CN116854820B - Pd-1非阻断性清除抗体及其用途 - Google Patents
Pd-1非阻断性清除抗体及其用途 Download PDFInfo
- Publication number
- CN116854820B CN116854820B CN202310759264.4A CN202310759264A CN116854820B CN 116854820 B CN116854820 B CN 116854820B CN 202310759264 A CN202310759264 A CN 202310759264A CN 116854820 B CN116854820 B CN 116854820B
- Authority
- CN
- China
- Prior art keywords
- antibody
- antigen
- cells
- binding fragment
- seq
- 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.)
- Active
Links
- 230000000903 blocking effect Effects 0.000 title claims abstract description 17
- 230000002000 scavenging effect Effects 0.000 title description 4
- 230000027455 binding Effects 0.000 claims abstract description 63
- 238000009739 binding Methods 0.000 claims abstract description 63
- 239000003814 drug Substances 0.000 claims abstract description 15
- 210000000056 organ Anatomy 0.000 claims abstract description 14
- 238000011282 treatment Methods 0.000 claims abstract description 10
- 239000000427 antigen Substances 0.000 claims description 55
- 108091007433 antigens Proteins 0.000 claims description 54
- 102000036639 antigens Human genes 0.000 claims description 54
- 239000012634 fragment Substances 0.000 claims description 46
- 241001529936 Murinae Species 0.000 claims description 22
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 19
- 239000013598 vector Substances 0.000 claims description 19
- 108091033319 polynucleotide Proteins 0.000 claims description 18
- 102000040430 polynucleotide Human genes 0.000 claims description 18
- 239000002157 polynucleotide Substances 0.000 claims description 18
- 239000008194 pharmaceutical composition Substances 0.000 claims description 9
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 55
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 45
- 238000002054 transplantation Methods 0.000 abstract description 22
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 17
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 10
- 101710089372 Programmed cell death protein 1 Proteins 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 239000003446 ligand Substances 0.000 abstract description 6
- 206010061218 Inflammation Diseases 0.000 abstract description 4
- 230000004054 inflammatory process Effects 0.000 abstract description 4
- 102100023990 60S ribosomal protein L17 Human genes 0.000 abstract 3
- 239000000243 solution Substances 0.000 description 24
- 238000005406 washing Methods 0.000 description 23
- 238000001514 detection method Methods 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 18
- 239000007788 liquid Substances 0.000 description 15
- 238000002965 ELISA Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 206010052779 Transplant rejections Diseases 0.000 description 9
- 238000011161 development Methods 0.000 description 9
- 230000018109 developmental process Effects 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 9
- 238000006467 substitution reaction Methods 0.000 description 9
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 8
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 238000007865 diluting Methods 0.000 description 8
- 210000004698 lymphocyte Anatomy 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 108010074708 B7-H1 Antigen Proteins 0.000 description 7
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 7
- 238000010009 beating Methods 0.000 description 7
- 102000048362 human PDCD1 Human genes 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 238000010494 dissociation reaction Methods 0.000 description 6
- 230000005593 dissociations Effects 0.000 description 6
- 239000011148 porous material Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- -1 small molecule compounds Chemical class 0.000 description 6
- 229940124597 therapeutic agent Drugs 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 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 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 5
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 5
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 210000000987 immune system Anatomy 0.000 description 5
- 229960003444 immunosuppressant agent Drugs 0.000 description 5
- 239000003018 immunosuppressive agent Substances 0.000 description 5
- 208000011580 syndromic disease Diseases 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 201000004624 Dermatitis Diseases 0.000 description 4
- 102000006395 Globulins Human genes 0.000 description 4
- 108010044091 Globulins Proteins 0.000 description 4
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 4
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 4
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 4
- 102100037850 Interferon gamma Human genes 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 4
- 230000003187 abdominal effect Effects 0.000 description 4
- 238000001994 activation Methods 0.000 description 4
- 210000000709 aorta Anatomy 0.000 description 4
- 210000001367 artery Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 230000001861 immunosuppressant effect Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 239000012266 salt solution Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000012089 stop solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 3
- 206010009900 Colitis ulcerative Diseases 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 206010018364 Glomerulonephritis Diseases 0.000 description 3
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 206010021245 Idiopathic thrombocytopenic purpura Diseases 0.000 description 3
- 208000028622 Immune thrombocytopenia Diseases 0.000 description 3
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 3
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 3
- 108091008874 T cell receptors Proteins 0.000 description 3
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 3
- 201000006704 Ulcerative Colitis Diseases 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 230000000961 alloantigen Effects 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000001363 autoimmune Effects 0.000 description 3
- 201000003710 autoimmune thrombocytopenic purpura Diseases 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 201000001981 dermatomyositis Diseases 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 229940046781 other immunosuppressants in atc Drugs 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 210000001147 pulmonary artery Anatomy 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 238000010188 recombinant method Methods 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 3
- YMHOBZXQZVXHBM-UHFFFAOYSA-N 2,5-dimethoxy-4-bromophenethylamine Chemical compound COC1=CC(CCN)=C(OC)C=C1Br YMHOBZXQZVXHBM-UHFFFAOYSA-N 0.000 description 2
- OBYNJKLOYWCXEP-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-4-isothiocyanatobenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C1=CC(N=C=S)=CC=C1C([O-])=O OBYNJKLOYWCXEP-UHFFFAOYSA-N 0.000 description 2
- 208000026872 Addison Disease Diseases 0.000 description 2
- 108091008875 B cell receptors Proteins 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 2
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 2
- 206010015218 Erythema multiforme Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 206010020850 Hyperthyroidism Diseases 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 206010029240 Neuritis Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 206010034277 Pemphigoid Diseases 0.000 description 2
- 201000011152 Pemphigus Diseases 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 206010036105 Polyneuropathy Diseases 0.000 description 2
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 2
- 201000004681 Psoriasis Diseases 0.000 description 2
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 2
- 201000009594 Systemic Scleroderma Diseases 0.000 description 2
- 206010042953 Systemic sclerosis Diseases 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 206010047115 Vasculitis Diseases 0.000 description 2
- 241000545067 Venus Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 2
- 230000000735 allogeneic effect Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 208000007502 anemia Diseases 0.000 description 2
- 239000002260 anti-inflammatory agent Substances 0.000 description 2
- 229940124599 anti-inflammatory drug Drugs 0.000 description 2
- 230000000781 anti-lymphocytic effect Effects 0.000 description 2
- 230000001494 anti-thymocyte effect Effects 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 2
- 210000004507 artificial chromosome Anatomy 0.000 description 2
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 239000008228 bacteriostatic water for injection Substances 0.000 description 2
- 229960004669 basiliximab Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000008366 buffered solution Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000011577 humanized mouse model Methods 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- 206010028417 myasthenia gravis Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 201000008383 nephritis Diseases 0.000 description 2
- 229960003301 nivolumab Drugs 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 201000001976 pemphigus vulgaris Diseases 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 208000005987 polymyositis Diseases 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 206010027654 Allergic conditions Diseases 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 208000003343 Antiphospholipid Syndrome Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 206010050245 Autoimmune thrombocytopenia Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000023328 Basedow disease Diseases 0.000 description 1
- 208000009137 Behcet syndrome Diseases 0.000 description 1
- 208000033386 Buerger disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010008748 Chorea Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 208000006313 Delayed Hypersensitivity Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 208000007465 Giant cell arteritis Diseases 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 208000024869 Goodpasture syndrome Diseases 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 208000015023 Graves' disease Diseases 0.000 description 1
- 208000001204 Hashimoto Disease Diseases 0.000 description 1
- 208000010496 Heart Arrest Diseases 0.000 description 1
- 201000004331 Henoch-Schoenlein purpura Diseases 0.000 description 1
- 206010019617 Henoch-Schonlein purpura Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical class OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010019755 Hepatitis chronic active Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101001047628 Homo sapiens Immunoglobulin kappa variable 2-29 Proteins 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 208000031814 IgA Vasculitis Diseases 0.000 description 1
- 208000010159 IgA glomerulonephritis Diseases 0.000 description 1
- 206010021263 IgA nephropathy Diseases 0.000 description 1
- 206010065042 Immune reconstitution inflammatory syndrome Diseases 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102100022949 Immunoglobulin kappa variable 2-29 Human genes 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 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 compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 208000005777 Lupus Nephritis Diseases 0.000 description 1
- 206010050551 Lupus-like syndrome Diseases 0.000 description 1
- 101710175243 Major antigen Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 1
- 208000010428 Muscle Weakness Diseases 0.000 description 1
- 206010028372 Muscular weakness Diseases 0.000 description 1
- 206010029132 Nephritis haemorrhagic Diseases 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010031252 Osteomyelitis Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 241000721454 Pemphigus Species 0.000 description 1
- 208000031845 Pernicious anaemia Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 208000033464 Reiter syndrome Diseases 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 208000022901 Reynold syndrome Diseases 0.000 description 1
- 208000016717 Reynolds syndrome Diseases 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 208000004732 Systemic Vasculitis Diseases 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 108010010056 Terlipressin Proteins 0.000 description 1
- 206010043540 Thromboangiitis obliterans Diseases 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 206010046851 Uveitis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 208000011341 adult acute respiratory distress syndrome Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 231100000540 amenorrhea Toxicity 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 201000004984 autoimmune cardiomyopathy Diseases 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000013357 binding ELISA Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 208000026555 breast adenosis Diseases 0.000 description 1
- 208000000594 bullous pemphigoid Diseases 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 229950007712 camrelizumab Drugs 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000004970 cd4 cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 208000012601 choreatic disease Diseases 0.000 description 1
- 208000016644 chronic atrophic gastritis Diseases 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000012085 chronic inflammatory response Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 201000003278 cryoglobulinemia Diseases 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 229960002806 daclizumab Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 210000001951 dura mater Anatomy 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 208000007475 hemolytic anemia Diseases 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000005931 immune cell recruitment Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 208000015446 immunoglobulin a vasculitis Diseases 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002350 laparotomy Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000023404 leukocyte cell-cell adhesion Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 208000037890 multiple organ injury Diseases 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 208000029264 myotonic syndrome Diseases 0.000 description 1
- 208000009928 nephrosis Diseases 0.000 description 1
- 231100001027 nephrosis Toxicity 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940029358 orthoclone okt3 Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 208000003154 papilloma Diseases 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 201000006292 polyarteritis nodosa Diseases 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 208000019629 polyneuritis Diseases 0.000 description 1
- 230000007824 polyneuropathy Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229940068977 polysorbate 20 Drugs 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 210000003492 pulmonary vein Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 208000002574 reactive arthritis Diseases 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229940115586 simulect Drugs 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000009168 stem cell therapy Methods 0.000 description 1
- 238000009580 stem-cell therapy Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 208000011317 telomere syndrome Diseases 0.000 description 1
- 206010043207 temporal arteritis Diseases 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- BENFXAYNYRLAIU-QSVFAHTRSA-N terlipressin Chemical compound NCCCC[C@@H](C(=O)NCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)CN)CSSC1 BENFXAYNYRLAIU-QSVFAHTRSA-N 0.000 description 1
- 229960003813 terlipressin Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 208000005057 thyrotoxicosis Diseases 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 229940121514 toripalimab Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 208000009999 tuberous sclerosis Diseases 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000002477 vacuolizing effect Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- 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
-
- 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
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
-
- 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
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
-
- 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
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
-
- 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
Abstract
本发明属于生物医药领域,具体涉及一种PD‑1非阻断性抗体及其应用。更具体的说,本发明涉及一种与PD‑1特异性高亲和力结合,但不阻断PD‑1与其配体结合的清除性抗体,达到清除自身反应性和同种异体反应性T细胞和B细胞的作用,以用于自身免疫性疾病、器官及细胞移植的排斥反应和炎症的治疗。
Description
技术领域
本发明属于生物医药领域,具体涉及一种PD-1非阻断性清除抗体及其用途。更具体的说,本发明涉及一种与PD-1特异性高亲和力结合,但不阻断PD-1与其配体结合的清除性抗体,达到清除自身反应性和同种异体反应性T细胞和B细胞的作用,以用于自身免疫性疾病、器官及细胞移植的排斥反应和炎症的治疗。
背景技术
程序性死亡受体-1(programmed cell death protein 1,PD-1,又称CD297)是CD28受体家族成员,其主要表达于活化的CD4+T细胞、CD8+T细胞、B细胞等,主要受T细胞受体(TCR)和B细胞受体(BCR)信号的诱导表达。PD-1分子有胞外区、跨膜区和胞内区构成,胞外区含有一个免疫球蛋白可变区IgV结构域以与其配体结合,胞内区含有一个免疫受体酪氨酸抑制基序(ITIM)和一个免疫受体酪氨酸转换基序(ITSM)以传递抑制性信号。PD-1有两个配体:PD-L1和PD-L2,均属于B7同源物,能与PD-1结合,但无法与其他CD28家族成员结合。PD-L1广泛表达于活化的T细胞、B细胞、巨噬细胞、树突状细胞及肿瘤细胞上,而PD-L2表达局限,主要在活化的T细胞和B细胞和少数肿瘤细胞。PD-1与其配体结合介导的免疫抑制作用是免疫耐受、肿瘤逃逸、移植耐受的重要机制。PD-1缺陷型动物形成多种自身免疫性疾病、包括自身免疫性心肌病和狼疮样综合征等。而PD-1信号介导的肿瘤中T细胞失能是肿瘤免疫逃逸的主要原因,阻断性PD-1抗体能阻断其与PD-L1和PD-L2结合,逆转T细胞失能,使T细胞的活化程度和杀伤能力显著增强,达到肿瘤的免疫治疗作用。目前已有多种PD-1阻断性抗体药物被批准用于肿瘤治疗:纳武利尤单抗(Nivolumab)、帕博利珠单抗(Pembrolizumab)、西米普利单抗(Cemiplimab)、特瑞普利单抗(Toripalimab)、信迪利单抗(Cindilimab)和卡瑞利珠单抗(Camrelizumab)。然而,临床使用发现,PD-1的阻断抗体可能诱发和加重自身免疫病及器官移植后的排斥反应。
反应性T细胞和B细胞介导的细胞免疫和体液免疫反应是自身免疫病和移植排斥反应的主要发病机制。自身免疫疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病,具体指自身免疫细胞主要是T和B细胞对自身免疫耐受异常,出现过度活化而导致的器官特异性(如慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、1型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎)或者系统性损害(多发性硬化症、系统性红斑狼疮、类风湿关节炎、系统性血管炎、银屑病、硬皮病、皮肌炎、溃疡性结肠炎等)。而器官及细胞移植排斥反应是免疫系统对外来异体抗原的一种免疫反应,引发排斥反应的主要抗原为主要组织相容性复合体(major histocompatibility complex,MHC),人类的MHC被称为HLA(human leukocyte antigen,HLA),即人白细胞抗原。排斥反应主要包括活化T细胞介导的细胞免疫应答和B细胞介导体液免疫应答,是导致移植物功能衰竭的主要原因。综合上述可知,自身免疫病及移植排斥反应主要是由活化的T细胞和B细胞介导的免疫反应。其具体机制包括:当T细胞的TCR识别到自身抗原或异体抗原后会活化,高表达CD2、ICOS、PD-1等共刺激及共抑制分子,并分化为效应性T细胞,包括Th1、Th2、Th9、Th17、Th22、滤泡样T细胞(Tfh)、细胞毒性T细胞(Tc)等,通过分泌促炎性细胞因子(IFN-γ、TNF-α、IL-2、IL-4、IL-17等)促进免疫细胞活化加重炎症反应、通过辅助B细胞活化和成熟分泌特异性抗体、通过分泌毒性蛋白(穿孔素和颗粒酶等)或者表达配体(Fas-FasL)诱导细胞死亡或调亡。免疫抑制剂是目前治疗自身免疫病和抑制排斥反应最重要的药物。
免疫抑制剂研发主要策略是抑制和清除自身反应性T细胞和B细胞,除了小分子化合物,目前临床常用的抗体药物主要包括:(1)抗胸腺细胞球蛋白(Antithymocyteglobulin,ATG)和抗淋巴细胞球蛋白(Antilymphocyte globulin,ALG):均是多克隆IgG组分,由已对人胸腺细胞或淋巴细胞免疫的各种动物(兔、马、山羊或猪)血清制备并收集而成,为强效免疫抑制剂。作用机制在于抑制经抗原识别后的淋巴细胞激活过程,并在补体协助下,对淋巴细胞产生细胞溶解作用。(2)抗人CD3抗体(Muromonab-CD3,商品名OrthocloneOKT3):是一种针对T淋巴细胞CD3复合物的抗体,其包括嵌合抗体及人源化抗体,其可以清除T细胞从而发挥较强的免疫抑制作用。(3)抗人CD25(高亲和力IL-2受体α链)抗体:包括人源化CD25单克隆抗体(daclizumab,xnapaxR)和人-鼠嵌合型CD25单克隆抗体(Basiliximab,simulect巴利昔单抗),能特异性的作用于活化的T细胞IL-2受体α链,通过竞争性的与IL-2受体结合,拮抗IL-2与其受体结合而达到抑制T细胞增殖作用。然而ATG/ALG或OKT3因为作用的靶细胞缺乏特异性,副作用较大,已经很少被用于临床,而CD25抗体只能抑制T细胞增殖,缺乏对特异性T细胞的清除作用,且存在影响抑制性T细胞——调节性T细胞(Treg)作用。
PD-1是T细胞和B细胞活化后表达的分子,目前上市的PD-1抗体均为阻断性抗体,且缺乏细胞毒作用,其竞争性结合PD-1,阻断其与PD-L1和PD-L2的结合以阻断T细胞负调控信号,增强T细胞的免疫功能及抗肿瘤作用,但同时会诱导和加重自身免疫病和移植排斥反应。而清除PD-1阳性的细胞则可能减轻自身免疫病及移植排斥反应,因此,本领域急需开发PD-1清除性抗体。
发明内容
为了解决上述问题,本发明提供一种PD-1非阻断性清除抗体及其用途。该新型的PD-1抗体——抗人PD-1非阻断性的高亲和力清除性抗体,其能高亲和力结合人PD-1,且对PD-1与PD-L1和PD-L2的结合无明显阻断效果,其清除性抗体在人源化PD-1小鼠的自身免疫病模型和心脏移植移植模型中均具有较好的保护效果,该抗体人源化的Fc段为具有ADCC和CDC作用的人IgG1,具有对表达PD-1细胞的清除作用。
特异性结合程序性死亡受体-1(PD-1)的抗体或其抗原结合片段
在一个方面,本发明提供了一种特异性结合PD-1的非阻断性清除抗体或其抗原结合片段,包含轻链可变区和重链可变区,所述轻链可变区包含分别如SEQ ID NO:1、2和3所示的LCDR1(SSVSSSY)、LCDR2(STS)和LCDR3(HQYHRSPLT),所述重链可变区包含分别如SEQID NO:4、5和6所示的HCDR1(GYTFTDYS)、HCDR2(IKIETGYP)和HCDR3(ARDYFGNYYYAMDY)。
更具体地,所述特异性结合PD-1的抗体或其抗原结合片段可以为鼠源抗体、嵌合抗体或人源化抗体中的任一种。
更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为鼠源抗体,其包含如SEQ ID NO:7所示的轻链和如SEQ ID NO:9所示的重链,此抗体在本文中命名为鼠源性抗体3D6-mIgG2a。更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为鼠源抗体,其包含由SEQ ID NO:8编码的轻链和由SEQ ID NO:10编码的重链。
更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为人源化抗体,并且其Fc段为具有ADCC和CDC作用的人IgG1的Fc段。
更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为人源化抗体,其包含如SEQ ID NO:11所示的轻链可变区和如SEQ ID NO:13所示的重链可变区。更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为人源化抗体,其包含由SEQ ID NO:12编码的轻链可变区和由SEQ ID NO:14编码的重链可变区。
更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为人源化抗体,其包含如SEQ ID NO:15所示的轻链和如SEQ ID NO:17所示的重链,此抗体在本文中命名为人源化抗体3D6-hIgG1。更具体地,所述特异性结合PD-1的抗体或其抗原结合片段为人源化抗体,其包含由SEQ ID NO:16编码的轻链和由SEQ ID NO:18编码的重链。
如本文中所使用的,术语"抗体"涵盖全长抗体(例如,IgG1或IgG4抗体)、其各种功能性片段(例如可仅包含抗原结合部分,如Fab、F(ab')2或scFv片段)以及经过修饰的抗体(例如人源化、糖基化等)。
如本文中所使用的,术语"单克隆抗体或mAb",指由单一的克隆细胞株得到的抗体,所述的细胞株不限于真核的,原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术,合成技术(如CDR移植),或其它现有技术进行重组得到。
如本文中所使用的,"抗体片段"和"抗原结合片段"可互换使用并意即抗体的抗原结合片段及抗体类似物,其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常,当基于摩尔来表示活性时,抗体片段保留至少10%的母体结合活性。优选地,抗体片段保留至少20%、50%、70%、80%、90%、95%或100%或更多的母体抗体对靶标的结合亲和力。抗体片段实例包括但不限于:Fab、Fab'、F(ab')2和Fv片段;双抗体;线性抗体(linearantibody);单链抗体分子,例如ScFv、单抗体(技术来自Genmab);纳米抗体(技术来自Domantis);结构域抗体(技术来自Ablynx);和由抗体片段形成的多特异性抗体。工程改造的抗体变体综述于Holliger等,2005;Nat Biotechnol,23:1126-1136中。
如本文中所使用的,术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在抗体中含有三个CDR,命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义,例如可按照Kabat编号系统、Chothia编号系统或IMGT编号系统中的定义。对于给定的抗体,本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且,不同编号系统之间的对应关系是本领域技术人员熟知的。
如本文中所使用的,术语“特异性结合”是指两分子间的非随机的结合反应,如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以由该相互作用的平衡解离常数(KD)表示。在本发明中,术语“KD”是指特定抗体-抗原相互作用的解离平衡常数,其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小,抗体-抗原结合越紧密,抗体与抗原之间的亲和力越高。
如本文中所使用的,术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人PD-1的单克隆抗体。制备时用PD-1抗原注射试验对象,然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中,所述的鼠源PD-1抗体或其抗原结合片段,可进一步包含鼠源κ、λ链或其变体的轻链恒定区,或进一步包含鼠源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。
如本文中所使用的,术语"嵌合抗体(Chimeric antibody)",是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体,可以减鼠源性抗体诱发的免疫应答反应。建立嵌合抗体,要先建立分泌鼠源性特异性单抗的杂交瘤,然后从小鼠杂交瘤细胞中克隆可变区基因,再根据需要克隆人抗体的恒定区基因,将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入载体中,最后在真核表达系统或原核表达系统中表达嵌合抗体分子。在本发明的一优选实施方案中,所述的PD-1嵌合抗体的抗体轻链可变区进一步包含鼠源,λ链或其变体的轻链FR区。所述PD-1嵌合抗体的抗体重链可变区进一步包含鼠源IgGl、IgG2、IgG3或IgG4或其变体的重链FR区。人抗体的恒定区可选自人源IgGl、IgG2、IgG3或IgG4或其变体的重链恒定区,优选具有ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的人源IgG1重链恒定区。
如本文中所使用的,术语"人源化抗体(antibody)",非人类(例如鼠)抗体的“人源化”形式为含有最小限度的来源于非人类免疫球蛋白序列的嵌合抗体。人源化抗体的大部分为人免疫球蛋白,其中抗体的高变区残基被具有所需特异性、亲和力的非人类物种高变区的残基置换,在某些情况下,人免疫球蛋白的Fv构架区残基被相应的非人类残基取代,此外,人源化抗体可包含不在受体抗体或供体抗体中存在的残基,进行这些修饰以进一步改进抗体性能。
应理解,本发明涵盖了如本文所述的PD-1特异性抗体或其抗原结合片段的变体,其与如本文所述的PD-1特异性抗体或其抗原结合片段的氨基酸序列的具有至少90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的序列同一性,并且基本保留了其所源自的抗体的生物学功能(例如特异性结合PD-1的生物活性)。
更具体地,所述变体与如本文所述的PD-1特异性抗体或其抗原结合片段相比差异仅在于一个或多个(例如,至多20个、至多15个、至多10个、至多5个或至多1个氨基酸的保守置换)氨基酸残基的保守置换。
如本文中所使用的,术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如,两个DNA分子的每一个中的某个位置都被腺嘌呤占据,或两个多肽的每一个中的某个位置都被赖氨酸占据),那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如,如果两个序列的10个位置中有6个匹配,那么这两个序列具有60%的同一性。例如,DNA序列CTGACT和CAGGTT共有50%的同一性(总共6个位置中有3个位置匹配)。通常,在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用,例如,可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48:443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.,4:11-17(1988))的算法,使用PAM120权重残基表(weight residuetable)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外,可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(JMoIBiol.48:444-453(1970))算法,使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的,术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如,可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换,例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质,包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如,赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如,苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如,酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此,优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见,例如,Brummell等人,Biochem.32:1180-1187(1993);Kobayashi等人Protein Eng.12(10):879-884(1999);和Burks等人Proc.NatlAcad.Set USA 94:412-417(1997),其通过引用并入本文)。
多核苷酸
在另一个方面,本发明还涉及一种多核苷酸,其编码如本文所述的抗体或其抗原结合片段。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
术语“编码多肽/蛋白/抗体的多核苷酸”可以是包括编码此多肽/蛋白/抗体的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与如本文所述的多核苷酸序列杂交且两个序列之间具有至少50%,优选地至少70%,更优选地至少80%,最优选至少90%同一性的多核苷酸,并且它们编码的多肽/蛋白/抗体具有基本上相同的功能和活性。本发明特别涉及可在严格条件下与本发明所述多核苷酸杂交的多核苷酸。在本发明中,“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,60℃;或(2)杂交时加有变性剂,如50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的同一性至少在90%以上,更优选95%以上时才发生杂交。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。
载体
在另一个方面,本发明还涉及一种载体,其包含如本文所述的多核苷酸。
如本文中所使用的,术语“载体(vector)”是指,可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时,载体称为表达载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒;噬菌粒;柯斯质粒;人工染色体,例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于,逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件,包括但不限于,启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。
宿主细胞
在另一个方面,本发明还涉及一种宿主细胞,其包含如本文所述的载体。
如本文中所使用的,术语“宿主细胞”是指,可用于导入载体的细胞,其包括但不限于,如大肠杆菌或枯草菌等的原核细胞,如酵母细胞或曲霉菌等的真菌细胞,如S2果蝇细胞或Sf9等的昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK 293细胞或其他人细胞等的动物细胞。宿主细胞可以包括单个细胞或细胞群体。
将载体导入宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔,脂质体包装等。
缀合物
在另一个方面,本发明还涉及一种缀合物,其包含如本文所述的抗体或其抗原结合片段以及偶联部分。
所述偶联部分选自可检测标记物、药物、毒素、细胞因子、放射性核素或酶。
在某些实施方案中,本发明的抗体或其抗原结合片段任选地通过接头与所述偶联部分缀合。
在某些实施方案中,所述偶联部分选自可检测标记物,例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。本发明所述的可检测标记物可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的,其实例包括但不限于,酶(例如,辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶,等)、放射性核素(例如,3H、125I、35S、14C或32P)、荧光染料(例如,异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa750))、发光物质(例如化学发光物质,如吖啶酯类化合物)、磁珠、测热标记物例如胶体金或有色玻璃或塑料(例如,聚苯乙烯、聚丙烯、乳胶,等)珠、以及用于结合上述标记物修饰的亲和素(例如,链霉亲和素)的生物素。在某些实施方案中,此类标记能够适用于免疫学检测(例如,酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中,可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段,以降低潜在的位阻。
在某些实施方案中,所述偶联部分选自药物,例如抗炎药物或免疫抑制剂。
在某些实施方案中,所述偶联部分选自另外的生物活性多肽。
药物组合物
在另一个方面,本发明还涉及一种药物组合物,其包含如本文所述的抗体或其抗原结合片段以及药学上可接受的载体、稀释剂或赋形剂。
在某些实施方案中,所述药物组合物还可以包含第二治疗剂。所述第二治疗剂是有利地与PD-1抗体组合的任意试剂。在一些实施方案中,所述第二治疗剂是其他抗炎药物或免疫抑制剂。
在某些实施方案中,在所述药物组合物中,如本文所述的抗体或其抗原结合片段与所述第二治疗剂可以作为分离的组分或作为混合的组分提供。因此,如本文所述的抗体或其抗原结合片段与所述第二治疗剂可以同时、分开或相继施用。
在一些实施方案中,所述药学上可接受的载体和/或赋形剂可以包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中,此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9%(w/v)NaCl)、葡萄糖溶液(例如5%葡萄糖)、含有表面活性剂的溶液(例如0.01%聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的如本文所述的抗体或其抗原结合片段与所述第二治疗剂。“预防有效量”是指足以预防、阻止或延迟疾病的发生的量。“治疗有效量”是指足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。治疗有效量可根据如下因素发生变化:待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄,体重和性别,药物的施用方式,以及同时施用的其他治疗等等。
治疗用途
在另一个方面,本发明还涉及如本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、缀合物或药物组合物在制备用于清除自身反应性和同种异体反应性T细胞和B细胞的药物中的用途。
在另一个方面,本发明还涉及如本文所述的抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、缀合物或药物组合物在制备用于治疗自身免疫性疾病、器官或细胞移植的排斥反应和/或炎症的药物中的用途。
自身反应性T细胞
如本文所用,自身反应性T细胞意指攻击自身组织、细胞及器官的T细胞。有时机体免疫系统不能将自身一个或多个组分识别为“自我”,并产生自身抗体及自身反应性T细胞,这些抗体及T细胞攻击自身细胞、组织和/或器官(误将自身正常组织细胞当做外源性抗原)。从而导致炎症和损伤,引起自身免疫性疾病。
同种异体反应性T细胞
如本文所用,同种异体反应性T细胞是介导移植排斥反应的重要细胞,同种异体的供、受者间MHC分子存在差异,供、受者的淋巴细胞通过直接识别对方淋巴细胞表面的异型MHC分子,从而发生增殖和活化。区别于对一般抗原的应答,由直接识别导致的排斥反应有两个特点:①速度快,因为无需经历抗原摄取、处理和加工;②强度大,因为每一个体中,具有同种抗原反应性的T细胞克隆占T细胞库总数的1%-l0%,而针对一般异源性抗原的T细胞克隆仅占总数的1/100000-1/10000。
器官移植排斥反应
本发明抗体或抗体片段可用于治疗器官移植排斥反应,其是在受者进行同种异体组织或器官移植后,外来的组织或器官等移植物作为一种“异己成分”被受者免疫系统识别,后者发起针对移植物的攻击、破坏和清除的免疫学反应。本发明抗体或其缀合物可单独使用,或者与其他免疫抑制剂联合使用,以治疗器官移植排斥反应,例如但不限于这些的一些实例,包括:心脏移植、肾移植、肺移植、肝移植、胰腺与胰岛移植、甲状旁腺移植、心肺联合移植、骨髓移植、角膜移植、小肠移植,手移植等,以及各类组织包括皮肤、脂肪、筋膜、肌腱、硬膜、血管、淋巴管、软骨和骨移植。
细胞移植排斥反应
细胞治疗是指将正常或生物工程改造过的人体细胞移植或输入患者体内,新输入的细胞可以替代受损细胞、或者具有更强的免疫杀伤功能,从而达到治疗疾病的目的。细胞治疗按照细胞种类可以分为干细胞治疗和免疫细胞治疗。本发明抗体或其缀合物可单独使用,或者与其他免疫抑制剂联合使用,以治疗细胞移植排斥反应。
自身免疫性疾病
本发明抗体或抗体片段可用于治疗自身免疫性疾病(autoimmune disease,AID),其是在一定条件下,免自身疫耐受被打破,免疫系统将对自身抗原产生病理性免疫应答,造成自身组织细胞损伤或功能异常。本发明抗体或其缀合物可单独使用,或者与其他免疫抑制剂联合使用,以治疗自身免疫性疾病,例如但不限于这些的一些实例,包括:免疫介导的血小板减少症,如急性特发性血小板减少性紫癜和慢性特发性血小板减少性紫癜、皮肌炎、西登哈姆氏舞蹈病、狼疮肾炎、风湿热、多内分泌腺综合征、过敏性紫癜、链球菌感染后性肾炎、结节性红斑、高安氏动脉炎(Takayasu's arteritis)、阿狄森病(Addison's disease)、多形红斑(erythemamultiforme)、结节性多动脉炎、强直性脊柱炎、古德帕斯丘综合征(Goodpasture'ssyndrome)、血栓闭塞性脉管炎、原发性胆汁性肝硬化、桥本氏甲状腺炎(Hashimoto'sthyroiditis)、甲状腺中毒症、慢性活动性肝炎、多发性肌炎/皮肌炎、多软骨炎、寻常型天疱疮、韦格纳氏肉牙肿病(Wegener's granulomatosis)、膜性肾病、肌肉萎缩性侧索硬化、脊髓痨、多肌痛、恶性贫血、急进性肾小球肾炎和纤维性肺泡炎;炎症性皮肤病,包括牛皮癣和皮肤炎(例如异位性皮肤炎);全身性硬皮病和硬化;发炎性肠病(如克罗恩病(Crohn'sdisease)和溃疡性结肠炎);呼吸窘迫综合征包括成人呼吸窘迫症候群;ARDS);皮肤炎;脑膜炎;脑炎;葡萄膜炎;结肠炎;肾小球肾炎;过敏性病状,如湿疹和哮喘以及涉及T细胞浸润和慢性发炎性反应的其它病状;动脉粥样硬化;白细胞粘附不足;类风湿性关节炎;全身性红斑性狼疮症(systemic lupus erythematosus,SLE);糖尿病(例如1型糖尿病或胰岛素依赖型糖尿病);多发性硬化:雷诺综合征(Reynaud's syndrome);自体免疫甲状腺炎;过敏性脑脊髓炎;斯耶格伦氏综合症(Sjorgen's syndrome);青少年型糖尿病:以及与由通常发现于肺结核结节病、多发性肌炎、肉芽肿病和血管炎中的细胞介素和T淋巴细胞介导的急性和迟发性超敏感性相关的免疫反应:恶性经闭(阿狄森病,Addison'sdisease);涉及白细胞渗出的疾病;中枢神经系统(CNS)发炎性病症;多重器官损伤综合征;溶血性贫血(包括但不限于冷球蛋白血症或库姆斯阳性贫血(Coombs positive anemia));重症肌无力;抗原-抗体复合物介导疾病:抗肾小球基底膜疾病:抗磷脂综合征;过敏性神经炎;格雷夫斯氏病(Graves'disease);朗伯一伊顿肌无力综合征(Lambert-Eaton肌无力综合征);大疱性类天疱疮;天疱疮;自身免疫多内分泌腺病;莱特尔氏病(Reiter'sdisease);全身肌强直综合征:贝切特氏病(Behcedisease);巨细胞动脉炎;免疫复合物肾炎;IgA肾病;1gM多发性神经病:免疫血小板减少性紫癜(immune thrombocytopenicpurpura,ITP)或自身免疫血小板减少等。
缩写
hPD-1 人PD-1蛋白
CDR 用Kabat编号系统界定的免疫球蛋白可变区中的互补决定区
EC50 产生50%功效和结合的浓度
ELISA 酶联免疫吸附测定
IL-2 白细胞介素-2
IC50 产生50%抑制的浓度
IgG 免疫球蛋白G
Kabat 由Elvin A Kabat倡导的免疫球蛋白比对及编号系统
FR 抗体构架区:将CDR区排除在外的免疫球蛋白可变区
HRP 辣根过氧化物酶
IFN-γ 干扰素γ
mAb 单克隆抗体
PCR 聚合酶链式反应
V区 在不同抗体之间序列可变的IgG链区段。其延伸到轻链的109位Kabat残基和重链的第113位残基
VH 免疫球蛋白重链可变区
VK 免疫球蛋白κ轻链可变区
Kd 平衡解离常数
Ka 结合速率常数
Kd 解离速率常数
附图说明
图1示出了制备hPD-1抗体的流程图。
图2示出了鼠源性抗体3D6-mIgG2a的SDS-PAGE鉴定结果。
图3示出了鼠源性抗体3D6-mIgG2a的ELISA结合检测结果。
图4示出了鼠源性抗体3D6-mIgG2a的FCM检测结果。
图5示出了鼠源性抗体3D6-mIgG2a的竞争ELISA检测结果。
图6示出了鼠源性抗体3D6-mIgG2a延长PD-1人源化小鼠心脏移植物生存期。
图7示出了鼠源性抗体3D6-mIgG2a的ELISPOT结果。
图8示出了人源化抗体质粒图。
图9示出了人源化抗体3D6-hIgG1的SDS-PAGE检测结果。
图10示出了人源化抗体3D6-hIgG1的BLI亲和力检测结果。
图11示出了人源化抗体3D6-hIgG1对混合淋巴细胞反应的作用。
图12示出了人源化抗体3D6-hIgG1的ADCC活性检测结果。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1:产生小鼠抗hPD-1抗体
为了产生针对人PD-1的小鼠抗体,用人PD-1免疫接种6-8周大的雌性Balb/c小鼠,通过如图1所示的方法收集抗hPD-1抗体。
小鼠的免疫接种
免疫原为重组人PD-1蛋白(ABclonal,RP00067),第一次免疫:100ug稀释的免疫原用等量的完全弗氏佐剂(CFA)乳化后皮下注射,随后第2-3次免疫用50ug稀释的免疫原用等量的不完全弗氏佐剂(IFA)乳化后皮下注射,100ug免疫原静脉注射冲击免疫3天后用ELISA法测定小鼠血清抗体效价,选取效价最高的小鼠脾细胞进行细胞融合。
细胞融合
采用处于对数生长期的Balb/c来源的骨髓瘤细胞sp2/0,用CD19的磁珠富集效价最高的已免疫小鼠脾脏中的B细胞,B细胞与骨髓瘤细胞以1:5-1:10混合,滴加37℃的50%PEG(pH 8.0)1ml,加入不完全培养基及其余终止液,离心弃上清后加入HAT培养基悬浮混匀,置于37℃、5%CO2恒温培养箱中进行培养。
结合ELISA初步筛选和亚克隆
融合7-10天内挑选杂交瘤细胞克隆,取培养上清进行ELISA结合检测:包被PD-1蛋白0.1ug/ml,1ug/ml及5ug/ml,100ul/孔,4℃包被过夜;板内液体甩净拍干,2%BSA 300ul每孔,密封后室温孵育1h;300ul每孔洗涤液,洗板2次,最后一次拍干;将培养上清稀释后加入对应孔板中,100ul每孔,室温2小时;重复洗板过程;稀释二抗至使用浓度,100ul每孔,室温1小时;重复洗板过程;将显色液A液和B液1:1混匀后,每孔加入200ul,室温避光孵育20min;每孔加入50ul终止液,立即在450nm波长处测量OD值。对阳性孔细胞进行有限稀释,每次有限稀释后5-6天测定ELISA值,挑取ELISA检测OD450阳性值较高的单克隆孔进行有限稀释,直至ELISA测定96孔板全板结果为阳性。
FCM结合筛选
使用处于对数生长期的MOLT-4细胞系,分至10万个细胞每管,加入10ul前述ELISA测定全板结果为阳性的单克隆细胞的培养上清,室温孵育30分钟,PBS洗一遍,离心,加入抗小鼠IgG的荧光二抗0.5ul每管,室温孵育30分钟,PBS后洗一遍后上机检测,选取平均荧光强度较高的几个克隆进行进一步筛选。
竞争ELISA筛选
包被PDCD1-Fc,100uL/孔,4℃包被过夜;板内液体甩净拍干,2%BSA,300μL/孔,密封后室温孵育1h;300μL/孔洗涤液,洗板2次,最后一次拍干;竞争物PD-L2-His稀释至0.2ug/mL及竞争物B7-H1-His稀释至3ug/mL,取稀释后样品(前述平均荧光强度较高的克隆)及竞争物各100ul加入至对应孔板中,混合均匀,室温反应2h;300μL/孔洗涤液,洗板3次,最后一次拍干;稀释HRP标记的抗His检测二抗至使用浓度,100μL/孔,混合均匀,室温孵育1h;洗板,将A液和B液按1:1混匀后,每孔加入200μL,室温避光孵育10min;每孔加入50μL终止液,立即在450nm波长处测量OD值。筛选出阻断PD-1与PD-L1/2结合效率较低的克隆3D6进行测序(其包含如SEQ ID NO:7所示的轻链和如SEQ ID NO:9所示的重链)及3D6-mIgG2a抗体的生产。
杂交瘤细胞基因提取及测序
将上述克隆3D6的杂交瘤细胞裂解后提取RNA,以提取的RNA为模板,采用义翘科技反转录试剂盒,分两步获得cDNA,以反转录获得的cDNA为模板,通过多条引物进行PCR扩增,将扩增的片段插入到表达载体上,测序得到含有正确序列的质粒。
细胞培养与抗体纯化
将1mL杂交瘤细胞转入100mL培养瓶中,定期加入一定量的1640培养基进行细胞扩增,培养10-12天。Protein A亲和层析柱用超纯水冲洗,然后用平衡缓冲液平衡;将培养后的杂交瘤细胞的上清上样至亲和层析柱,上样完毕后,用平衡缓冲液淋洗。洗脱缓冲液洗脱,收集洗脱峰,用Tris缓冲液中和。脱盐至PBS7.4。制样,凝胶电泳鉴定,判断胶图条带大小是否正确,SDS-PAGE鉴定其纯度。结果显示:抗体获得成功表达,纯度大于90%(图2)。
实施例2:对生产出的鼠源性抗体3D6-mIgG2a再次进行结合能力及阻断活性的检测结合ELISA检测
包被PD-1蛋白0.1ug/ml,1ug/ml及5ug/ml,100ul/孔,4℃包被过夜;板内液体甩净拍干,2%BSA 300ul每孔,密封后室温孵育1h;300ul每孔洗涤液,洗板2次,最后一次拍干;抗体稀释至1ug/ml加入对应孔板中,100ul每孔,室温2小时;重复洗板过程;稀释二抗至使用浓度,100ul每孔,室温1小时;重复洗板过程;将显色液A液和B液1:1混匀后,每孔加入200ul,室温避光孵育20min;每孔加入50ul终止液,立即在450nm波长处测量OD值(图3),结果显示该抗体3D6-mIgG2a(即图3中的3D6表示)与PD-1蛋白结合良好。
FCM检测
使用处于对数生长期的MOLT-4细胞系,分至10万个细胞每管,加入抗体稀释至1ug/ml,室温孵育30分钟,PBS洗一遍,离心,加入抗小鼠IgG的荧光二抗0.5ul每管,室温孵育30分钟,PBS后洗一遍后上机检测(图4),结果显示该抗体3D6-mIgG2a(即图4中的3D6表示)与表达人PD-1的细胞结合良好。
竞争ELISA检测
包被PDCD1-Fc,100uL/孔,4℃包被过夜;板内液体甩净拍干,2%BSA,300μL/孔,密封后室温孵育1h;300μL/孔洗涤液,洗板2次,最后一次拍干;竞争物PD-L2-His稀释至0.2ug/mL及竞争物B7-H1-His稀释至3ug/mL,加入0.1ug/ml和5ug/ml抗体及竞争物各100ul加入至对应孔板中,混合均匀,室温反应2h;300μL/孔洗涤液,洗板3次,最后一次拍干;稀释HRP标记的抗His检测二抗至使用浓度,100μL/孔,混合均匀,室温孵育1h;洗板,将A液和B液按1:1混匀后,每孔加入200μL,室温避光孵育10min;每孔加入50μL终止液,立即在450nm波长处测量OD值(图5),结果显示该抗体3D6-mIgG2a(即图5中的3D6表示)几乎不阻断PD-1与PD-L1/2结合。
实施例3:PD-1人源化小鼠心脏移植模型检测3D6-mIgG2a抗体的体内效果
麻醉小鼠,将BALB/c小鼠的心脏移植到B6 hPD-1(集萃药康,T003095)受体小鼠的腹部。打开供体小鼠腹部,经下腔静脉注入1ml肝素盐水,1分钟后打开胸廓,立即用冰肝素盐水滴在心脏表面至心脏停止跳动,用6-0的丝线结扎上下腔静脉,摘除胸腺,暴露主动脉和肺动脉,用维纳斯剪剪开主动脉及肺动脉,用2ml冰肝素盐水经主动脉冲洗心脏至无血色,将肺静脉统一结扎之后将心脏整体取下,放入冰肝素盐水中备用。打开受体小鼠腹腔,分离暴露腹腔动静脉,结扎分支血管,分别在近端和远端阻断腹腔动静脉,用维纳斯剪在动静脉正面分别开一个小口,用10-0的丝线将供心的主动脉端侧连续缝合至腹腔动脉,将肺动脉端侧连续缝合至腹腔静脉,开放循环,待心脏复跳后,关腹,补液。将抗体(500μg)在移植后第0、3、6天腹腔注射到受体小鼠体内,随后250μg每5天一次,每天通过触摸心脏跳动来判断排斥情况,当心脏完全停止跳动时认为发生了完全的排斥,并通过剖腹手术证实。3天以内的心脏停跳则判断为技术失误(小于5%),从后续分析中剔除(图6),结果显示,该抗体显著延长心脏移植物生存期。
实施例4:ELISPOT检测同种异体反应性T细胞数量
构建BALB/c小鼠的心脏移植到B6 hPD-1受体小鼠的心脏移植模型,并予以抗体(腹腔注射500ug,第0、3、6天注射)治疗,在移植后第6天,取受体脾脏研磨成单细胞悬液,使用T细胞阴选试剂盒(美天旎,130-095-130)将其中的T细胞分选出来并与刺激细胞BALB/cAPCs 1:1混合,以2x105个/孔接种在小鼠IFN-γ预包被ELISpot板子上,37℃孵育16小时。在4℃下用冷ddH2O裂解10分钟后,用洗涤缓冲液洗涤6次,然后在37℃下与生物素化的抗IFN-γ孵育1小时。洗涤之后,与链霉亲和素-HRP在37℃下孵育1小时。重复洗涤过程后,在37℃下用AEC溶液孵育10分钟,然后用冷ddH2O终止。利用RAWspot技术在单细胞水平上进行计数,使用Mabtech IRIS FluoroSpot/ELISpot阅读器对结果进行分析(图7),结果显示该抗体显著减少了受体脾脏中的同种异体反应性T细胞数量。
实施例5:鼠源抗体的人源化及人源化抗体的亲和力检测
通过对3D6进行抗体序列分析,3D结构建模及CDR结构分析,人源化设计,随后进行基因合成、载体构建(图8)、HEK293瞬转表达纯化,获得了人源化抗体3D6-hIgG1(其包含如SEQ ID NO:15所示的轻链和如SEQ ID NO:17所示的重链),并通过SDS-PAGE检测了目的蛋白纯度(图9),结果显示:条带大小位置正确,纯度为98.7%。具体的人源化抗体构建方法可根据本领域许多文献公示的方法进行。
利用BLI测定人源化抗体与抗原蛋白亲和力
首先将ForteBio Octet RED384系统的生物传感器浸入缓冲液中进行平衡,浸入5ug/ml的人源化抗体3D6-hIgG1溶液中,再浸入含有梯度稀释的hPD-1蛋白溶液(0.125μg/ml、0.25μg/ml、0.5μg/ml、1μg/ml、2μg/ml)中,将已结合待测抗体的传感器浸入缓冲液中进行解离,待测抗体从生物传感器表面脱落导致膜层厚度的减少。通过对实验过程中生物传感器生物膜层厚度的实时监控,可以得到待测样品的动力学常数,结果表明,人源化抗体3D6-hIgG1的亲和力为5.29E-10,仍然保持着较高的亲和力(图10)。
实施例6:人源化抗体3D6-hIgG1不增强混合淋巴细胞反应
取健康人外周血,用淋巴细胞分离液(天津市灏阳生物科技有限限公司)分离外周血单个核细胞,进一步采用单核细胞分离试剂盒(美天旎公司)分离单核细胞,加入25ng/mlGM-CSF及50ng/ml IL-4接种至96孔板,37℃培养7天诱导树突状细胞。实验第7天,取另外一份健康人外周血用淋巴细胞分离液分离外周血单个核细胞,进一步采用CD4细胞分离试剂盒(美天旎公司)分离CD4阳性T细胞,调整细胞浓度并接种至前述诱导树突状细胞的培养板中,各加入1ug/ml hIgG1同种型阴性对照抗体(hIgG1 isotype)、纳武利尤单抗阳性对照抗体(Nivolumab)及3D6-hIgG1,常规培养5天后收集上清,使用人IFN-gamma ELISA试剂盒(ABclonal RK00015)检测各上清中的IFN-γ浓度,结果显示,PD-1阻断性抗体纳武利尤单抗相比hIgG1同种型阴性对照抗体显著增加混合淋巴细胞反应中IFN-γ分泌量,而3D6-hIgG1则并不增加IFN-γ分泌量(图11)。
实施例7:人源化抗体3D6-hIgG1的体外ADCC活性检测
取健康人外周血用淋巴细胞分离液分离外周血单个核细胞,进一步采用NK细胞分离试剂盒(美天旎公司)分离NK细胞,并在体外用IL-2刺激培养,将体外活化的CD4阳性T细胞用PKH26细胞连接试剂盒(Sigma)标记,随后与NK细胞以1:5的比例种板,加入10倍稀释梯度(0.001ug/ml、0.01ug/ml、0.1ug/ml、1ug/ml、10ug/ml)的3D6-hIgG1和hIgG1同种型抗体(hIgG1 isotype)并在37℃孵育4小时,通过流式细胞仪分析活的CD4阳性T细胞比例,计算裂解比例。结果显示,随着3D6-hIgG1抗体浓度的增加,CD4阳性T细胞的裂解比例逐渐增加,当3D6-hIgG1浓度为0.074ug/ml时,有约50%的CD4阳性T细胞发生了裂解(图12)。
在说明书的描述中,参考术语“一个实施方案”、“具体实施方案”、“实例”等的描述意指结合该实施方案或实例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施方案或实例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施方案或实例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施实施方案或实例中以合适的方式结合。
以上内容仅仅是对本发明所作的举例和说明,所属本技术领域的技术人员对所描述的具体实施方案做各种各样的修改或补充或采用类似的方式替代,只要不偏离发明或者超越本权利要求书所定义的范围,均应属于本发明的保护范围。
需要说明的是,本发明的说明书及其附图中给出了本发明的较佳的实施例,但是,本发明可以通过许多不同的形式来实现,并不限于本说明书所描述的实施例,这些实施例不作为对本发明内容的额外限制,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。并且,上述各技术特征继续相互组合,形成未在上面列举的各种实施例,均视为本发明说明书记载的范围;进一步地,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (12)
1.一种特异性结合PD-1的非阻断性清除抗体或其抗原结合片段,其特征在于,包含轻链可变区和重链可变区,所述轻链可变区包含分别如SEQ ID NO:1、2和3所示的LCDR1、LCDR2和LCDR3,所述重链可变区包含分别如SEQ ID NO:4、5和6所示的HCDR1、HCDR2和HCDR3。
2.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段为鼠源抗体。
3.根据权利要求2所述的抗体或其抗原结合片段,其特征在于,所述鼠源抗体由如SEQID NO:7所示的轻链和如SEQ ID NO:9所示的重链组成。
4.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体或其抗原结合片段为人源化抗体。
5.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述人源化抗体的Fc段为具有ADCC和CDC作用的人IgG1的Fc段。
6.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述人源化抗体包含如SEQ ID NO:11所示的轻链可变区和如SEQ ID NO:13所示的重链可变区。
7.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述人源化抗体由如SEQ ID NO:15所示的轻链和如SEQ ID NO:17所示的重链组成。
8.一种多核苷酸,其特征在于,所述多核苷酸编码权利要求1-7中任一项所述的抗体或其抗原结合片段。
9.一种载体,其特征在于,包含权利要求8所述的多核苷酸。
10.一种宿主细胞,其特征在于,包含权利要求9所述的载体。
11.一种药物组合物,其特征在于,包含权利要求1-7中任一项所述的抗体或其抗原结合片段以及药学上可接受的载体、稀释剂或赋形剂。
12.一种根据权利要求1-7中任一项所述的抗体或其抗原结合片段、权利要求8所述的多核苷酸、权利要求9所述的载体、权利要求10所述的宿主细胞或权利要求11所述的药物组合物在制备用于治疗器官或细胞移植的排斥反应的药物中的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310759264.4A CN116854820B (zh) | 2023-06-26 | 2023-06-26 | Pd-1非阻断性清除抗体及其用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310759264.4A CN116854820B (zh) | 2023-06-26 | 2023-06-26 | Pd-1非阻断性清除抗体及其用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116854820A CN116854820A (zh) | 2023-10-10 |
CN116854820B true CN116854820B (zh) | 2024-02-06 |
Family
ID=88226058
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310759264.4A Active CN116854820B (zh) | 2023-06-26 | 2023-06-26 | Pd-1非阻断性清除抗体及其用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116854820B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AR105520A1 (es) * | 2015-07-30 | 2017-10-11 | Macrogenics Inc | Moléculas de unión a pd-1 y métodos de uso de las mismas |
CN110997712A (zh) * | 2017-06-05 | 2020-04-10 | 詹森生物科技公司 | 特异性结合pd-1的抗体及其使用方法 |
CN112204052A (zh) * | 2018-04-05 | 2021-01-08 | 诺华股份有限公司 | 针对癌症的三特异性结合分子及其用途 |
CN114316045A (zh) * | 2020-09-29 | 2022-04-12 | 锋宏生物医药科技(昆山)有限公司 | 抗pd-l1抗体及其用途 |
CN116178545A (zh) * | 2021-09-24 | 2023-05-30 | 广东菲鹏制药股份有限公司 | 一种抗人pd-l1人源化抗体或其抗原结合片段及其应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2017355401A1 (en) * | 2016-11-02 | 2019-05-02 | Jounce Therapeutics, Inc. | Antibodies to PD-1 and uses thereof |
-
2023
- 2023-06-26 CN CN202310759264.4A patent/CN116854820B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AR105520A1 (es) * | 2015-07-30 | 2017-10-11 | Macrogenics Inc | Moléculas de unión a pd-1 y métodos de uso de las mismas |
CN110997712A (zh) * | 2017-06-05 | 2020-04-10 | 詹森生物科技公司 | 特异性结合pd-1的抗体及其使用方法 |
CN112204052A (zh) * | 2018-04-05 | 2021-01-08 | 诺华股份有限公司 | 针对癌症的三特异性结合分子及其用途 |
CN114316045A (zh) * | 2020-09-29 | 2022-04-12 | 锋宏生物医药科技(昆山)有限公司 | 抗pd-l1抗体及其用途 |
CN116178545A (zh) * | 2021-09-24 | 2023-05-30 | 广东菲鹏制药股份有限公司 | 一种抗人pd-l1人源化抗体或其抗原结合片段及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN116854820A (zh) | 2023-10-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210009688A1 (en) | Anti-pd-1 antibody and use thereof | |
CN110950953B (zh) | 抗b7-h3的单克隆抗体及其在细胞治疗中的应用 | |
JP7361610B2 (ja) | 抗pd-l1抗体及びその使用 | |
WO2017201766A1 (zh) | 抗人pd-1人源化单克隆抗体及其应用 | |
WO2017197667A1 (zh) | 抗人pd-l1人源化单克隆抗体及其应用 | |
JP2022088411A (ja) | Pd-1アゴニスト抗体およびその使用 | |
US20200325236A1 (en) | Agonistic 4-1bb monoclonal antibody | |
KR20180037949A (ko) | Cd70 및 cd3에 대한 항체 구조체 | |
US11639388B2 (en) | CD3 antigen binding fragment and application thereof | |
KR20190065183A (ko) | Pd-1 항체 | |
CN111344304B (zh) | 新型抗cd40抗体及其用途 | |
TW201922784A (zh) | 4﹘1bb抗體及其製備方法和應用 | |
CN112500485B (zh) | 一种抗b7-h3抗体及其应用 | |
US10774144B2 (en) | Human programmed cell death 1 receptor antibody, method of preparing same, and use thereof | |
TW201938586A (zh) | 雙特異性抗體產品之連續製造製程 | |
CN113151186B (zh) | 抗人cd271的单克隆抗体及用途 | |
RU2261723C2 (ru) | Молчащие анти-cd28-антитела и их применение | |
JP7078237B1 (ja) | 抗tspan8-抗cd3二重特異性抗体及び抗tspan8抗体 | |
AU2002226086A1 (en) | Silensed anti-CD28 antibodies and use thereof | |
WO2023109976A2 (zh) | Ox40抗体及其医药用途 | |
CN116854820B (zh) | Pd-1非阻断性清除抗体及其用途 | |
CN113754770A (zh) | 一种特异性结合人ctla4的抗体及包含其的药物和试剂盒 | |
CN115785269B (zh) | 抗pd-l1的抗体及其应用 | |
CN115947855B (zh) | 抗cd24抗体的制备及其用途 | |
CN114213538B (zh) | Cd44抗体、嵌合抗原受体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |