal transduction upon engagement of its ligands (PD-L1 and/or PD-L2). At present, the structure of mouse PD-1 and the co-crystal structure of mouse PD-1 with human PD-L1 have been clarified (Zhang, X. et al., Immunity 20: 337-347 (2004); Lin et al., Proc.Natl.Acad.Sci.USA 105: 3011-6(2008)).
- PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain that is responsible for ligand binding and a cytoplasmic tail that is responsible for the binding of signal transduction molecules.
- the cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, an ITIM (immunoreceptor tyrosine-based inhibition motif) and an ITSM (immunoreceptor tyrosine-based switch motif).
- PD-1 plays an important role in tumor immune evasion mechanism.
- Tumor immunotherapy which uses the body's own immune system to fight cancer, is a breakthrough in cancer treatment.
- the tumor microenvironment may protect tumor cells from effective immune damage, so how to break the tumor microenvironment becomes the focus of anti-tumor research.
- Existing research results have identified the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and may be induced by IFN ⁇ in most PD-L1-negative tumor cell lines), and presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc.Natl.Acad.Sci.U.S.A.
- PD-1 on tumor infiltrating lymphocytes
- PD-L1 on tumor cells
- Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer as well as tumors of head and neck, etc.
- blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-1 and PD-L1 have become popular targets for development of tumor immunotherapy drugs.
- Bispecific antibodies refer to antibody molecules that can specifically bind to two antigens or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that may be used to treat various inflammatory diseases, cancers and other diseases. Although a large number of new bispecific antibody structural forms have been reported recently, the main technical difficulty in producing bispecific antibodies lies in obtaining the correct paired molecules.
- bispecific antibodies all have mismatch problems, so one or more by-products or aggregates caused by mismatches will be produced, thereby affecting the yield, purity and physical and chemical stability of the bispecific antibodies of interest, and further affecting the safety and efficiency of the bispecific antibodies in vivo.
- the present invention provides an antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the antibody that binds to human PD-L1.
- the first object of the present invention is to provide an antibody that binds to human PD-L1 or an antigen-binding fragment thereof.
- the second object of the present invention is to provide an isolated nucleotide encoding the antibody that binds to human PD-L1 or the antigen-binding fragment thereof.
- the third object of the present invention is to provide an expression vector comprising the nucleotide.
- the fourth object of the present invention is to provide a host cell comprising the expression vector.
- the fifth object of the present invention is to provide a method of preparing the antibody that binds to human PD-L1 or the antigen-binding fragment thereof.
- the sixth object of the present invention is to provide a pharmaceutical composition comprising the antibody that binds to human PD-L1 or the antigen-binding fragment thereof.
- the seventh object of the present invention is to provide the use of the antibody that binds to human PD-L1 or the antigen-binding fragment thereof or the pharmaceutical composition in the preparation of a medicine for the treatment of PD-L1 overexpression diseases.
- the eighth object of the present invention is to provide a method of treating PD-L1 overexpression diseases with the antibody that binds to human PD-L1 or the antigen-binding fragment thereof or the pharmaceutical composition.
- the ninth object of the present invention is to provide a tetravalent bispecific antibody against PD-1 and PD-L1.
- the tenth object of the present invention is to provide an isolated nucleotide encoding the tetravalent bispecific antibody.
- the eleventh object of the present invention is to provide an expression vector comprising the nucleotide.
- the twelfth object of the present invention is to provide a host cell comprising the expression vector.
- the thirteenth object of the present invention is to provide a method of preparing the tetravalent bispecific antibody.
- the fourteenth object of the present invention is to provide a pharmaceutical composition comprising the tetravalent bispecific antibody.
- the fifteenth object of the present invention is to provide the use of the tetravalent bispecific antibody or the pharmaceutical composition in the preparation of a medicine for the treatment of cancer.
- the sixteenth object of the present invention is to provide a method of treating cancer with the tetravalent bispecific antibody or the pharmaceutical composition.
- the first aspect of the present invention provides an antibody that binds to human PD-L1 or the antigen-binding fragment thereof, comprising:
- the antibody is a monoclonal antibody or a polyclonal antibody.
- the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
- the antigen-binding fragment comprises a Fab fragment, a F(ab′)2 fragment, a Fv fragment or a single chain antibody.
- the antibody that binds to human PD-L1 or the antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 10.
- the antibody that binds to human PD-L1 or the antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 13, and a light chain having an amino acid sequence as shown in SEQ ID NO: 15.
- the second aspect of the present invention provides an isolated nucleotide, which encodes the antibody that binds to human PD-L1 or the antigen-binding fragment thereof as described above.
- nucleotide sequence encoding the heavy chain of the antibody that binds to human PD-L1 or the antigen-binding fragment thereof is shown in SEQ ID NO: 14, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 16.
- the third aspect of the present invention provides an expression vector, which comprises the nucleotides as described above.
- the fourth aspect of the present invention provides a host cell, which comprises the expression vector as described above.
- the fifth aspect of the present invention provides a method of preparing the antibody that binds to human PD-L1 or the antigen-binding fragment thereof, which comprises the following steps:
- step b) isolating and purifying the antibody that binds to human PD-L1 or the antigen-binding fragment thereof of step a).
- the sixth aspect of the present invention provides a pharmaceutical composition, which comprises the antibody that binds to human PD-L1 or the antigen-binding fragment thereof as described above and a pharmaceutically acceptable carrier.
- the seventh aspect of the present invention provides the use of the antibody that binds to human PD-L1 or the antigen-binding fragment thereof, or of the pharmaceutical composition as described above, in the preparation of a medicine for the treatment of PD-L1 overexpression diseases.
- the PD-L1 overexpression disease is cancer.
- the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- the eighth aspect of the present invention provides a method of treating PD-L1 overexpression diseases, which comprises administering the antibody that binds to human PD-L1 or the antigen-binding fragment thereof or the pharmaceutical composition as described above to a subject in need.
- the PD-L1 overexpression disease is cancer.
- the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- the ninth aspect of the present invention provides a tetravalent bispecific antibody against PD-1 and PD-L1, comprising two polypeptide chains and four common light chains, wherein the polypeptide chains have an amino acid sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 31, and the common light chains have the amino acid sequence as shown in SEQ ID NO: 15.
- the tenth aspect of the present invention provides an isolated nucleotide, which encodes the tetravalent bispecific antibody.
- the nucleotide encodes the polypeptide chains and the common light chains, wherein the nucleotide sequence encoding the polypeptide chains is as shown in SEQ ID NO: 30 or SEQ ID NO: 32, the nucleotide sequence encoding the common light chains is as shown in SEQ ID NO:16.
- the eleventh aspect of the present invention provides an expression vector, which comprises the nucleotide as described above.
- the twelfth aspect of the present invention provides a host cell, which comprises the expression vector as described above.
- the thirteenth aspect of the present invention provides a method of preparing the tetravalent bispecific antibody, which comprises the following steps:
- the fourteenth aspect of the present invention provides a pharmaceutical composition, which comprises the tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier.
- the fifteenth aspect of the present invention provides the use of the tetravalent bispecific antibody or the pharmaceutical composition as described above in the preparation of a medicine for the treatment of cancer.
- the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- the sixteenth aspect of the present invention provides a method of treating cancers, which comprises administering the tetravalent bispecific antibody or the pharmaceutical composition as described above to a subject in need.
- the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- the present invention provides an antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the antibody that binds to human PD-L1.
- the tetravalent bispecific antibody of the present invention requires no Fc modification, has no mismatch problems, and the preparation method thereof is simple. Its biological activities and physical and chemical properties are similar with or even better than those of the monoclonal antibodies.
- FIG. 1 is a schematic diagram of the structure of the bispecific antibody of the present invention, where VH-A represents the heavy chain variable region of Anti-PDL1 or 609, VH-B represents the heavy chain variable region of 609 or Anti-PDL1, and VL represents the light chain variable region of the common light chains, CH1, CH2, and CH3 are the three domains of the heavy chain constant region, CL is the light chain constant region of the common light chains, and the line between the two heavy chains represents the disulfide bond, the line between the heavy chain and the light chain also represents the disulfide bond, the line between the CH1 near the N-terminus of the polypeptide chain and the VH-A represents the artificially designed linker, and the line between the CH1 near the C-terminus of the polypeptide chain and the CH2 represents the natural linker and hinge region of the antibody (if the heavy chain is human IgG4 subtype, the hinge region contains the S228P mutation, according to the EU numbering).
- VH-A represents the heavy chain variable region of
- FIG. 2 shows the ELISA results of the relative affinity of Anti-PDL1 to PD-L1.
- FIG. 3 shows results of the ability of Anti-PDL1 to block the interaction of PD-1/PD-L1.
- FIGS. 4A and 4B show the evaluation results of the ability of Anti-PDL1 to enhance MLR.
- FIGS. 5A and 5B show the ELISA results of Anti-PDL1 and 609 and their hybrid antibodies.
- FIGS. 6A and 6B show the ELISA results of PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4.
- FIGS. 7A ⁇ 7 D show the evaluation results of the ability of 609-Fab-PDL1-IgG4 to enhance MLR.
- FIGS. 8A and 8B show the pharmacokinetic results of 609-Fab-PDL1-IgG4.
- FIGS. 9A and 9B show the HPLC-SEC patterns of 609-Fab-PDL1-IgG4.
- FIGS. 10A ⁇ 10 D show the CE-SDS patterns of 609-Fab-PDL1-IgG4.
- FIGS. 11A and 11B show the HPLC-IEC patterns of 609-Fab-PDL1-IgG4.
- FIGS. 12A and 12B show the DSC patterns of 609-Fab-PDL1-IgG4.
- FIG. 13 shows the mass spectrum pattern of 609-Fab-PDL1-IgG4.
- FIG. 14 shows the anti-tumor effect of the bispecific antibody 609-Fab-PDL1-IgG4 in mice.
- the terms “antibody (abbreviated as Ab)” and “immunoglobulin G (abbreviated as IgG)” are heterotetrameric glycoproteins of about 150,000 daltons with identical structural characteristics, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable region (VH) followed by constant regions. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3.
- Each light chain has a variable region (VL) at one end and a constant region at its other end, the light chain constant region includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region the heavy chain, and the light chain variable region is paired with the variable region of the heavy chain.
- the constant regions are not involved directly in binding an antibody to an antigen, but they exhibit various effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC).
- the heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes; the light chain constant region includes ⁇ (Kappa) or ⁇ (Lambda).
- the heavy chain and light chain of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.
- the antibody of the present invention comprises monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two antibodies (such as bispecific antibodies), antigen-binding fragments of antibodies, etc.
- the antibody of the present invention comprises murine antibodies, chimeric antibodies, humanized antibodies, etc.
- BsAb bispecific antibody
- the term “monoclonal antibody (mAb)” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies contained in the population are the same, except for a few possible naturally occurring mutations.
- Monoclonal antibodies target a single antigen site with high specificity.
- polyclonal antibody preparations usually a mixture having different antibodies directed against different antigen determinants
- each monoclonal antibody is directed against a single determinant on the antigen.
- the benefit of monoclonal antibodies is that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins.
- the modifier “monoclonal” indicates the characteristics of an antibody, which is obtained from a substantially uniform antibody population, and should not be interpreted as requiring any special method to produce the antibody.
- murine antibody refers to an antibody derived from rats or mice, preferably mice.
- the murine antibody of the present invention is obtained by immunizing mice with the extracellular domain of human PD-L1 as an antigen and screening hybridoma cells.
- chimeric antibody refers to an antibody that comprises heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having mouse heavy and light chain variable regions linked to human constant region.
- humanized antibody means that the CDRs are derived from a non-human (preferably, mouse) antibody, while the remaining parts (including framework regions and constant regions) are derived from human antibody.
- framework region residues may be altered to preserve the binding affinity.
- the term “antigen-binding fragment” refers to a fragment of an antibody capable of specifically binding to an epitope of human PD-L1.
- the antigen-binding fragments of the present invention include Fab fragments, F(ab′)2 fragments, Fv fragments, and single chain antibodies (scFv).
- the Fab fragment is composed of the domains which are the VH and CH1 of the heavy chain of the antibody, and the VL and CL of the light chain of the antibody.
- the F(ab′)2 fragment is a fragment produced by digesting the antibody with pepsin.
- the Fv fragment is composed of dimers in which the heavy chain variable region and the light chain variable region of the antibody are closely and non-covalently related.
- the single-chain antibody (scFv) is an antibody in which the heavy chain variable region and the light chain variable region of the antibody are linked by a short peptide (linker) of 15-20 amino acids.
- Fc is a fragment crystallizable (Fc), which is composed of the CH2 and CH3 domains of an antibody.
- the Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.
- variable refers to the fact that certain portions of the variable regions differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen.
- variability is not evenly distributed through the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain variable regions and the heavy chain variable regions.
- CDRs complementarity determining regions
- FR framework regions
- the variable regions of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of the ⁇ -sheet structure.
- the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).
- the terms “anti-/against” and “binding” refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against.
- the antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10 ⁇ 7 M, for example, less than about 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M or less.
- KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
- SPR surface plasmon resonance
- the term “valency” refers to the presence of a specified number of antigen binding sites in an antibody molecule.
- the bispecific antibody of the present invention has four antigen binding sites and is tetravalent.
- the antigen binding site includes a heavy chain variable region (VH) and a light chain variable region (VL).
- epitope refers to a polypeptide determinant that specifically binds to an antibody.
- the epitope of the present invention is a region of an antigen that is bound by an antibody.
- the term “common light chain” refers to a light chain comprising the same light chain variable region and light chain constant region, which can pair with the heavy chain of a first antibody that binds to a first antigen to form a binding site that specifically binds to the first antigen, and can also pair with the heavy chain of a second antibody that binds to a second antigen to form a binding site that specifically binds to the second antigen. Further, the light chain variable region of the common light chain and the heavy chain variable region of the first antibody form the first antigen binding site, and the light chain variable region of the common light chain and the heavy chain variable region of the second antibody form the second antigen binding site.
- expression vector may be pTT5, pSECtag series, pCGS3 series, pCDNA series vectors, as well as other vectors used in mammalian expression systems, etc.
- the expression vector comprises a fusion DNA sequence connected with appropriate transcription and translation regulatory sequences.
- the term “host cell” refers to a cell suitable for expressing the expression vector as described above, which may be a eukaryotic cell, for example, mammalian or insect host cell culture system may be used to express the fusion protein of the present invention, CHO (Chinese hamster Ovary), HEK293, COS, BHK, as well as derived cells of the above-mentioned cells are applicable to the present invention.
- a eukaryotic cell for example, mammalian or insect host cell culture system may be used to express the fusion protein of the present invention, CHO (Chinese hamster Ovary), HEK293, COS, BHK, as well as derived cells of the above-mentioned cells are applicable to the present invention.
- the term “pharmaceutical composition” means that the antibody or antigen-binding fragment thereof that binds to human PD-L1 or the tetravalent bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition, so as to exert a therapeutic effect more stably.
- These preparations can ensure the conformational integrity of the amino acid core sequences of the antibody or antigen-binding fragment thereof that binds to human PD-L1 disclosed in the present invention, and meanwhile, protect the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).
- the target genes are constructed into the expression vector pcDNA4, and the constructed expression vectors or combination of expression vectors are transferred into FreeStyleTM 293-F cells (hereinafter referred to as HEK293F, purchased from Thermo Fisher Scientific) using PEI (Polyethylenimine), to express an antibody or recombinant protein.
- HEK293F cells are cultured in Free Style 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days and the cell supernatant are collected, and then the antibody or recombinant protein are purified by ProteinA affinity chromatography or nickel affinity chromatography.
- MLR mixed lymphocyte reaction
- the induced dendritic cells (10 4 /well) and the isolated CD4 + T cells (10 5 /well) are mixed in proportion and then inoculated into a 96-well plate, 150 ⁇ l per well; a few hours later, 50 ⁇ l of serially diluted antibody is added into the above 96-well plate; the 96-well plate is incubated in a 37° C. cell incubator for 3 days.
- AIM-V medium purchased from Thermo Fisher Scientific
- the secretion of IL-2 and IFN- ⁇ is detected in accordance with standard operating procedures.
- IL-2 and IFN- ⁇ are detected using double-antibody sandwich ELISA (related paired antibodies are purchased from BD Biosciences).
- OD450 values are read with a microplate reader (SpectraMax 190). Graphing is performed by GraphPad Prism6 and EC50 values are calculated.
- Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Due to changes such as post-translational modification, aggregation, and degradation, antibodies are heterogeneous in their biochemical and biophysical properties. Variants, aggregates, and degraded fragments are commonly observed when bispecific antibodies are analyzed by separation techniques, and their presence may compromise safety and effectiveness. Aggregates, degraded fragments and incompletely assembled molecules are prone to appear during production and storage of an antibody. In the present invention, high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) is used to detect the content of the above impurities in a sample.
- HPLC-SEC high-performance liquid chromatography-size exclusion chromatography
- the molecular weight of the aggregate is larger than that of the monomer, so the retention time of the corresponding peak is shorter; the molecular weight of the degraded fragment or the incompletely assembled molecule is smaller than that of the monomer, so the retention time of the corresponding peak is longer.
- Chromatograph used for HPLC-SEC Dionex Ultimate 3000; the method for the preparation of mobile phase is as follows: an appropriate amount of 20 mM sodium dihydrogen phosphate mother liquor is adjusted with 20 mM disodium hydrogen phosphate to a pH of 6.8 ⁇ 0.1; injection amount: 20 ⁇ g; chromatograph column: TSK G3000SWXL, specification: 7.8 ⁇ 300 mm 5 ⁇ m; flow rate: 0.5 ml/min, elution time: 30 min; column temperature: 25° C., sample room temperature: 10° C.; detection wavelength: 214 nm.
- charge variants can be separated and analyzed based on the charge.
- Commonly used analysis methods include cation exchange chromatography (CEX) and anion exchange chromatography (AEX).
- CEX cation exchange chromatography
- AEX anion exchange chromatography
- the acidic species are the variants that eluted earlier than the main peak of CEX or later than the main peak of AEX, while the basic species are the variants that eluted later than the main peak of CEX or earlier than the main peak of AEX.
- the peaks corresponding to the acidic species and the basic species are called acidic peaks and basic peaks, respectively.
- Charge variants are easily generated during the production and storage of antibodies.
- high-performance liquid chromatography-ion exchange chromatography HPLC-IEC is used to analyze the charge heterogeneity of the samples.
- CE-SDS Capillary Electrophoresis-Sodium Dodecyl Sulfate
- CE is divided into two types: non-reduced and reduced; for the former, when the sample is denatured, the reducing agent DTT is not needed to destroy the disulfide bond in the molecule; for the latter, when the sample is denatured, the reducing agent DTT is needed to destroy the disulfide bond in the molecule.
- Non-reduced and reduced CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively.
- the capillary electrophoresis instrument used is ProteomeLabTM PA800 plus (Beckman Coulter), equipped with a UV 214 nm detector, capillary model: Bare Fused-Silica Capillary, specification: 30.7 cm ⁇ 50 ⁇ m, effective length: 20.5 cm; other related reagents: purchased from Beckman Coulter.
- the key parameters of the instrument are set as follows: temperature of capillary and sample chamber: 20 ⁇ 2° C., separation voltage: 15 kV.
- Differential Scanning calorimeter reflects the thermal stability of the sample mainly by detecting the heat change in biomolecules in a controlled heating or cooling process.
- the unfolding of the protein sample will absorb heat, and the supplementary energy required to eliminate the temperature difference in the sample pool will be recorded by the device.
- These heat changes will form a peak shape in the spectrum.
- the peak top temperature corresponding to the unfolding of the protein sample is taken as the melting temperature Tm.
- Tm is an important indicator of protein thermal stability. The higher the Tm is, the better the stability of the protein is.
- the antibody is deglycosylated by treating with PNGase F and endoglycosidase F2.
- UPLC-XEVO G2 Q-TOF liquid chromatography-mass spectrometry system Waters is used for the analysis and identification of molecular weight of the sample.
- Mobile phase A is HPLC-grade water containing 0.1% trifluoroacetic acid (TFA).
- Mobile phase B is acetonitrile containing 0.1% TFA.
- the chromatographic column used is Mass PREPTM Micro Desalting Column (specification 2.1 ⁇ 5 mm).
- the source of genes encoding the extracellular regions of PD-1 and PD-L1 was described in WO2018/137576A1.
- the ends of the extracellular region coding genes of PD-1 and PD-L1 were connected to the polyhistidine coding sequence, respectively, and then the recombinant genes were cloned into the pcDNA4 expression vector, and the recombinant proteins were expressed and purified, respectively.
- the obtained recombinant proteins were named PD1-His and PD-L1-His, respectively.
- the ends of the extracellular region coding genes of PD-1 and PD-L1 were connected to the Fc segment coding sequence of human IgG1, respectively, and then the recombinant genes were cloned into the pcDNA4 expression vector, and the recombinant proteins were expressed and purified, respectively.
- the obtained recombinant proteins were named PD1-ECD-hFc and PD-L1-ECD-hFc, respectively.
- the above PD-L1-ECD-hFc was used as an antigen to immunize Balb/c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.).
- the methods of immunizing mice, detecting titer and screening hybridoma clones are described in Example 2 of WO2018/137576A1.
- the method of screening positive clones of hybridomas by ELISA is as follows: An ELISA plate was coated with the above PD-L1-His with a coating concentration of 10 ng/well, and blocked with PBST (KH 2 PO 4 0.2 g, Na 2 HPO 4 .12H 2 O 2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-20 0.5 ml, added with pure water to 1 L) containing 1% bovine serum albumin (BSA).
- PBST KH 2 PO 4 0.2 g, Na 2 HPO 4 .12H 2 O 2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-20 0.5 ml, added with pure water to 1 L
- BSA bovine serum albumin
- the antibody to be tested was serially diluted, and then transferred to the above plate coated with the recombinant protein, incubated at room temperature for half an hour and then the plate was washed; an appropriately diluted HRP (Horseradish Peroxidase)-labeled goat anti-mouse antibody (Fc-Specific) (purchased from Sigma) was added, incubated at room temperature for half an hour and then the plate was washed; 100 ⁇ l of chromogenic solution (chromogenic substrate solution A: sodium acetate-trihydrate 13.6 g, citric acid-monohydrate 1.6 g, 30% hydrogen peroxide 0.3 ml, pure water 500 ml; chromogenic substrate solution B: disodium ethylenediaminetetraacetic acid 0.2 g, citric acid-monohydrate 0.95 g, glycerol 50 ml, 0.15 g TMB dissolved in 3 ml DMSO, pure water 500 ml; A and B mixed in equal volume before use
- the positive hybridoma clones were selected for expansion in a 24-well plate and subcloned by limiting dilution method.
- a monoclonal hybridoma cell line stably expressing the target antibody was obtained by the above-mentioned method, and these clones were amplified.
- the above hybridoma cell line was cultured in serum-free medium Hybridoma-SFM (purchased from Thermo Fisher Scientific) for 7 days, and then the murine anti-human PD-L1 monoclonal antibody was purified from the culture supernatant by Protein A/G affinity chromatography. After purification, several murine monoclonal antibodies that can bind to human PD-L1 were obtained. The relative affinity of the murine monoclonal antibodies to human PD-L1 was evaluated by ELISA. Finally, the clone M8 with the highest relative affinity was selected for further development.
- Step 1 Determination of the Variable Region Sequence of Murine Anti-Human PD-L1 Monoclonal Antibody
- RNAs were extracted from the M8 hybridoma monoclonal cell line using Trizol, and mRNAs were reverse transcribed into cDNAs using a reverse transcription kit.
- primer combination reported in the literature (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dubel; the sequences of the primer combination from page 323), the genes of light chain variable region and heavy chain variable region of M8 were amplified by PCR, and then the PCR products were cloned into the pMD18-T vector, and the variable region genes were sequenced and analyzed.
- the amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody M8 were analyzed, and the complementarity-determining regions and frame regions of the antibody M8 were determined according to Kabat rule.
- the amino acid sequences of the heavy chain CDRs of the antibody M8 are: H-CDR1: SYGVH (SEQ ID NO: 1), H-CDR2: LIWSGGGTDYNAAFIS (SEQ ID NO: 2) and H-CDR3: QLGLRAMDY (SEQ ID NO: 3), the amino acid sequences of CDRs of the light chain are: L-CDR1: RASQSIGTTIH (SEQ ID NO: 4), L-CDR2: YASESVS (SEQ ID NO: 5) and L-CDR3: QQSNSWPLT (SEQ ID NO: 6).
- Step 2 Humanization of Murine Anti-Human PD-L1 Monoclonal Antibody
- the homology comparison of the heavy chain variable region of the murine antibody M8 with the human IgG germline sequence was performed at https://www.ncbi.nlm.nih.gov/igblast/. Selecting IGHV4-59*01 as the heavy chain CDR grafting template, the heavy chain CDRs of the murine antibody M8 were transplanted into the framework regions of IGHV4-59*01, and WGQGTSVTVSS (SEQ ID NO: 7) was added following the H-CDR3 as the fourth framework region, to obtain a CDR-grafted heavy chain variable region sequence. Similarly, the homology comparison of the light chain variable region of the murine antibody M8 with the human IgG germline sequence was performed.
- IGKV6-21*01 As the light chain CDR grafting template, the light chain CDRs of the murine antibody M8 were transplanted into the framework regions of IGKV6-21*01, and FGAGTKLEIK (SEQ ID NO: 8) was added following the L-CDR3 as the fourth framework region, to obtain a CDR-grafted light chain variable region sequence.
- FGAGTKLEIK SEQ ID NO: 8
- some amino acid sites were subjected to mutation. When mutation was performed, the amino acid sequence was numbered by Kabat rule and the position of each site was indicated by Kabat numbering.
- E at position 6 was mutated to Q
- P at position 9 was mutated to G
- E at position 16 was mutated to Q
- T at position 17 was mutated to S
- G at position 27 was mutated to F
- I at position 29 was mutated to L
- I at position 37 was mutated to V
- a at position 61 was mutated to P
- a at position 62 was mutated to S
- F at position 63 was mutated to L
- I at position 64 was mutated to K
- V at position 67 was mutated to L
- V at position 71 was mutated to R
- F at position 78 was mutated to V
- L at position 80 was mutated to F
- L at position 82 was mutated to I
- V at position 82C was mutated to L.
- Q at position 11 was mutated to L
- E at position 53 was mutated to Q
- V at position 53 was mutated to Q
- V at position 53
- the above heavy chain variable region and light chain variable region with mutation sites were defined as humanized heavy chain variable region and light chain variable region (SEQ ID NOs: 9 and 10), respectively.
- the DNAs encoding the humanized heavy chain and light chain variable regions were synthesized by Shanghai Sango Biotech Co., Ltd.
- the synthesized humanized heavy chain variable region was connected to the human IgG1 heavy chain constant region (SEQ ID NO: 11) to obtain a full-length humanized heavy chain gene, named Anti-PDL1-HC (SEQ ID NOs: 13 and 14).
- the humanized light chain variable region was connected to the human Kappa chain constant region (SEQ ID NO: 12) to obtain a full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NOs: 15 and 16).
- the Anti-PDL1-HC and Anti-PDL1-LC genes were constructed into the pcDNA4 expression vector, respectively, and the resulting heavy and light chain expression vectors were transferred together into HEK293F cells by PEI transfection method to express the antibody, and the antibody was purified using Protein A affinity chromatography. The obtained antibody was named Anti-PDL1.
- the final Anti-PDL1 antibody comprises the heavy chain CDRs having the amino acid sequences: H-CDR1: SYGVH (SEQ ID NO: 17), H-CDR2: LIWSGGGTDYNPSLKS (SEQ ID NO: 18) and H-CDR3: QLGLRAMDY (SEQ ID NO: 19); the light chain CDRs having the amino acid sequences: L-CDR1: RASQSIGTTIH (SEQ ID NO: 20), L-CDR2: YASQSFS (SEQ ID NO: 21) and L-CDR3: QQSNSWPLT (SEQ ID NO: 22).
- the sequences of heavy chain variable region and light chain variable region of the positive control antibody Atezolizumab were obtained from WHO Drug Information, Vol. 29, No. 3, 2015 (SEQ ID NOs: 23 and 24).
- the DNAs encoding the above variable regions were synthesized by Shanghai Sango Biotech Co., Ltd.
- Atezolizumab-VH The heavy chain variable region of Atezolizumab (Atezolizumab-VH) was connected to the human IgG1 heavy chain constant region (SEQ ID NO: 11) to obtain a full-length heavy chain gene, named Atezolizumab-HC; the light chain variable region of Atezolizumab (Atezolizumab-VL) was connected to the human Kappa light chain constant region (SEQ ID NO: 12) to obtain a full-length light chain gene, named Atezolizumab-LC.
- Atezolizumab-HC and Atezolizumab-LC were constructed into the pcDNA4 expression vector, respectively, and the antibody was expressed and purified. The obtained antibody was named Atezolizumab-IgG1.
- An ELISA plate was coated with the above PD-L1-His with a coating concentration of 10 ng/well, and blocked with PBST containing 1% BSA.
- the antibody to be tested was serially diluted, and then transferred to the above plate coated with recombinant protein, incubated at room temperature for half an hour and then the plate was washed; an appropriately diluted HRP-labeled goat anti-mouse antibody (Fc-Specific) (purchased from Sigma) was added, incubated at room temperature for half an hour and then the plate was washed; 100 ⁇ l of chromogenic solution with TMB as a substrate was added to each well, incubated at room temperature for 1 ⁇ 5 min; 50 ⁇ l of stop solution (2M H 2 SO 4 ) was added to stop the reaction.
- OD450 values were read with a microplate reader (SpectraMax 190), and graphing and data analysis were performed using GraphPad Prism6, and EC50 values were calculated.
- both Anti-PDL1 and Atezolizumab-IgG1 can effectively bind to PD-L1-His, with EC50s of 0.1018 nM and 0.09351 nM, respectively, and their apparent affinities are equivalent.
- the isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.
- PD-L1-ECD-hFc was biotinylated with Biotin N-hydroxysuccinimide ester (purchased from Sigma, Catalog No./Specification: H1759-100MG).
- Human PD-1-ECD-hFc was diluted to 2 ⁇ g/ml with sodium carbonate buffer (1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 dissolved in 1 L of pure water), and added into a 96-well ELISA plate with a multichannel pipette at 100 ⁇ l/well, incubated at room temperature for 4 h; washed once with PBST, blocked with PBST containing 1% BSA at 200 ⁇ l/well, incubated at room temperature for 2 h; the blocking solution was discarded, the plate was patted dry, and placed at 4° C.
- the biotinylated PD-L1-ECD-hFc was diluted to 500 ng/ml with PBST solution containing 1% BSA in a 96-well plate; the anti-human PD-L1 antibody was serially diluted with the above protein solution; the above diluted antibody and the biotinylated PD-L1-ECD-hFc were mixed and transferred to the above ELISA plate coated with human PD1-ECD-hFc, and incubated at room temperature for 1 hour; the plate was washed 3 times with PBST; Streptavidin-HRP (purchased from BD Biosciences) diluted 1:1000 in PBST with 1% BSA was added, and incubated at room temperature for 45 min; the plate was washed 3 times with PBST; chromogenic solution (with TMB as a substrate) was added at 100 ⁇ l/well, incubated at room temperature for 1 ⁇ 5 min; stop solution (2M H 2 SO 4 ) was added at
- both Anti-PDL1 and Atezolizumab-IgG1 can effectively block the interaction between PD-1 and PD-L1, with IC50s of 1.366 nM and 1.471 nM, respectively, and their blocking abilities are equivalent.
- the isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.
- both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR to secrete IL-2, with EC50s of 0.306 nM and 0.29 nM, respectively.
- both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR to secrete IFN- ⁇ , with EC50s of 0.1464 nM and 0.1294 nM, respectively.
- the isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1.
- MAb1-25-Hu (hereinafter referred to as 609) is a humanized anti-human PD-1 monoclonal antibody, the sequences of heavy chain variable region and light chain variable region of which are described in WO2018/137576A1.
- Humanized heavy chain variable region and light chain variable region (SEQ ID NOs: 25 and 26) were connected to the human IgG4 (S228P) heavy chain constant region (SEQ ID NO: 27) and Kappa light chain constant region (SEQ ID NO: 12), respectively, to finally obtain the heavy and light chain amino acid sequences of the complete humanized monoclonal antibody mAb1-25-Hu (609).
- Anti-PDL1 is a humanized monoclonal antibody of anti-human PD-L1, and its sequence is shown in Example 1.3.
- BLAST Basic Local Alignment Search Tool
- the heavy chain and light chain genes of Anti-PDL1 were named Anti-PDL1-HC and Anti-PDL1-LC, and the heavy chain and light chain genes of 609 were named 609-HC and 609-LC, respectively. They were constructed into the pcDNA4 expression vector, respectively.
- the above heavy chain and light chain expression vectors were combined in the following manner: Anti-PDL1-HC+Anti-PDL1-LC, 609-HC+609-LC, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC, and the antibodies were expressed and purified.
- the obtained antibodies were named Anti-PDL1, 609, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC, respectively.
- ELISA plates were coated with the above PD1-ECD-hFc and PD-L1-ECD-hFc with a coating concentration of 10 ng/well, respectively.
- the plates were blocked with PBST containing 1% BSA.
- the antibodies to be tested were serially diluted, then transferred to the above plates coated with recombinant proteins, incubated at room temperature for half an hour and then the plates were washed; an appropriately diluted HRP-labeled goat anti-human antibody (Fc-Specific) (purchased from Sisgma) was added, incubated at room temperature for half an hour and then the plates were washed; chromogenic solution with TMB as a substrate was added at 100 ⁇ l/well, incubated at room temperature for 1 ⁇ 5 min; 50 ⁇ l of stop solution (2M H 2 SO 4 ) was added to stop the reaction.
- OD450 values were read with a microplate reader (SpectraMax 190). Graphing and data analysis were performed using GraphPad
- 609 and 609-HC+Anti-PDL1-LC can effectively bind to PD1-ECD-hFc, with EC50s of 0.2001 nM and 0.2435 nM, respectively; while Anti-PDL1 and Anti-PDL1-HC+609-LC cannot binds to PD1-ECD-hFc.
- Anti-PDL1 can effectively bind to PD-L1-ECD-hFc, with an EC50 of 0.1246 nM
- 609, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC cannot effectively bind to PD1-ECD-hFc.
- Anti-PDL1-LC SEQ ID NOs: 15 and 16 was selected as the common light chain to construct a bispecific antibody.
- the heavy chain variable region of Anti-PDL1 was connected to the CH1 domain of human IgG4, and then connected to the heavy chain variable region of 609 through an artificial linker (the linker used here is three GGGGS in series, SEQ ID NO: 28), and finally connected to the heavy chain constant region of human IgG4 (CH1+CH2+CH3, with S228P mutation in the hinge region).
- the long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this program was named PDL1-Fab-609-IgG4 (SEQ ID NOs: 29 and 30).
- the heavy chain variable region of 609 was connected to the CH1 domain of human IgG4, and then connected to the heavy chain variable region of Anti-PDL1 through an artificial linker (the linker used here is three GGGGS in series, SEQ ID NO: 28), and finally connected to the heavy chain constant region of human IgG4 (CH1+CH2+CH3, with S228P mutation in the hinge region).
- the long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this program was named 609-Fab-PDL1-IgG4 (SEQ ID NOs: 31 and 32).
- the above sequences were constructed into the pcDNA4 expression vector, respectively, the expression vectors of PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 were combined with the Anti-PDL1-LC expression vector, and the antibodies were expressed and purified.
- the obtained antibodies were named PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4, respectively (for brevity, here only the name of the heavy chain is used as the name of the antibody).
- the determination method by ELISA refers to the description in Example 1.3.
- FIG. 6A 609-HC+Anti-PDL1-LC, PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can effectively bind to PD1-His, with EC50s of 0.3821 nM, 5.308 nM and 0.4213 nM, respectively.
- FIG. 6B Anti-PDL1, PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can effectively bind to PD-L1-His, with EC50s of 0.1204 nM, 0.1400 nM and 0.1350 nM, respectively.
- the isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1.
- the above results show that PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can bind to both PD-1 and PD-L1, indicating that they are bispecific antibodies.
- the results of A and C are from the same MLR experiment, and the results of B and D are from another independent MLR experiment, where the isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1.
- the isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1.
- FIGS. 7A and 7B Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IL-2, with EC50s of 0.306 nM, 0.5384 nM, and 0.1023 nM in FIG. 7A , and EC50s of 0.1016 nM, 0.6819 nM, and 0.1259 nM in FIG. 7B , respectively.
- Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IFN- ⁇ , with EC50s of 0.5119 nM, 1.21 nM and 0.1675 nM in FIG. 7C , and E50s of 0.1464 nM, 1.29 nM and 0.05491 nM in FIG. 7D , respectively.
- FIGS. 7C and 7D show that Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IFN- ⁇ , with EC50s of 0.5119 nM, 1.21 nM and 0.1675 nM in FIG. 7C , and E50s of 0.1464 nM, 1.29 nM and 0.05491 nM in FIG. 7D , respectively.
- 7A and 7B show that at a same concentration, compared to the monoclonal antibody Anti-PDL1 or 609-HC+Anti-PDL1-LC, 609-Fab-PDL1-IgG4 can stimulate MLR to secrete more IL-2.
- the affinity of the above antibodies to PD-1 or PD-L1 was determined by Biacore 8K (GE healthcare).
- Biacore 8K the chip coupled with Protein A/G was used to capture various antibodies, respectively, and then the recombinant protein PD1-His or PD-L1-His was injected to obtain the binding-dissociation curve, which was eluted with 6M guanidine hydrochloride regeneration buffer for next cycle.
- Data were analyzed using the Biacore 8K Evaluation Software. The results are shown in Table 2.
- 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC have very similar binding constants (Kon) and dissociation constants (Koff) for PD-1, and have basically equivalent equilibrium dissociation constants (KD), with KDs of 2.57E ⁇ 08 and 3.49E ⁇ 08, respectively.
- 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC also have very similar binding constants (Kon) and dissociation constants (Koff) for PD-L1, and have basically equivalent equilibrium dissociation constants (KD), with KDs of 6.08E ⁇ 10 and 8.43E ⁇ 10, respectively.
- the equilibrium dissociation constant (KD) is inversely proportional to the affinity.
- SD rats Male-Dawley rats (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used for the pharmacokinetic study of 609-Fab-PDL1-IgG4. There were five rats in each group, weighing about 200 g. Each rat was administered 1 mg of antibody by intravenous injection; blood was taken from the orbit at a specific time after the administration, and the blood was spontaneously coagulated and then centrifuged to obtain the serum.
- the method of measuring the concentration of the target antibody in the serum is as follows: ELISA plates were coated with PD1-His and PD-L1-His, respectively, with the coating concentrations of 20 ng/well and 10 ng/well, respectively. The ELISA plates were blocked with PBST containing 1% bovine serum albumin.
- the half-life of 609-Fab-PDL1-IgG4 was 365 hours (15.2 days).
- the half-life of 609-Fab-PDL1-IgG4 was 446 hours (18.6 days), and the half-life of the monoclonal antibody Anti-PDL1 was 361 hours (15.0 days).
- the above results indicate that 609-Fab-PDL1-IgG4 has similar pharmacokinetic properties to the monoclonal antibody Anti-PDL1.
- FIG. 9A shows the HPLC-SEC pattern of the monoclonal antibody 609, in which there are 3 obvious peaks, Peak 1, Peak 2 and Peak 3, accounting for 0.7%, 0.3% and 99.0%, respectively. Among them, the retention time of Peak1 and Peak 2 was less than that of Peak 3, indicating that Peak1 and Peak 2 may be produced by aggregates, and the sum of them accounts for 1.0%; there was no peak that may represent degraded fragments or incompletely assembled molecules in the pattern.
- FIG. 9B shows the HPLC-SEC pattern of 609-Fab-PDL1-IgG4, in which there are 3 obvious peaks, Peak 1, Peak 2, and Peak3, accounting for 0.2%, 99.5%, and 0.3%, respectively. The retention time of Peak1 was less than that of Peak 2, indicating that Peak 1 may be produced by aggregates; the retention time of Peak3 was more than that of Peak 2, indicating that Peak 3 may be produced by degraded fragments or incompletely assembled molecules.
- FIGS. 10A and 10B show the NR-CE-SDS and R-CE-SDS patterns of 609 mAb, respectively
- FIGS. 10C and 10D show the NR-CE-SDS and R-CE-SDS patterns of 609-Fab-PDL1-IgG4, respectively.
- Peak 8 the main peak in NR-CE-SDS of 609, accounted for 98.92%
- Peak 9 the main peak in NR-CE-SDS of 609-Fab-PDL1-IgG4, accounted for 97.70%.
- Peak 4 (corresponding to the light chain) and Peak 8 (corresponding to the heavy chain), the main peaks in R-CE-SDS of 609, accounted for 32.03% and 66.99%, respectively, and the ratio of the two peak areas was 1:2.09.
- Peak 3 (corresponding to the light chain) and Peak 9 (corresponding to the heavy chain), the main peaks in R-CE-SDS of 609-Fab-PDL1-IgG4, accounted for 38.73% and 58.78%, respectively, and the ratio of the two peak areas was 2:3.03.
- FIGS. 11A and 11B show the HPLC-IEC patterns of 609 and 609-Fab-PDL1-IgG4, respectively. Their main peaks accounted for 82.95% and 92.70%, respectively. The results show that the charge heterogeneity of 609-Fab-PDL1-IgG4 is better than that of the 609 mAb.
- FIGS. 12A and 12B show the DSC patterns of 609 and 609-Fab-PDL1-IgG4, respectively.
- TmOnset represents the temperature at which the protein begins to unfold or denature
- Tm corresponds to the peak top temperature.
- the TmOnset and Tm of 609 were 63.68° C. and 72.36° C.
- the TmOnset and Tm of 609-Fab-PDL1-IgG4 were 64.22° C. and 76.25° C., respectively.
- the above results indicate that 609 and 609-Fab-PDL1-IgG4 have very similar thermal stability.
- Each molecule of 609-Fab-PDL1-IgG4 contains two long heavy chains and four light chains.
- the calculation of the expected molecular weight took into account the modification of the C-terminal lysine residue of the heavy chain, being 238,099 Da. As shown in FIG. 13 , the measured molecular weight was 238,100 Da, which only differs by 1 Da from the expected molecular weight. The above results indicate that the molecular structure of 609-Fab-PDL1-IgG4 is consistent with expectations.
- Human peripheral blood mononuclear cells were used to rebuild the human immune system in NSG mice, and a human lung cancer NCI-H292 subcutaneous xenograft model was established on the mice.
- the mouse model had both T cells expressing human PD-1 and human tumor cells expressing human PD-L1, so it can be used to evaluate the anti-tumor activity of the bispecific antibody against PD-1 and PD-L1 in vivo.
- the specific procedures are as follows: human non-small cell lung cancer NCI-H292 cells (ATCC® CRL-1848TM) cultured in vitro were collected, the cell suspension was adjusted to a concentration of 1 ⁇ 10 8 /ml, and then mixed with Matrigel (purchased from BD Biosciences, Cat.
- mice inoculated with mixed cells were randomly divided into groups according to their body weight, with 10 mice in each group.
- mice in each group are as follows: Control group, injected with saline; Opdivo group, injected with 10 mg/kg of anti-PD-1 positive control antibody Opdivo (produced by Bristol-Myers Squibb); Tecentriq group, injected with 10 mg/kg of anti-PD-L1 positive control antibody Tecentriq (produced by Roche Pharmaceuticals); 609-Fab-PDL1-IgG4 group, injected with 16 mg/kg of 609-Fab-PDL1-IgG4. Taking into account the difference in molecular weight between bispecific antibodies and monoclonal antibodies, the dose of the drug in this experiment was provided according to the rule of equal amount of substance. Subsequently, the drugs were administered according to the above-designed plan, twice a week for a total of 8 times, and the tumor volumes were measured twice a week. Finally, the growth curve of tumor of each group determined over time is shown in FIG. 14 .
- 609-Fab-PDL1-IgG4 can inhibit tumor growth more effectively.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
- The present application is a continuation of U.S. patent application Ser. No. 17/615,475, filed on Nov. 30, 2021, which is the national stage of International Patent Application Serial No. PCT/CN2021/088154, filed on Apr. 19, 2021, which in turn claims the benefit and priority of International Patent Application Serial No. PCT/CN2020/090442, filed on May 15, 2020. The contents of the three aforementioned applications are hereby incorporated by reference in their entirety.
- The present invention relates to the field of antibodies. More particularly, the present invention discloses a tetravalent bispecific antibody against PD-1 and PD-L1.
- Human programmed cell death receptor-1 (PD-1) is a type I membrane protein of 288 amino acids and is one of the known major immune checkpoints (Blank et al, 2005, Cancer Immunotherapy, 54:307-314). PD-1 is expressed in activated T lymphocytes, and it binds to the ligands PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2) to inhibit the activity of T lymphocytes and related cellular immune responses in vivo. PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, tissue cells of lung, liver, heart and the like. Numerous studies have shown that the interaction between PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in vivo, but also the main mechanism and cause of PD-L1 expression-positive tumor cells to evade immune surveillance. By blocking the negative regulation of cancer cells on the PD-1/PD-L1 signaling pathway and activating the immune system, T cell-related tumor-specific cellular immune responses can be promoted, thereby opening a new door for tumor treatment—tumor immunotherapy.
- PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Studies have shown that PD-1 negatively regulates antigen receptor signal transduction upon engagement of its ligands (PD-L1 and/or PD-L2). At present, the structure of mouse PD-1 and the co-crystal structure of mouse PD-1 with human PD-L1 have been clarified (Zhang, X. et al., Immunity 20: 337-347 (2004); Lin et al., Proc.Natl.Acad.Sci.USA 105: 3011-6(2008)). PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain that is responsible for ligand binding and a cytoplasmic tail that is responsible for the binding of signal transduction molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, an ITIM (immunoreceptor tyrosine-based inhibition motif) and an ITSM (immunoreceptor tyrosine-based switch motif).
- PD-1 plays an important role in tumor immune evasion mechanism. Tumor immunotherapy, which uses the body's own immune system to fight cancer, is a breakthrough in cancer treatment. However, the tumor microenvironment may protect tumor cells from effective immune damage, so how to break the tumor microenvironment becomes the focus of anti-tumor research. Existing research results have identified the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and may be induced by IFNγ in most PD-L1-negative tumor cell lines), and presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc.Natl.Acad.Sci.U.S.A. 99: 12293-12297(2002); Strome S. E. et al., Cancer Res., 63: 6501-6505(2003). Through immunohistochemical assessment of biopsies, the expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found in many primary human tumors. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer as well as tumors of head and neck, etc. Thus, blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-1 and PD-L1 have become popular targets for development of tumor immunotherapy drugs.
- Bispecific antibodies refer to antibody molecules that can specifically bind to two antigens or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that may be used to treat various inflammatory diseases, cancers and other diseases. Although a large number of new bispecific antibody structural forms have been reported recently, the main technical difficulty in producing bispecific antibodies lies in obtaining the correct paired molecules. The current forms of bispecific antibodies all have mismatch problems, so one or more by-products or aggregates caused by mismatches will be produced, thereby affecting the yield, purity and physical and chemical stability of the bispecific antibodies of interest, and further affecting the safety and efficiency of the bispecific antibodies in vivo.
- The present invention provides an antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the antibody that binds to human PD-L1.
- Therefore, the first object of the present invention is to provide an antibody that binds to human PD-L1 or an antigen-binding fragment thereof.
- The second object of the present invention is to provide an isolated nucleotide encoding the antibody that binds to human PD-L1 or the antigen-binding fragment thereof.
- The third object of the present invention is to provide an expression vector comprising the nucleotide.
- The fourth object of the present invention is to provide a host cell comprising the expression vector.
- The fifth object of the present invention is to provide a method of preparing the antibody that binds to human PD-L1 or the antigen-binding fragment thereof.
- The sixth object of the present invention is to provide a pharmaceutical composition comprising the antibody that binds to human PD-L1 or the antigen-binding fragment thereof.
- The seventh object of the present invention is to provide the use of the antibody that binds to human PD-L1 or the antigen-binding fragment thereof or the pharmaceutical composition in the preparation of a medicine for the treatment of PD-L1 overexpression diseases.
- The eighth object of the present invention is to provide a method of treating PD-L1 overexpression diseases with the antibody that binds to human PD-L1 or the antigen-binding fragment thereof or the pharmaceutical composition.
- The ninth object of the present invention is to provide a tetravalent bispecific antibody against PD-1 and PD-L1.
- The tenth object of the present invention is to provide an isolated nucleotide encoding the tetravalent bispecific antibody.
- The eleventh object of the present invention is to provide an expression vector comprising the nucleotide.
- The twelfth object of the present invention is to provide a host cell comprising the expression vector.
- The thirteenth object of the present invention is to provide a method of preparing the tetravalent bispecific antibody.
- The fourteenth object of the present invention is to provide a pharmaceutical composition comprising the tetravalent bispecific antibody.
- The fifteenth object of the present invention is to provide the use of the tetravalent bispecific antibody or the pharmaceutical composition in the preparation of a medicine for the treatment of cancer.
- The sixteenth object of the present invention is to provide a method of treating cancer with the tetravalent bispecific antibody or the pharmaceutical composition.
- In order to achieve the above objects, the present invention provides the following technical solutions:
- The first aspect of the present invention provides an antibody that binds to human PD-L1 or the antigen-binding fragment thereof, comprising:
- (a) heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, the H-CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, the H-CDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and the H-CDR3 having an amino acid sequence as shown in SEQ ID NO:19, and
- (b) light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3, the L-CDR1 having an amino acid sequence as shown in SEQ ID NO: 20, the L-CDR2 having an amino acid sequence as shown in SEQ ID NO: 21, and the L-CDR3 having an amino acid sequence as shown in SEQ ID NO: 22.
- According to the present invention, the antibody is a monoclonal antibody or a polyclonal antibody.
- According to the present invention, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
- According to the present invention, the antigen-binding fragment comprises a Fab fragment, a F(ab′)2 fragment, a Fv fragment or a single chain antibody.
- According to the present invention, the antibody that binds to human PD-L1 or the antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 9, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 10.
- According to the present invention, the antibody that binds to human PD-L1 or the antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 13, and a light chain having an amino acid sequence as shown in SEQ ID NO: 15.
- The second aspect of the present invention provides an isolated nucleotide, which encodes the antibody that binds to human PD-L1 or the antigen-binding fragment thereof as described above.
- According to the present invention, the nucleotide sequence encoding the heavy chain of the antibody that binds to human PD-L1 or the antigen-binding fragment thereof is shown in SEQ ID NO: 14, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 16.
- The third aspect of the present invention provides an expression vector, which comprises the nucleotides as described above.
- The fourth aspect of the present invention provides a host cell, which comprises the expression vector as described above.
- The fifth aspect of the present invention provides a method of preparing the antibody that binds to human PD-L1 or the antigen-binding fragment thereof, which comprises the following steps:
- a) culturing the host cell as described above under expression conditions, to express the antibody that binds to human PD-L1 or the antigen-binding fragment thereof;
- b) isolating and purifying the antibody that binds to human PD-L1 or the antigen-binding fragment thereof of step a).
- The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the antibody that binds to human PD-L1 or the antigen-binding fragment thereof as described above and a pharmaceutically acceptable carrier.
- The seventh aspect of the present invention provides the use of the antibody that binds to human PD-L1 or the antigen-binding fragment thereof, or of the pharmaceutical composition as described above, in the preparation of a medicine for the treatment of PD-L1 overexpression diseases.
- According to the present invention, the PD-L1 overexpression disease is cancer. Preferably, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- The eighth aspect of the present invention provides a method of treating PD-L1 overexpression diseases, which comprises administering the antibody that binds to human PD-L1 or the antigen-binding fragment thereof or the pharmaceutical composition as described above to a subject in need.
- According to the present invention, the PD-L1 overexpression disease is cancer. Preferably, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- The ninth aspect of the present invention provides a tetravalent bispecific antibody against PD-1 and PD-L1, comprising two polypeptide chains and four common light chains, wherein the polypeptide chains have an amino acid sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 31, and the common light chains have the amino acid sequence as shown in SEQ ID NO: 15.
- The tenth aspect of the present invention provides an isolated nucleotide, which encodes the tetravalent bispecific antibody.
- According to a preferred embodiment of the present invention, the nucleotide encodes the polypeptide chains and the common light chains, wherein the nucleotide sequence encoding the polypeptide chains is as shown in SEQ ID NO: 30 or SEQ ID NO: 32, the nucleotide sequence encoding the common light chains is as shown in SEQ ID NO:16.
- The eleventh aspect of the present invention provides an expression vector, which comprises the nucleotide as described above.
- The twelfth aspect of the present invention provides a host cell, which comprises the expression vector as described above.
- The thirteenth aspect of the present invention provides a method of preparing the tetravalent bispecific antibody, which comprises the following steps:
- a) culturing the host cell as described above under expression conditions, to express the tetravalent bispecific antibody;
- b) isolating and purifying the tetravalent bispecific antibody of step a).
- The fourteenth aspect of the present invention provides a pharmaceutical composition, which comprises the tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier.
- The fifteenth aspect of the present invention provides the use of the tetravalent bispecific antibody or the pharmaceutical composition as described above in the preparation of a medicine for the treatment of cancer.
- According to the present invention, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- The sixteenth aspect of the present invention provides a method of treating cancers, which comprises administering the tetravalent bispecific antibody or the pharmaceutical composition as described above to a subject in need.
- According to the present invention, the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases, etc.
- Benefit Effect:
- The present invention provides an antibody that binds to human PD-L1, and a tetravalent bispecific antibody against PD-1 and PD-L1 constructed based on the antibody that binds to human PD-L1. The tetravalent bispecific antibody of the present invention requires no Fc modification, has no mismatch problems, and the preparation method thereof is simple. Its biological activities and physical and chemical properties are similar with or even better than those of the monoclonal antibodies.
-
FIG. 1 is a schematic diagram of the structure of the bispecific antibody of the present invention, where VH-A represents the heavy chain variable region of Anti-PDL1 or 609, VH-B represents the heavy chain variable region of 609 or Anti-PDL1, and VL represents the light chain variable region of the common light chains, CH1, CH2, and CH3 are the three domains of the heavy chain constant region, CL is the light chain constant region of the common light chains, and the line between the two heavy chains represents the disulfide bond, the line between the heavy chain and the light chain also represents the disulfide bond, the line between the CH1 near the N-terminus of the polypeptide chain and the VH-A represents the artificially designed linker, and the line between the CH1 near the C-terminus of the polypeptide chain and the CH2 represents the natural linker and hinge region of the antibody (if the heavy chain is human IgG4 subtype, the hinge region contains the S228P mutation, according to the EU numbering). -
FIG. 2 shows the ELISA results of the relative affinity of Anti-PDL1 to PD-L1. -
FIG. 3 shows results of the ability of Anti-PDL1 to block the interaction of PD-1/PD-L1. -
FIGS. 4A and 4B show the evaluation results of the ability of Anti-PDL1 to enhance MLR. -
FIGS. 5A and 5B show the ELISA results of Anti-PDL1 and 609 and their hybrid antibodies. -
FIGS. 6A and 6B show the ELISA results of PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4. -
FIGS. 7A ˜7D show the evaluation results of the ability of 609-Fab-PDL1-IgG4 to enhance MLR. -
FIGS. 8A and 8B show the pharmacokinetic results of 609-Fab-PDL1-IgG4. -
FIGS. 9A and 9B show the HPLC-SEC patterns of 609-Fab-PDL1-IgG4. -
FIGS. 10A ˜10D show the CE-SDS patterns of 609-Fab-PDL1-IgG4. -
FIGS. 11A and 11B show the HPLC-IEC patterns of 609-Fab-PDL1-IgG4. -
FIGS. 12A and 12B show the DSC patterns of 609-Fab-PDL1-IgG4. -
FIG. 13 shows the mass spectrum pattern of 609-Fab-PDL1-IgG4. -
FIG. 14 shows the anti-tumor effect of the bispecific antibody 609-Fab-PDL1-IgG4 in mice. - The sequence information involved in the present invention is summarized in Table 1.
-
TABLE 1 Sequence information of the antibodies of the present invention SEQ ID NO: Sequence name 1 Amino acid sequence of the heavy chain complementarity determining region H-CDR1 of murine M8 antibody 2 Amino acid sequence of the heavy chain complementarity determining region H-CDR2 of murine M8 antibody 3 Amino acid sequence of the heavy chain complementarity determining region H-CDR3 of murine M8 antibody 4 Amino acid sequence of the light chain complementarity determining region L-CDR1 of murine M8 antibody 5 Amino acid sequence of the light chain complementarity determining region L-CDR2 of murine M8 antibody 6 Amino acid sequence of the light chain complementarity determining region L-CDR3 of murine M8 antibody 7 the fourth framework region of heavy chain (WGQGTSVTVSS) 8 the fourth framework region of light chain (FGAGTKLEIK) 9 Amino acid sequence of the heavy chain variable region of Anti-PDL1 10 Amino acid sequence of the light chain variable region of Anti-PDL1 11 Amino acid sequence of the heavy chain constant region of human IgG1 12 Amino acid sequence of human Kappa light chain constant region 13 Amino acid sequence of heavy chain of Anti-PDL1 14 Nucleotide sequence of heavy chain of Anti-PDL1 15 Amino acid sequence of light chain of Anti-PDL1 16 Nucleotide sequence of light chain of Anti-PDL1 17 Amino acid sequence of heavy chain complementarity determining region H-CDR1 of Anti-PDL1 18 Amino acid sequence of heavy chain complementarity determining region H-CDR2 of Anti-PDL1 19 Amino acid sequence of heavy chain complementarity determining region H-CDR3 of Anti-PDL1 20 Amino acid sequence of light chain complementarity determining region L-CDR1 of Anti-PDL1 21 Amino acid sequence of light chain complementarity determining region L-CDR2 of Anti-PDL1 22 Amino acid sequence of light chain complementarity determining region L-CDR3 of Anti-PDL1 23 Amino acid sequence of heavy chain variable region of Atezolizumab 24 Amino acid sequence of light chain variable region of Atezolizumab 25 Amino acid sequence of heavy chain variable region of mAb1- 25-Hu (609) 26 Amino acid sequence of light chain variable region of mAb1- 25-Hu (609) 27 Amino acid sequence of heavy chain constant region of IgG4 (S228P) 28 Linker (GGGGSGGGGSGGGGS) 29 Amino acid sequence of PDL1-Fab-609-IgG4 30 Nucleotide sequence of PDL1-Fab-609-IgG4 31 Amino acid sequence of 609-Fab-PDL1-IgG4 32 Nucleotide sequence of 609-Fab-PDL1-IgG4 - In the present invention, the terms “antibody (abbreviated as Ab)” and “immunoglobulin G (abbreviated as IgG)” are heterotetrameric glycoproteins of about 150,000 daltons with identical structural characteristics, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable region (VH) followed by constant regions. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at its other end, the light chain constant region includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region the heavy chain, and the light chain variable region is paired with the variable region of the heavy chain. The constant regions are not involved directly in binding an antibody to an antigen, but they exhibit various effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions. The antibody of the present invention comprises monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two antibodies (such as bispecific antibodies), antigen-binding fragments of antibodies, etc. The antibody of the present invention comprises murine antibodies, chimeric antibodies, humanized antibodies, etc.
- In the present invention, the term “bispecific antibody (BsAb)” refers to an antibody molecule that can specifically bind two antigens (targets) or two epitopes at the same time.
- In the present invention, the term “monoclonal antibody (mAb)” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies contained in the population are the same, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigen site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture having different antibodies directed against different antigen determinants), each monoclonal antibody is directed against a single determinant on the antigen. Besides their specificity, the benefit of monoclonal antibodies is that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the characteristics of an antibody, which is obtained from a substantially uniform antibody population, and should not be interpreted as requiring any special method to produce the antibody.
- In the present invention, the term “murine antibody” refers to an antibody derived from rats or mice, preferably mice. The murine antibody of the present invention is obtained by immunizing mice with the extracellular domain of human PD-L1 as an antigen and screening hybridoma cells.
- In the present invention, the term “chimeric antibody” refers to an antibody that comprises heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having mouse heavy and light chain variable regions linked to human constant region.
- In the present invention, the term “humanized antibody” means that the CDRs are derived from a non-human (preferably, mouse) antibody, while the remaining parts (including framework regions and constant regions) are derived from human antibody. In addition, framework region residues may be altered to preserve the binding affinity.
- In the present invention, the term “antigen-binding fragment” refers to a fragment of an antibody capable of specifically binding to an epitope of human PD-L1. Examples of the antigen-binding fragments of the present invention include Fab fragments, F(ab′)2 fragments, Fv fragments, and single chain antibodies (scFv). The Fab fragment is composed of the domains which are the VH and CH1 of the heavy chain of the antibody, and the VL and CL of the light chain of the antibody. The F(ab′)2 fragment is a fragment produced by digesting the antibody with pepsin. The Fv fragment is composed of dimers in which the heavy chain variable region and the light chain variable region of the antibody are closely and non-covalently related. The single-chain antibody (scFv) is an antibody in which the heavy chain variable region and the light chain variable region of the antibody are linked by a short peptide (linker) of 15-20 amino acids.
- In the present invention, the term “Fc” is a fragment crystallizable (Fc), which is composed of the CH2 and CH3 domains of an antibody. The Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.
- In the present invention, the term “variable” refers to the fact that certain portions of the variable regions differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain variable regions and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).
- In the present invention, the terms “anti-/against” and “binding” refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against. Generally, the antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10−7M, for example, less than about 10−8M, 10−9M, 10−10M, 10−11M or less. In the present invention, the term “KD” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant is, the tighter the antibody-antigen binding is, and the higher the affinity between the antibody and the antigen is. For example, surface plasmon resonance (abbreviated as SPR) is used to measure the binding affinity of antibody to antigen in BIACORE instrument or ELISA is used to measure the relative binding affinity of antibody to antigen.
- In the present invention, the term “valency” refers to the presence of a specified number of antigen binding sites in an antibody molecule. Preferably, the bispecific antibody of the present invention has four antigen binding sites and is tetravalent. In the present invention, the antigen binding site includes a heavy chain variable region (VH) and a light chain variable region (VL).
- In the present invention, the term “epitope” refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.
- In the present invention, the term “common light chain” refers to a light chain comprising the same light chain variable region and light chain constant region, which can pair with the heavy chain of a first antibody that binds to a first antigen to form a binding site that specifically binds to the first antigen, and can also pair with the heavy chain of a second antibody that binds to a second antigen to form a binding site that specifically binds to the second antigen. Further, the light chain variable region of the common light chain and the heavy chain variable region of the first antibody form the first antigen binding site, and the light chain variable region of the common light chain and the heavy chain variable region of the second antibody form the second antigen binding site.
- In the present invention, the term “expression vector” may be pTT5, pSECtag series, pCGS3 series, pCDNA series vectors, as well as other vectors used in mammalian expression systems, etc. The expression vector comprises a fusion DNA sequence connected with appropriate transcription and translation regulatory sequences.
- In the present invention, the term “host cell” refers to a cell suitable for expressing the expression vector as described above, which may be a eukaryotic cell, for example, mammalian or insect host cell culture system may be used to express the fusion protein of the present invention, CHO (Chinese hamster Ovary), HEK293, COS, BHK, as well as derived cells of the above-mentioned cells are applicable to the present invention.
- In the present invention, the term “pharmaceutical composition” means that the antibody or antigen-binding fragment thereof that binds to human PD-L1 or the tetravalent bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition, so as to exert a therapeutic effect more stably. These preparations can ensure the conformational integrity of the amino acid core sequences of the antibody or antigen-binding fragment thereof that binds to human PD-L1 disclosed in the present invention, and meanwhile, protect the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).
- The protein expression and purification methods used in the following examples are described as follows: the target genes are constructed into the expression vector pcDNA4, and the constructed expression vectors or combination of expression vectors are transferred into FreeStyle™ 293-F cells (hereinafter referred to as HEK293F, purchased from Thermo Fisher Scientific) using PEI (Polyethylenimine), to express an antibody or recombinant protein. HEK293F cells are cultured in Free Style 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days and the cell supernatant are collected, and then the antibody or recombinant protein are purified by ProteinA affinity chromatography or nickel affinity chromatography.
- The mixed lymphocyte reaction (MLR) method used in the following examples is described as follows: Peripheral blood mononuclear cells (PBMC) are separated from human blood using Histopaque (purchased from Sigma), and then the monocytes in PBMC are separated by adherence method, and then the monocytes are induced with IL-4 (25 ng/ml) and GM-CSF (25 ng/ml) to differentiate into dendritic cells. Seven days later, the above-induced dendritic cells are digested and collected. PBMCs are separated from the blood of other donors by the above method, and then CD4+ T cells are separated from PBMCs with MACS magnet and CD4 MicroBeads (purchased from Miltenyibiotec). The induced dendritic cells (104/well) and the isolated CD4+ T cells (105/well) are mixed in proportion and then inoculated into a 96-well plate, 150 μl per well; a few hours later, 50 μl of serially diluted antibody is added into the above 96-well plate; the 96-well plate is incubated in a 37° C. cell incubator for 3 days. During the above experiment, AIM-V medium (purchased from Thermo Fisher Scientific) is used to culture the cells. Then, the secretion of IL-2 and IFN-γ is detected in accordance with standard operating procedures. IL-2 and IFN-γ are detected using double-antibody sandwich ELISA (related paired antibodies are purchased from BD Biosciences). OD450 values are read with a microplate reader (SpectraMax 190). Graphing is performed by GraphPad Prism6 and EC50 values are calculated.
- The methods for detecting physical and chemical properties used in the following examples are described as follows:
- HPLC-SEC
- Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Due to changes such as post-translational modification, aggregation, and degradation, antibodies are heterogeneous in their biochemical and biophysical properties. Variants, aggregates, and degraded fragments are commonly observed when bispecific antibodies are analyzed by separation techniques, and their presence may compromise safety and effectiveness. Aggregates, degraded fragments and incompletely assembled molecules are prone to appear during production and storage of an antibody. In the present invention, high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) is used to detect the content of the above impurities in a sample. The molecular weight of the aggregate is larger than that of the monomer, so the retention time of the corresponding peak is shorter; the molecular weight of the degraded fragment or the incompletely assembled molecule is smaller than that of the monomer, so the retention time of the corresponding peak is longer. Chromatograph used for HPLC-SEC:
Dionex Ultimate 3000; the method for the preparation of mobile phase is as follows: an appropriate amount of 20 mM sodium dihydrogen phosphate mother liquor is adjusted with 20 mM disodium hydrogen phosphate to a pH of 6.8±0.1; injection amount: 20 μg; chromatograph column: TSK G3000SWXL, specification: 7.8×300mm 5 μm; flow rate: 0.5 ml/min, elution time: 30 min; column temperature: 25° C., sample room temperature: 10° C.; detection wavelength: 214 nm. - HPLC-IEC
- Many post-translational modifications (such as N-glycosylation, C-terminal lysine residue modification, N-terminal glutamine or glutamate cyclization, asparagine deamidation, aspartic acid isomerization, amino acid residue oxidation, etc.) will directly or indirectly change the surface charge of the antibody, leading to the generation of charge heterogeneity. The charge variants can be separated and analyzed based on the charge. Commonly used analysis methods include cation exchange chromatography (CEX) and anion exchange chromatography (AEX). When analyzed by a chromatography-based method, acidic species and basic species are defined based on their retention time relative to the main peak. The acidic species are the variants that eluted earlier than the main peak of CEX or later than the main peak of AEX, while the basic species are the variants that eluted later than the main peak of CEX or earlier than the main peak of AEX. The peaks corresponding to the acidic species and the basic species are called acidic peaks and basic peaks, respectively. Charge variants are easily generated during the production and storage of antibodies. Here, high-performance liquid chromatography-ion exchange chromatography (HPLC-IEC) is used to analyze the charge heterogeneity of the samples. Chromatograph used in HPLC-IEC:
Dionex Ultimate 3000; mobile phase A: 20 mM PB pH 6.3, mobile phase B: 20 mM PB+200 mM NaCl pH 6.3, the mixing ratio of the two mobile phases changes with time according to the preset program, flow rate 1.0 ml/min; chromatographic column: Thermo Propac™ WCX-10; column temperature: 30° C., sample room temperature: 10° C.; injection amount: 20 μg; detection wavelength: 214 nm. - CE-SDS
- In the present invention, CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) is used to analyze the content of degraded fragments or incompletely assembled molecules in the sample. CE is divided into two types: non-reduced and reduced; for the former, when the sample is denatured, the reducing agent DTT is not needed to destroy the disulfide bond in the molecule; for the latter, when the sample is denatured, the reducing agent DTT is needed to destroy the disulfide bond in the molecule. Non-reduced and reduced CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively. The capillary electrophoresis instrument used is ProteomeLab™ PA800 plus (Beckman Coulter), equipped with a UV 214 nm detector, capillary model: Bare Fused-Silica Capillary, specification: 30.7 cm×50 μm, effective length: 20.5 cm; other related reagents: purchased from Beckman Coulter. The key parameters of the instrument are set as follows: temperature of capillary and sample chamber: 20±2° C., separation voltage: 15 kV.
- DSC
- Differential Scanning calorimeter (DSC) reflects the thermal stability of the sample mainly by detecting the heat change in biomolecules in a controlled heating or cooling process. By heating, the unfolding of the protein sample will absorb heat, and the supplementary energy required to eliminate the temperature difference in the sample pool will be recorded by the device. These heat changes will form a peak shape in the spectrum. The peak top temperature corresponding to the unfolding of the protein sample is taken as the melting temperature Tm. Tm is an important indicator of protein thermal stability. The higher the Tm is, the better the stability of the protein is.
- Molecular Weight Detection
- The antibody is deglycosylated by treating with PNGase F and endoglycosidase F2. UPLC-XEVO G2 Q-TOF liquid chromatography-mass spectrometry system (Waters) is used for the analysis and identification of molecular weight of the sample. Mobile phase A is HPLC-grade water containing 0.1% trifluoroacetic acid (TFA). Mobile phase B is acetonitrile containing 0.1% TFA. In the method of detecting the complete molecular weight, the chromatographic column used is Mass PREP™ Micro Desalting Column (specification 2.1×5 mm). The key parameters are set as follows: column temperature: 80° C.; flow rate of mobile phase: 0.2 mL/min; mobile phase gradient: mobile phase B rises from 5% to 90% within 1.5 min; ESI source temperature: 130° C. BiopharmaLynx v1.2 (Waters) is used to control the liquid chromatography-mass spectrometry system and collect data, and the mass spectrum signals are deconvoluted with BiopharmaLynx v1.2.
- The following examples are used to further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such as those methods of constructing expression vectors and preparing plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of transfecting plasmids into host cells. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.
- The source of genes encoding the extracellular regions of PD-1 and PD-L1 was described in WO2018/137576A1. Using gene recombination technology, the ends of the extracellular region coding genes of PD-1 and PD-L1 were connected to the polyhistidine coding sequence, respectively, and then the recombinant genes were cloned into the pcDNA4 expression vector, and the recombinant proteins were expressed and purified, respectively. The obtained recombinant proteins were named PD1-His and PD-L1-His, respectively. Using gene recombination technology, the ends of the extracellular region coding genes of PD-1 and PD-L1 were connected to the Fc segment coding sequence of human IgG1, respectively, and then the recombinant genes were cloned into the pcDNA4 expression vector, and the recombinant proteins were expressed and purified, respectively. The obtained recombinant proteins were named PD1-ECD-hFc and PD-L1-ECD-hFc, respectively.
- The above PD-L1-ECD-hFc was used as an antigen to immunize Balb/c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.). The methods of immunizing mice, detecting titer and screening hybridoma clones are described in Example 2 of WO2018/137576A1. The method of screening positive clones of hybridomas by ELISA is as follows: An ELISA plate was coated with the above PD-L1-His with a coating concentration of 10 ng/well, and blocked with PBST (KH2PO4 0.2 g, Na2HPO4.12H2O 2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-20 0.5 ml, added with pure water to 1 L) containing 1% bovine serum albumin (BSA). The antibody to be tested was serially diluted, and then transferred to the above plate coated with the recombinant protein, incubated at room temperature for half an hour and then the plate was washed; an appropriately diluted HRP (Horseradish Peroxidase)-labeled goat anti-mouse antibody (Fc-Specific) (purchased from Sigma) was added, incubated at room temperature for half an hour and then the plate was washed; 100 μl of chromogenic solution (chromogenic substrate solution A: sodium acetate-trihydrate 13.6 g, citric acid-monohydrate 1.6 g, 30% hydrogen peroxide 0.3 ml,
pure water 500 ml; chromogenic substrate solution B: disodium ethylenediaminetetraacetic acid 0.2 g, citric acid-monohydrate 0.95 g, glycerol 50 ml, 0.15 g TMB dissolved in 3 ml DMSO,pure water 500 ml; A and B mixed in equal volume before use) with TMB (3,3′,5,5′-Tetramethylbenzidine) as a substrate was added to each well, incubated at room temperature for 1˜5 min; 50 μl of stop solution (2M H2SO4) was added to stop the reaction. OD450 values were read with a microplate reader (SpectraMax 190). - The positive hybridoma clones were selected for expansion in a 24-well plate and subcloned by limiting dilution method. A monoclonal hybridoma cell line stably expressing the target antibody was obtained by the above-mentioned method, and these clones were amplified. The above hybridoma cell line was cultured in serum-free medium Hybridoma-SFM (purchased from Thermo Fisher Scientific) for 7 days, and then the murine anti-human PD-L1 monoclonal antibody was purified from the culture supernatant by Protein A/G affinity chromatography. After purification, several murine monoclonal antibodies that can bind to human PD-L1 were obtained. The relative affinity of the murine monoclonal antibodies to human PD-L1 was evaluated by ELISA. Finally, the clone M8 with the highest relative affinity was selected for further development.
- Total RNAs were extracted from the M8 hybridoma monoclonal cell line using Trizol, and mRNAs were reverse transcribed into cDNAs using a reverse transcription kit. By the primer combination reported in the literature (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dubel; the sequences of the primer combination from page 323), the genes of light chain variable region and heavy chain variable region of M8 were amplified by PCR, and then the PCR products were cloned into the pMD18-T vector, and the variable region genes were sequenced and analyzed.
- The amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody M8 were analyzed, and the complementarity-determining regions and frame regions of the antibody M8 were determined according to Kabat rule. The amino acid sequences of the heavy chain CDRs of the antibody M8 are: H-CDR1: SYGVH (SEQ ID NO: 1), H-CDR2: LIWSGGGTDYNAAFIS (SEQ ID NO: 2) and H-CDR3: QLGLRAMDY (SEQ ID NO: 3), the amino acid sequences of CDRs of the light chain are: L-CDR1: RASQSIGTTIH (SEQ ID NO: 4), L-CDR2: YASESVS (SEQ ID NO: 5) and L-CDR3: QQSNSWPLT (SEQ ID NO: 6).
- The homology comparison of the heavy chain variable region of the murine antibody M8 with the human IgG germline sequence was performed at https://www.ncbi.nlm.nih.gov/igblast/. Selecting IGHV4-59*01 as the heavy chain CDR grafting template, the heavy chain CDRs of the murine antibody M8 were transplanted into the framework regions of IGHV4-59*01, and WGQGTSVTVSS (SEQ ID NO: 7) was added following the H-CDR3 as the fourth framework region, to obtain a CDR-grafted heavy chain variable region sequence. Similarly, the homology comparison of the light chain variable region of the murine antibody M8 with the human IgG germline sequence was performed. Selecting IGKV6-21*01 as the light chain CDR grafting template, the light chain CDRs of the murine antibody M8 were transplanted into the framework regions of IGKV6-21*01, and FGAGTKLEIK (SEQ ID NO: 8) was added following the L-CDR3 as the fourth framework region, to obtain a CDR-grafted light chain variable region sequence. On the basis of the CDR-grafted variable regions, some amino acid sites were subjected to mutation. When mutation was performed, the amino acid sequence was numbered by Kabat rule and the position of each site was indicated by Kabat numbering.
- Preferably, for the CDR-grafted heavy chain variable region, according to Kabat numbering, E at
position 6 was mutated to Q, P at position 9 was mutated to G, E at position 16 was mutated to Q, T at position 17 was mutated to S, G atposition 27 was mutated to F, I at position 29 was mutated to L, I at position 37 was mutated to V, A at position 61 was mutated to P, A at position 62 was mutated to S, F at position 63 was mutated to L, I at position 64 was mutated to K, V at position 67 was mutated to L, V at position 71 was mutated to R, F at position 78 was mutated to V, L atposition 80 was mutated to F, L at position 82 was mutated to I, V at position 82C was mutated to L. For the CDR-grafted light chain variable region, Q atposition 11 was mutated to L, E at position 53 was mutated to Q, V at position 55 was mutated to F, and L at position 78 was mutated to V. - The above heavy chain variable region and light chain variable region with mutation sites were defined as humanized heavy chain variable region and light chain variable region (SEQ ID NOs: 9 and 10), respectively. The DNAs encoding the humanized heavy chain and light chain variable regions were synthesized by Shanghai Sango Biotech Co., Ltd. The synthesized humanized heavy chain variable region was connected to the human IgG1 heavy chain constant region (SEQ ID NO: 11) to obtain a full-length humanized heavy chain gene, named Anti-PDL1-HC (SEQ ID NOs: 13 and 14). The humanized light chain variable region was connected to the human Kappa chain constant region (SEQ ID NO: 12) to obtain a full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NOs: 15 and 16). The Anti-PDL1-HC and Anti-PDL1-LC genes were constructed into the pcDNA4 expression vector, respectively, and the resulting heavy and light chain expression vectors were transferred together into HEK293F cells by PEI transfection method to express the antibody, and the antibody was purified using Protein A affinity chromatography. The obtained antibody was named Anti-PDL1.
- The final Anti-PDL1 antibody comprises the heavy chain CDRs having the amino acid sequences: H-CDR1: SYGVH (SEQ ID NO: 17), H-CDR2: LIWSGGGTDYNPSLKS (SEQ ID NO: 18) and H-CDR3: QLGLRAMDY (SEQ ID NO: 19); the light chain CDRs having the amino acid sequences: L-CDR1: RASQSIGTTIH (SEQ ID NO: 20), L-CDR2: YASQSFS (SEQ ID NO: 21) and L-CDR3: QQSNSWPLT (SEQ ID NO: 22).
- The sequences of heavy chain variable region and light chain variable region of the positive control antibody Atezolizumab were obtained from WHO Drug Information, Vol. 29, No. 3, 2015 (SEQ ID NOs: 23 and 24). The DNAs encoding the above variable regions were synthesized by Shanghai Sango Biotech Co., Ltd. The heavy chain variable region of Atezolizumab (Atezolizumab-VH) was connected to the human IgG1 heavy chain constant region (SEQ ID NO: 11) to obtain a full-length heavy chain gene, named Atezolizumab-HC; the light chain variable region of Atezolizumab (Atezolizumab-VL) was connected to the human Kappa light chain constant region (SEQ ID NO: 12) to obtain a full-length light chain gene, named Atezolizumab-LC. Atezolizumab-HC and Atezolizumab-LC were constructed into the pcDNA4 expression vector, respectively, and the antibody was expressed and purified. The obtained antibody was named Atezolizumab-IgG1.
- An ELISA plate was coated with the above PD-L1-His with a coating concentration of 10 ng/well, and blocked with PBST containing 1% BSA. The antibody to be tested was serially diluted, and then transferred to the above plate coated with recombinant protein, incubated at room temperature for half an hour and then the plate was washed; an appropriately diluted HRP-labeled goat anti-mouse antibody (Fc-Specific) (purchased from Sigma) was added, incubated at room temperature for half an hour and then the plate was washed; 100 μl of chromogenic solution with TMB as a substrate was added to each well, incubated at room temperature for 1˜5 min; 50 μl of stop solution (2M H2SO4) was added to stop the reaction. OD450 values were read with a microplate reader (SpectraMax 190), and graphing and data analysis were performed using GraphPad Prism6, and EC50 values were calculated.
- As shown in
FIG. 2 , both Anti-PDL1 and Atezolizumab-IgG1 can effectively bind to PD-L1-His, with EC50s of 0.1018 nM and 0.09351 nM, respectively, and their apparent affinities are equivalent. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1. - PD-L1-ECD-hFc was biotinylated with Biotin N-hydroxysuccinimide ester (purchased from Sigma, Catalog No./Specification: H1759-100MG). Human PD-1-ECD-hFc was diluted to 2 μg/ml with sodium carbonate buffer (1.59 g Na2CO3 and 2.93 g NaHCO3 dissolved in 1 L of pure water), and added into a 96-well ELISA plate with a multichannel pipette at 100 μl/well, incubated at room temperature for 4 h; washed once with PBST, blocked with PBST containing 1% BSA at 200 μl/well, incubated at room temperature for 2 h; the blocking solution was discarded, the plate was patted dry, and placed at 4° C. for later use. The biotinylated PD-L1-ECD-hFc was diluted to 500 ng/ml with PBST solution containing 1% BSA in a 96-well plate; the anti-human PD-L1 antibody was serially diluted with the above protein solution; the above diluted antibody and the biotinylated PD-L1-ECD-hFc were mixed and transferred to the above ELISA plate coated with human PD1-ECD-hFc, and incubated at room temperature for 1 hour; the plate was washed 3 times with PBST; Streptavidin-HRP (purchased from BD Biosciences) diluted 1:1000 in PBST with 1% BSA was added, and incubated at room temperature for 45 min; the plate was washed 3 times with PBST; chromogenic solution (with TMB as a substrate) was added at 100 μl/well, incubated at room temperature for 1˜5 min; stop solution (2M H2SO4) was added at 50 μl/well to stop the chromogenic reaction. OD450 values were read with a microplate reader (SpectraMax 190). Graphing and data analysis were performed using GraphPad Prism6, and IC50 values were calculated.
- As shown in
FIG. 3 , both Anti-PDL1 and Atezolizumab-IgG1 can effectively block the interaction between PD-1 and PD-L1, with IC50s of 1.366 nM and 1.471 nM, respectively, and their blocking abilities are equivalent. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1. - As shown in
FIG. 4A , both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR to secrete IL-2, with EC50s of 0.306 nM and 0.29 nM, respectively. As shown inFIG. 4B , both Anti-PDL1 and Atezolizumab-IgG1 can effectively stimulate MLR to secrete IFN-γ, with EC50s of 0.1464 nM and 0.1294 nM, respectively. The isotype control antibody is a human IgG1 antibody that does not bind to human PD-L1. - MAb1-25-Hu (hereinafter referred to as 609) is a humanized anti-human PD-1 monoclonal antibody, the sequences of heavy chain variable region and light chain variable region of which are described in WO2018/137576A1. Humanized heavy chain variable region and light chain variable region (SEQ ID NOs: 25 and 26) were connected to the human IgG4 (S228P) heavy chain constant region (SEQ ID NO: 27) and Kappa light chain constant region (SEQ ID NO: 12), respectively, to finally obtain the heavy and light chain amino acid sequences of the complete humanized monoclonal antibody mAb1-25-Hu (609).
- Anti-PDL1 is a humanized monoclonal antibody of anti-human PD-L1, and its sequence is shown in Example 1.3.
- BLAST (Basic Local Alignment Search Tool) was used to compare the amino acid sequences of the light chain variable region of Anti-PDL1 and the light chain variable region of 609. The results show that between them, the identical amino acids accounted for 74% (Identities) and the amino acids with similar properties accounted for 86% (Positives).
- The heavy chain and light chain genes of Anti-PDL1 were named Anti-PDL1-HC and Anti-PDL1-LC, and the heavy chain and light chain genes of 609 were named 609-HC and 609-LC, respectively. They were constructed into the pcDNA4 expression vector, respectively. The above heavy chain and light chain expression vectors were combined in the following manner: Anti-PDL1-HC+Anti-PDL1-LC, 609-HC+609-LC, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC, and the antibodies were expressed and purified. The obtained antibodies were named Anti-PDL1, 609, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC, respectively.
- ELISA plates were coated with the above PD1-ECD-hFc and PD-L1-ECD-hFc with a coating concentration of 10 ng/well, respectively. The plates were blocked with PBST containing 1% BSA. The antibodies to be tested were serially diluted, then transferred to the above plates coated with recombinant proteins, incubated at room temperature for half an hour and then the plates were washed; an appropriately diluted HRP-labeled goat anti-human antibody (Fc-Specific) (purchased from Sisgma) was added, incubated at room temperature for half an hour and then the plates were washed; chromogenic solution with TMB as a substrate was added at 100 μl/well, incubated at room temperature for 1˜5 min; 50 μl of stop solution (2M H2SO4) was added to stop the reaction. OD450 values were read with a microplate reader (SpectraMax 190). Graphing and data analysis were performed using GraphPad Prism6, and EC50 values were calculated.
- As shown in
FIG. 5A , 609 and 609-HC+Anti-PDL1-LC can effectively bind to PD1-ECD-hFc, with EC50s of 0.2001 nM and 0.2435 nM, respectively; while Anti-PDL1 and Anti-PDL1-HC+609-LC cannot binds to PD1-ECD-hFc. As shown inFIG. 5B , Anti-PDL1 can effectively bind to PD-L1-ECD-hFc, with an EC50 of 0.1246 nM, while 609, Anti-PDL1-HC+609-LC and 609-HC+Anti-PDL1-LC cannot effectively bind to PD1-ECD-hFc. Here, Anti-PDL1-LC (SEQ ID NOs: 15 and 16) was selected as the common light chain to construct a bispecific antibody. - The heavy chain variable region of Anti-PDL1 was connected to the CH1 domain of human IgG4, and then connected to the heavy chain variable region of 609 through an artificial linker (the linker used here is three GGGGS in series, SEQ ID NO: 28), and finally connected to the heavy chain constant region of human IgG4 (CH1+CH2+CH3, with S228P mutation in the hinge region). The long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this program was named PDL1-Fab-609-IgG4 (SEQ ID NOs: 29 and 30). Similarly, the heavy chain variable region of 609 was connected to the CH1 domain of human IgG4, and then connected to the heavy chain variable region of Anti-PDL1 through an artificial linker (the linker used here is three GGGGS in series, SEQ ID NO: 28), and finally connected to the heavy chain constant region of human IgG4 (CH1+CH2+CH3, with S228P mutation in the hinge region). The long heavy chain gene containing two heavy chain variable regions and two CH1 domains constructed by this program was named 609-Fab-PDL1-IgG4 (SEQ ID NOs: 31 and 32).
- The above sequences were constructed into the pcDNA4 expression vector, respectively, the expression vectors of PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 were combined with the Anti-PDL1-LC expression vector, and the antibodies were expressed and purified. The obtained antibodies were named PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4, respectively (for brevity, here only the name of the heavy chain is used as the name of the antibody).
- The determination method by ELISA refers to the description in Example 1.3.
- As shown in
FIG. 6A , 609-HC+Anti-PDL1-LC, PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can effectively bind to PD1-His, with EC50s of 0.3821 nM, 5.308 nM and 0.4213 nM, respectively. As shown inFIG. 6B , Anti-PDL1, PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can effectively bind to PD-L1-His, with EC50s of 0.1204 nM, 0.1400 nM and 0.1350 nM, respectively. The isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1. The above results show that PDL1-Fab-609-IgG4 and 609-Fab-PDL1-IgG4 can bind to both PD-1 and PD-L1, indicating that they are bispecific antibodies. - The results of A and C are from the same MLR experiment, and the results of B and D are from another independent MLR experiment, where the isotype control antibody is a human IgG4 antibody that binds neither PD-1 nor PD-L1. As shown in
FIGS. 7A and 7B , Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IL-2, with EC50s of 0.306 nM, 0.5384 nM, and 0.1023 nM inFIG. 7A , and EC50s of 0.1016 nM, 0.6819 nM, and 0.1259 nM inFIG. 7B , respectively. In addition, as shown inFIGS. 7C and 7D , Anti-PDL1, 609-HC+Anti-PDL1-LC and 609-Fab-PDL1-IgG4 can effectively stimulate MLR to secrete IFN-γ, with EC50s of 0.5119 nM, 1.21 nM and 0.1675 nM inFIG. 7C , and E50s of 0.1464 nM, 1.29 nM and 0.05491 nM inFIG. 7D , respectively. In addition,FIGS. 7A and 7B show that at a same concentration, compared to the monoclonal antibody Anti-PDL1 or 609-HC+Anti-PDL1-LC, 609-Fab-PDL1-IgG4 can stimulate MLR to secrete more IL-2. - Herein, the affinity of the above antibodies to PD-1 or PD-L1 was determined by Biacore 8K (GE healthcare). On Biacore 8K, the chip coupled with Protein A/G was used to capture various antibodies, respectively, and then the recombinant protein PD1-His or PD-L1-His was injected to obtain the binding-dissociation curve, which was eluted with 6M guanidine hydrochloride regeneration buffer for next cycle. Data were analyzed using the Biacore 8K Evaluation Software. The results are shown in Table 2.
-
TABLE 2-1 Binding and dissociation kinetic parameter and equilibrium dissociation constant for PD-1 Sample name Kon (1/Ms) Koff (1/s) KD (M) 609-Fab-PDL1-IgG4 1.73E+05 4.43E−03 2.57E−08 609-HC + Anti-PDL1-LC 1.26E+05 4.39E−03 3.49E−08 -
TABLE 2-2 Binding and dissociation kinetic parameter and equilibrium dissociation constant for PD-L1 Sample name Kon (1/Ms) Koff (1/s) KD (M) 609-Fab-PDL1-IgG4 1.85E+06 1.12E−03 6.08E−10 Anti-PDL1 1.63E+06 1.38E−03 8.43E−10 - The experimental results show that 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC have very similar binding constants (Kon) and dissociation constants (Koff) for PD-1, and have basically equivalent equilibrium dissociation constants (KD), with KDs of 2.57E−08 and 3.49E−08, respectively. 609-Fab-PDL1-IgG4 and 609-HC+Anti-PDL1-LC also have very similar binding constants (Kon) and dissociation constants (Koff) for PD-L1, and have basically equivalent equilibrium dissociation constants (KD), with KDs of 6.08E−10 and 8.43E−10, respectively. The equilibrium dissociation constant (KD) is inversely proportional to the affinity.
- In this example, SD (Sprague-Dawley) rats (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used for the pharmacokinetic study of 609-Fab-PDL1-IgG4. There were five rats in each group, weighing about 200 g. Each rat was administered 1 mg of antibody by intravenous injection; blood was taken from the orbit at a specific time after the administration, and the blood was spontaneously coagulated and then centrifuged to obtain the serum.
- The method of measuring the concentration of the target antibody in the serum is as follows: ELISA plates were coated with PD1-His and PD-L1-His, respectively, with the coating concentrations of 20 ng/well and 10 ng/well, respectively. The ELISA plates were blocked with PBST containing 1% bovine serum albumin. An appropriately diluted rat serum was transferred to the above ELISA plates coated with PD1-His and PD-L1-His, incubated at room temperature for 1 hour, and the plates were washed, and then HRP-labeled goat anti-human (Fc-Specific) antibody (purchased from Sigma; this antibody has been treated with species cross-adsorption and does not recognize rat antibodies) was added, incubated at room temperature for half an hour, and the plates were washed; chromogenic solution with TMB as a substrate was added at 100 μl/well, incubated at room temperature for 1˜5 min; 50 μl of stop solution (2M H2SO4) was added to stop the reaction. OD450 values were read with a microplate reader, and the OD450 was converted into antibody serum concentration using standard curve. Graphing and data analysis were performed using GraphPad Prism6. The half-life of the antibody in rats was calculated using the Phoenix software.
- According to
FIG. 8A , the half-life of 609-Fab-PDL1-IgG4 was 365 hours (15.2 days). According toFIG. 8B , the half-life of 609-Fab-PDL1-IgG4 was 446 hours (18.6 days), and the half-life of the monoclonal antibody Anti-PDL1 was 361 hours (15.0 days). The above results indicate that 609-Fab-PDL1-IgG4 has similar pharmacokinetic properties to the monoclonal antibody Anti-PDL1. -
FIG. 9A shows the HPLC-SEC pattern of themonoclonal antibody 609, in which there are 3 obvious peaks, Peak 1,Peak 2 andPeak 3, accounting for 0.7%, 0.3% and 99.0%, respectively. Among them, the retention time of Peak1 andPeak 2 was less than that ofPeak 3, indicating that Peak1 andPeak 2 may be produced by aggregates, and the sum of them accounts for 1.0%; there was no peak that may represent degraded fragments or incompletely assembled molecules in the pattern.FIG. 9B shows the HPLC-SEC pattern of 609-Fab-PDL1-IgG4, in which there are 3 obvious peaks, Peak 1,Peak 2, and Peak3, accounting for 0.2%, 99.5%, and 0.3%, respectively. The retention time of Peak1 was less than that ofPeak 2, indicating that Peak 1 may be produced by aggregates; the retention time of Peak3 was more than that ofPeak 2, indicating thatPeak 3 may be produced by degraded fragments or incompletely assembled molecules. -
FIGS. 10A and 10B show the NR-CE-SDS and R-CE-SDS patterns of 609 mAb, respectively, andFIGS. 10C and 10D show the NR-CE-SDS and R-CE-SDS patterns of 609-Fab-PDL1-IgG4, respectively.Peak 8, the main peak in NR-CE-SDS of 609, accounted for 98.92%, and Peak 9, the main peak in NR-CE-SDS of 609-Fab-PDL1-IgG4, accounted for 97.70%. Peak 4 (corresponding to the light chain) and Peak 8 (corresponding to the heavy chain), the main peaks in R-CE-SDS of 609, accounted for 32.03% and 66.99%, respectively, and the ratio of the two peak areas was 1:2.09. Peak 3 (corresponding to the light chain) and Peak 9 (corresponding to the heavy chain), the main peaks in R-CE-SDS of 609-Fab-PDL1-IgG4, accounted for 38.73% and 58.78%, respectively, and the ratio of the two peak areas was 2:3.03. In NR-CE-SDS, the proportions of the main peaks of the 609 mAb and 609-Fab-PDL1-IgG4 were very similar; in R-CE-SDS, the peak area ratios of the light chain and heavy chain of the 609 mAb and 609-Fab-PDL1-IgG4 were consistent with expectations. -
FIGS. 11A and 11B show the HPLC-IEC patterns of 609 and 609-Fab-PDL1-IgG4, respectively. Their main peaks accounted for 82.95% and 92.70%, respectively. The results show that the charge heterogeneity of 609-Fab-PDL1-IgG4 is better than that of the 609 mAb. -
FIGS. 12A and 12B show the DSC patterns of 609 and 609-Fab-PDL1-IgG4, respectively. Among them, TmOnset represents the temperature at which the protein begins to unfold or denature, and Tm corresponds to the peak top temperature. The TmOnset and Tm of 609 were 63.68° C. and 72.36° C., and the TmOnset and Tm of 609-Fab-PDL1-IgG4 were 64.22° C. and 76.25° C., respectively. The above results indicate that 609 and 609-Fab-PDL1-IgG4 have very similar thermal stability. - Each molecule of 609-Fab-PDL1-IgG4 contains two long heavy chains and four light chains. The calculation of the expected molecular weight took into account the modification of the C-terminal lysine residue of the heavy chain, being 238,099 Da. As shown in
FIG. 13 , the measured molecular weight was 238,100 Da, which only differs by 1 Da from the expected molecular weight. The above results indicate that the molecular structure of 609-Fab-PDL1-IgG4 is consistent with expectations. - Human peripheral blood mononuclear cells (PBMC) were used to rebuild the human immune system in NSG mice, and a human lung cancer NCI-H292 subcutaneous xenograft model was established on the mice. The mouse model had both T cells expressing human PD-1 and human tumor cells expressing human PD-L1, so it can be used to evaluate the anti-tumor activity of the bispecific antibody against PD-1 and PD-L1 in vivo. The specific procedures are as follows: human non-small cell lung cancer NCI-H292 cells (ATCC® CRL-1848™) cultured in vitro were collected, the cell suspension was adjusted to a concentration of 1×108/ml, and then mixed with Matrigel (purchased from BD Biosciences, Cat. No.: 356234) in equal volume. The purchased PBMC (purchased from Allcells, Cat. No.: PB005-C) was resuscitated in vitro, and the PBMC cells were resuspended in PBS, and the PBMC suspension was adjusted to a concentration of 1×107/ml. The mixed tumor cell suspension and the PBMC suspension were mixed in equal volume. Under sterile conditions, 200 μl of the mixed cell suspension was inoculated subcutaneously on the right upper back of M-NSG mice (purchased from Shanghai Model Organisms Center, Inc.). On the same day, the mice inoculated with mixed cells were randomly divided into groups according to their body weight, with 10 mice in each group. The drug treatment of mice in each group is as follows: Control group, injected with saline; Opdivo group, injected with 10 mg/kg of anti-PD-1 positive control antibody Opdivo (produced by Bristol-Myers Squibb); Tecentriq group, injected with 10 mg/kg of anti-PD-L1 positive control antibody Tecentriq (produced by Roche Pharmaceuticals); 609-Fab-PDL1-IgG4 group, injected with 16 mg/kg of 609-Fab-PDL1-IgG4. Taking into account the difference in molecular weight between bispecific antibodies and monoclonal antibodies, the dose of the drug in this experiment was provided according to the rule of equal amount of substance. Subsequently, the drugs were administered according to the above-designed plan, twice a week for a total of 8 times, and the tumor volumes were measured twice a week. Finally, the growth curve of tumor of each group determined over time is shown in
FIG. 14 . - The results show that at the end of the experiment on the 30th day, the tumor inhibition rates of Opdivo, Tecentriq and 609-Fab-PDL1-IgG4 were 50.5%, 84.4% and 96.0%, respectively (tumor inhibition rate=(average volume of control group-average volume of experimental group)/average volume of control group×100%). Compared to Opdivo and Tecentriq, 609-Fab-PDL1-IgG4 can inhibit tumor growth more effectively.
Claims (18)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/574,960 US20220135686A1 (en) | 2020-05-15 | 2022-01-13 | Antibody that binds to human pd-l1 |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2020/090442 WO2021226984A1 (en) | 2020-05-15 | 2020-05-15 | Tetravalent bispecific antibody against pd-1 and pd-l1 |
CNPCT/CN2020/090442 | 2020-05-15 | ||
US17/615,475 US20230076124A1 (en) | 2020-05-15 | 2021-04-19 | A tetravalent bispecific antibody against pd-1 and pd-l1 |
PCT/CN2021/088154 WO2021227782A1 (en) | 2020-05-15 | 2021-04-19 | Anti-pd-1 and pd-l1 tetravalent bispecific antibody |
US17/574,960 US20220135686A1 (en) | 2020-05-15 | 2022-01-13 | Antibody that binds to human pd-l1 |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2021/088154 Continuation WO2021227782A1 (en) | 2020-05-15 | 2021-04-19 | Anti-pd-1 and pd-l1 tetravalent bispecific antibody |
US17/615,475 Continuation US20230076124A1 (en) | 2020-05-15 | 2021-04-19 | A tetravalent bispecific antibody against pd-1 and pd-l1 |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220135686A1 true US20220135686A1 (en) | 2022-05-05 |
Family
ID=81384514
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/574,960 Pending US20220135686A1 (en) | 2020-05-15 | 2022-01-13 | Antibody that binds to human pd-l1 |
Country Status (1)
Country | Link |
---|---|
US (1) | US20220135686A1 (en) |
-
2022
- 2022-01-13 US US17/574,960 patent/US20220135686A1/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11254746B2 (en) | Anti-PD-1 monoclonal antibody, and preparation method therefor and application thereof | |
CN106519034B (en) | anti-PD-1 antibodies and uses thereof | |
US20230076124A1 (en) | A tetravalent bispecific antibody against pd-1 and pd-l1 | |
CN102448987A (en) | Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody | |
EP4047018B1 (en) | Tetravalent bispecific antibody against pd-1 and vegf, preparation method therefor, and use thereof | |
EP4039704A1 (en) | Anti-pd-1 antibody and use thereof | |
WO2021244392A1 (en) | Anti-pd1×pdl1 bispecific antibody | |
US20220135686A1 (en) | Antibody that binds to human pd-l1 | |
EP4070816A1 (en) | Anti-gdf15 antibody | |
US20220169728A1 (en) | Bispecific antibodies and uses thereof | |
CN113754774A (en) | Tetravalent bispecific antibody for resisting PD-L1 and EGFR | |
CN113754771A (en) | Bispecific antibody for resisting PDL1 x EGFR | |
WO2022228545A1 (en) | Antibodies and variants thereof against human 4-1bb | |
WO2023078450A1 (en) | Multispecific antibodies and uses thereof | |
US20220127362A1 (en) | Tetravalent bispecific antibody against pd-1/lag-3, preparation method therefor, and use thereof |
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 |
|
AS | Assignment |
Owner name: ZEDA BIOPHARMACEUTICALS, INC., VIRGIN ISLANDS, BRITISH Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SUNSHINE GUOJIAN PHARMACEUTICAL (SHANGHAI) CO., LTD.;REEL/FRAME:063255/0468 Effective date: 20220623 |
|
AS | Assignment |
Owner name: SUNSHINE GUOJIAN PHARMACEUTICAL (SHANGHAI) CO., LTD., CHINA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ZHU, ZHENPING;ZHAO, JIE;HUANG, HAOMIN;AND OTHERS;SIGNING DATES FROM 20211124 TO 20211125;REEL/FRAME:067045/0539 |