WO2024012513A1 - Anticorps, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée - Google Patents
Anticorps, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée Download PDFInfo
- Publication number
- WO2024012513A1 WO2024012513A1 PCT/CN2023/107132 CN2023107132W WO2024012513A1 WO 2024012513 A1 WO2024012513 A1 WO 2024012513A1 CN 2023107132 W CN2023107132 W CN 2023107132W WO 2024012513 A1 WO2024012513 A1 WO 2024012513A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- antibody
- variable region
- chain variable
- light chain
- Prior art date
Links
- 230000027455 binding Effects 0.000 title claims abstract description 117
- 239000012634 fragment Substances 0.000 title claims abstract description 86
- 239000000427 antigen Substances 0.000 title claims abstract description 84
- 108091007433 antigens Proteins 0.000 title claims abstract description 84
- 102000036639 antigens Human genes 0.000 title claims abstract description 84
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 7
- 201000011510 cancer Diseases 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 81
- 238000000034 method Methods 0.000 claims description 27
- 239000013598 vector Substances 0.000 claims description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 17
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 16
- 150000007523 nucleic acids Chemical class 0.000 claims description 15
- 108020004707 nucleic acids Proteins 0.000 claims description 14
- 102000039446 nucleic acids Human genes 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 12
- 230000004913 activation Effects 0.000 claims description 10
- 241000282567 Macaca fascicularis Species 0.000 claims description 8
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 208000035475 disorder Diseases 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 5
- 241000282560 Macaca mulatta Species 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 208000023275 Autoimmune disease Diseases 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000001404 mediated effect Effects 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 2
- 241001529936 Murinae Species 0.000 claims description 2
- 230000001363 autoimmune Effects 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 238000003306 harvesting Methods 0.000 claims description 2
- 208000027866 inflammatory disease Diseases 0.000 claims description 2
- 210000004962 mammalian cell Anatomy 0.000 claims description 2
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 230000006044 T cell activation Effects 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 13
- 238000011282 treatment Methods 0.000 abstract description 13
- 208000035473 Communicable disease Diseases 0.000 abstract description 2
- 208000026278 immune system disease Diseases 0.000 abstract 1
- 230000004936 stimulating effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 20
- 210000004408 hybridoma Anatomy 0.000 description 19
- 230000035772 mutation Effects 0.000 description 16
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 12
- 238000003556 assay Methods 0.000 description 12
- 238000000684 flow cytometry Methods 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 10
- 238000001994 activation Methods 0.000 description 9
- 210000004602 germ cell Anatomy 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 241000894007 species Species 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 7
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 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 6
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 6
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 6
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 102000018358 immunoglobulin Human genes 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 241000283707 Capra Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 238000010494 dissociation reaction Methods 0.000 description 5
- 230000005593 dissociations Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 241000288906 Primates Species 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 210000004986 primary T-cell Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 101000642536 Apis mellifera Venom serine protease 34 Proteins 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 229940127174 UCHT1 Drugs 0.000 description 3
- 230000009830 antibody antigen interaction Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 241000282579 Pan Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000000833 heterodimer Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 238000007702 DNA assembly Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101100438932 Homo sapiens CD3D gene Proteins 0.000 description 1
- 101100438942 Homo sapiens CD3E gene Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 102220492414 Ribulose-phosphate 3-epimerase_H35A_mutation Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004970 cd4 cell Anatomy 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000007481 next generation sequencing Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 102220313740 rs1553127237 Human genes 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 238000013391 scatchard analysis Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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
- C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to novel antibodies or antigen-binding fragments that are specific for human CD3 expressed on T cell.
- the body's immune system serves as a defense against infection, injury and cancer.
- the humoral system is mediated by soluble factors, named antibodies, which neutralize products recognized as being foreign by the body.
- the cellular system involves cells, such as T cells and macrophages, which remove and neutralize foreign invaders.
- CD3 Cluster of differentiation 3
- TCR T cell receptor complex
- Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules.
- Anti-CD3 antibodies have been proposed for therapies involving the activation of T cells.
- Anti-CD3 antibodies have been used for the treatment of proliferative disorders such as cancer and for the treatment of autoimmune diseases.
- OKT3 reacts with the chimpanzees CD3, but not with CD3 homologs of other primates such as rhesus monkeys.
- the species specificity of CD3 monoclonal antibody is a significant obstacle to its development as an antibody drug for the treatment of human diseases. Any new drug candidate must undergo rigorous preclinical verification before it can be used in human patients for clinical trials.
- the pre-clinical safety test is to administer the candidate drug to related species, preferably non-human primates.
- chimpanzees are considered endangered species, and the use of such animals for drug safety testing is highly restricted.
- the species described in the art that are suitable for safety evaluation testing may be rhesus monkeys, particularly cynomolgus monkeys.
- CD3 antibodies that lack primate species-specific cross-reactivity are difficult to provide effective preclinical safety assessment data.
- SP34 is one of the very few antibodies that can bind to a variety of primate CD3 (such as human and cynomolgus CD3) .
- SP-34 recognizes an epitope present on solely the ⁇ chain of CD3 while the antibody UCHT1 recognize epitopes formed by ⁇ and ⁇ chains. In vivo, the ⁇ chain can also form a CD3 ⁇ / ⁇ heterodimer.
- Several antibodies directed against human CD3 ⁇ / ⁇ have been developed in the past, but the success of these attempts so far has been limited.
- novel CD3 binding molecules in particular novel anti-CD3 antibodies, which exhibit the desired affinity and potency profile, but which additionally are cross-reactive with other species, in particular with non-human primates such as cynomolgus monkeys, both in vitro and in a cellular context.
- An object of the present invention is to provide a novel antibody or an antigen-binding fragment of the antibody (hereinafter, also referred to as an antibody, etc. ) which binds to human CD3 and to cynomolgus monkey CD3, a molecule comprising the antibody, and a pharmaceutical composition having cytotoxic activity, etc. which includes the antibody, etc. or the molecule as an active ingredient.
- an antibody also referred to as an antibody, etc.
- the present inventors have conducted extensive research to achieve this object and have realized the present invention by developing a novel anti-CD3 antibody and a molecule comprising this antibody.
- the present invention encompasses the following aspects:
- the present disclosure provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising one or more the CDR region sequences selected from the following or a mutant sequence thereof:
- An antibody heavy chain variable region comprising one or more HCDR as shown in the sequences selected from SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05 and SEQ ID NO: 06; and an antibody light chain variable region comprising one or more LCDR as shown in the sequences selected from SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09 (X 1 QX 2 TX 3 FPYT) , SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; wherein X1 is M, Q, T or A, X2 is L or A, X3 is H or Q.
- the antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and the antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 as shown in SEQ ID NO: 01 or SEQ ID NO: 04; b) HCDR2 as shown in SEQ ID NO: 02 or SEQ ID NO: 05; c) HCDR3 as shown in SEQ ID NO: 03 or SEQ ID NO: 06; d) LCDR1 as shown in SEQ ID NO: 07 or SEQ ID NO: 10; e) LCDR2 as shown in SEQ ID NO: 08 or SEQ ID NO: 11; f) LCDR3 as shown in SEQ ID NO: 09 or SEQ ID NO: 12.
- an anti-CD3 antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 02, and HCDR3 as shown in SEQ ID NO: 03, respectively; or HCDR1 as shown in SEQ ID NO: 04, HCDR2 as shown in SEQ ID NO: 05, and HCDR3 as shown in SEQ ID NO: 06 respectively.
- an anti-CD3 antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO: 07, LCDR2 as shown in SEQ ID NO: 08, and LCDR3 as shown in SEQ ID NO: 09, respectively; or LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO: 12, respectively.
- the anti-CD3 antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 02 and SEQ ID NO: 03, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 07, SEQ ID NO: 08 and SEQ ID NO: 09, respectively; or b) a heavy chain variable region sequence comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 05 and SEQ ID NO: 06, respectively; and a light chain variable region sequence comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
- the anti-CD3 antibody or antigen-binding fragment is selected from murine antibody, chimeric antibody, humanized antibody, human antibody or the antigen-binding fragment thereof.
- the anti-CD3 antibody or antigen-binding fragment comprises: a) the heavy chain variable region as shown in SEQ ID NO: 13, or having at least 80%, 85%, 90%, 95% or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 18, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; or b) the heavy chain variable region as shown in SEQ ID NO: 17, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith; and/or the light chain variable region as shown in SEQ ID NO: 31, or having at least 80%, 85%, 90%, 95%or 99%sequence identity therewith.
- the anti-CD3 antibody or antigen-binding fragment comprises: a) the heavy chain variable region having the sequence selected from any one of SEQ ID NO: 13, 14, 15 and 16; and/or the light chain variable region having the sequence selected from any one of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30; or b) the heavy chain variable region as shown in SEQ ID NO: 17; and/or the light chain variable region as shown in SEQ ID NO: 31.
- the anti-CD3 antibody or antigen-binding fragment comprises: a) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 18; or b) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 31; or c) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 19; or d) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 20; or e) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 21; or f) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 22; or g) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ ID NO: 23; or h) the heavy chain variable region of SEQ ID NO: 13 and the light chain variable region of SEQ
- the anti-CD3 antibody or antigen-binding fragment comprises: a) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 38; or b) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 51; or c) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 39; or d) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 40; or e) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 41; or f) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 42; or g) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 43; or h) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 44; or i) the heavy chain of SEQ ID NO: 34 and the light chain of SEQ ID NO: 45; or
- the anti-CD3 antibody is a full-length antibody, further comprising human antibody constant regions; preferably, the heavy chain constant region of the human antibody constant regions is selected from constant regions of human IgG1, IgG2, IgG3 and IgG4 and conventional variants thereof, and the light chain constant region of the human antibody constant regions is selected from ⁇ and ⁇ chain constant regions of human antibody and conventional variants thereof; more preferably the full-length antibody comprises a human antibody heavy chain constant region of SEQ ID NO: 32 and a human light chain constant region of SEQ ID NO: 33.
- the CD3 antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, variable fragment (Fv) , single chain variable fragment (scFv) , dimerized domain V (diabody) , disulfide stabilized Fv (dsFv) , and any CDR-containing peptides.
- the anti-CD3 antibody or antigen-binding fragment binds to human CD3 with an affinity of a K D value of 1 ⁇ 10 -5 M to 1 ⁇ 10 -12 M.
- the present disclosure provides an isolated antibody or the antigen-binding fragment thereof that competing with the above-mentioned antibody or the antigen-binding fragment to bind to human CD3.
- the above-mentioned antibody or the antigen-binding fragment thereof having at least one of the following characteristics: a) binding to human CD3 with an affinity of a K D value of 1 ⁇ 10 -7 M to 1 ⁇ 10 -12 M; b) cross-reacting with CD3 of cynomolgus monkey or rhesus monkey; c) increased activation of T cells.
- the present disclosure provides an isolated nucleic acid molecule encoding any antibody or the antigen-binding fragment.
- the present disclosure also provides a recombinant vector comprising the above-mentioned isolated nucleic acid molecule.
- the present disclosure also provides a host cell transformed with the above-mentioned recombinant vector, wherein the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
- the present disclosure also provides a method for producing the above-mentioned antibody or the antigen-binding fragment in a medium to produce and accumulate the antibody or the antigen-binding fragment thereof, and harvesting the antibody or the antigen-binding fragment thereof from the culture.
- the present disclosure also provides a method for immunologically detecting or measuring CD3, wherein the method comprises detecting the CD3 by contacting with the above-mentioned antibody or the antigen-binding fragment.
- the present disclosure also provides a method for diagnosing a disease related to a human CD3 positive cell, wherein the method comprises a detecting or measuring the CD3 or CD3 positive cell by contacting with the above-mentioned antibody or the antigen-binding fragment.
- the present disclosure provides a pharmaceutical composition, which comprises a therapeutically effective amount of the above-mentioned antibody or the antigen-binding fragment, and one or more pharmaceutically acceptable carriers, diluents or excipients.
- the present disclosure also provides a method of treating a CD3-mediated disease or disorder in a subject in need thereof, comprising administering to the subject the above-mentioned antibody or the antigen-binding fragment, or the above-mentioned pharmaceutical composition, wherein the disease or disorder is selected from the group consisting of cancer, autoimmune and inflammatory diseases
- the invention obtained fully human antibody variable region using transgenic mice with human antibody variable genes, and the obtained antibodies have a series of desired characteristics:
- variable region sequences are different from the existing antibody
- the obtained antibodies have capacity of binding to human CD3
- the invention has obtained antibodies with different sequences, which can specifically bind to CD3, including to cross-reactive with other species, in particular with non-human primates such as cynomolgus monkeys.
- FIG. 1 Mice immunized using RIMMS protocol were tail-bleeded, and serum titers were measured against Jurkat E6.1 cells by flow cytometry (BD Fortessa X20) at 1/50 dilution. The cell binding is shown as histogram of normalized count across different fluorescence intensity. UCHT1 was used as positive control. Serums from unimmunized mice were used as negative control.
- Figure 2 Representative binding histograms from flow cytometry analysis of 13A1 hybridoma clone.
- the solid line represents the binding to Jurkat E6.1 cell and the dash line represents the binding to J. RT-T3.5. cell.
- F2B was used as positive control for CD3 binding and the secondary antibody-only was used as negative control.
- FIG. 4 Graphs showing the T cell activation activity of selected hybridoma clone supernatants using NFAT reporter assay.
- F2B and OKT3 are anti-CD3 mAbs that were used as positive controls.
- FIG. 8 Graphs showing the T cell activation activities of indicated recombinant antibodies using NFAT reporter assay. 13A1 VH1-VL1 and 13A1 VH1-VL2 activities were shown in panel A. Activities of 13A1 VH1-VL1 and its light chain mutants or heavy chain mutants were shown in (B) , (C) and (D) .
- Figure 9 Human PBMC activation by the anti-CD3 antibodies as measured by cell surface CD69 amount measured by flow cytometry analysis.
- FIG. 10 T cell activation upon binding of 13A1 VH1-VL1 and its mutation, as measured by up-regulation of the activation marker CD25 on CD4 (A) or CD8 T cells (C) , respectively the activation marker CD69 on either CD4 (B) or CD8 T cells (D) .
- CD3 and CD3 antigen are used interchangeably herein, and include any variants, isoforms and species homologs of human CD3 which are naturally expressed by cells or are expressed on cells transfected with the CD3 gene.
- an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
- Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
- Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
- the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
- CDR complementarity determining regions
- Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
- the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
- antigen-binding fragment of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD3) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
- binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) , and (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
- a Fab fragment a monovalent fragment consisting of the VL, VH, CL and
- the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al. (1988) Science 242 : 423-426 ; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85 : 5879-5883) .
- Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
- human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
- the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
- the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
- recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below) , (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
- Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
- such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
- CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
- One of the most commonly used definitions for the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.
- the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of the light chain variable domain (LCDR1, LCDR2, LCDR3 or L1, L2, L3) , as well as CDR1, CDR2 and CDR3 of heavy chain variable domain (HCDR1, HCDR2, HCDR3 or H1, H2, H3) .
- Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
- Exemplary conventions that can be used to identify the boundaries of CDRs including, e.g., Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
- Note 1 some of these definitions (particularly for Chothia loops) vary depending on the individual publication examined; Note2: any of the numbering schemes can be used for these CDR defintions, except the contact definition uses the Chothia or Martin (enhanced Chothia) definition; Note 3: the end of the Chothia HCDR1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places the insertions at H35A and H35B.
- Constant modification or “conservative replacement or substitution” refers to substitutions of amino acids in a protein with other amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc. ) , such that the changes can be frequently made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition) ) . In addition, substitutions with structurally or functionally similar amino acids are unlikely to disrupt biological activity.
- nucleic acid molecule refers to a DNA molecule and a RNA molecule.
- the nucleic acid molecule may be single stranded or double stranded but is preferably a double stranded DNA.
- a nucleic acid is “effectively linked” when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
- the preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it comprises the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-length sequence synthesis.
- the base sequence encoding the amino acid sequence of the protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog.
- the homolog of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked.
- the vector is a “plasmid” that refers to a circular double stranded DNA loop into which additional DNA segment can be ligated.
- the vector is a viral vector, wherein an additional DNA segment can be ligated into viral genome.
- the vectors disclosed herein are capable of self-replicating in a host cell into which they have been introduced (for example, a bacterial vector having a bacterial replication origin and a episomal mammalian vector) or can be integrated into the genome of a host cell upon introduction into host cell, thereby is replicated along with the host genome (e.g., a non-episomal mammalian vector) .
- the recombinant expression vector can be obtained by conventional methods in the art, that is, by connecting the nucleic acid molecule of the present invention to various expression vectors, thus being constructed.
- the expression vector is one of a variety of conventional vectors in the art, as long as it can carry the above-mentioned nucleic acid molecule.
- the vector preferably includes: various plasmids, cosmids, phage or virus vectors and the like.
- transfectoma includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.
- sequence of the DNA molecule for the antibody or a fragment thereof according to the present invention can be obtained by conventional techniques, for example, methods such as PCR amplification or genomic library screening.
- sequences encoding light chain and heavy chain can be fused together, to form a single-chain antibody.
- the relevant sequence can be obtained in bulk using a recombination method. This is usually carried out by cloning the sequence into a vector, transforming a cell with the vector, and then separating the relevant sequence from the proliferated host cell by conventional methods.
- a relevant sequence can be synthesized artificially, especially when the fragment is short in length.
- several small fragments are synthesized first, and then are linked together to obtain a fragment with a long sequence.
- DNA sequence encoding the antibody of the present invention (or fragments thereof, or derivatives thereof) completely by chemical synthesis.
- the DNA sequence can then be introduced into a variety of existing DNA molecules (or, for example, vectors) and cells known in the art.
- mutations can also be introduced into the protein sequences of the present invention by chemical synthesis.
- the host cell obtained is cultured.
- the antibody of the present invention is purified by using conventional immunoglobulin purification steps, for example, the conventional separation and purification means well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
- the monoclonal antibody obtained can be identified by conventional means.
- the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) ) .
- the binding affinity of a monoclonal antibody can be determined by, for example, the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980) ) .
- the antibody according to the present invention can be expressed in a cell or on the cell membrane, or is secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to its physical, chemical, and other properties. These methods are well known to those skilled in the art. The examples of these methods comprise, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation) , centrifugation, cell lysis by osmosis, ultrasonic treatment, supercentrifugation, molecular sieve chromatography (gel chromatography) , adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) , and any other liquid chromatography, and the combination thereof.
- protein precipitant such as salt precipitation
- centrifugation such as salt precipitation
- cell lysis by osmosis cell lysis by osmosis
- ultrasonic treatment supercentrifugation
- molecular sieve chromatography gel
- k d (sec -1 )
- k off value the term “k d” (sec -1 ) , as used herein, is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the k off value.
- k a (M -1 x sec -1 ) , as used herein, is intended to refer to the association rate constant of a particular antibody-antigen interaction.
- K D (M) , as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.
- binding refers to the binding of an antibody to an epitope on a predetermined antigen.
- “Pharmaceutical composition" is intended to refer to a mixture containing one or more of the compounds or a physiological/pharmaceutically acceptable salt or prodrug thereof described herein with other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.
- the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and exerts the biological activity.
- administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact with an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.
- administering can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contacting the cell with a reagent, as well as contacting a fluid with a reagent, wherein the fluid is in contact with the cell.
- administering and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell.
- Treatment when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
- the present disclosure includes a medicament for treating a disease associated with CD3, comprising an antibody or an antigen-binding fragment thereof of the present disclosure as an active ingredient.
- the molecules of the present disclosure are very useful for those who suffer a tumor, cancer or infectious disease when in preparations and formulations suitable for therapeutic applications.
- the present disclosure relates to a method for immunologically detecting or measuring CD3, a reagent for immunologically detecting or measuring CD3, a method for immunologically detecting or measuring cells expressing CD3, and a diagnostic reagent for diagnosis of disease related to CD3 positive cells, comprising the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human CD3, as an active ingredient.
- the method for detecting or determining the amount of CD3 may be any known method.
- it includes immunodetection or assay.
- the immunodetection or assay is a method of detecting or determining the amount of antibody or antigen by using labeled antigen or antibody.
- immunodetection or assay include a radioactive substance labeled immunological antibody method (RIA) , an enzyme immunoassay (EIA or ELISA) , a fluorescent immunoassay (FIA) , a luminescent immunoassay, a western blotting method, physicochemical methods, etc.
- the above-mentioned diseases related to CD3 positive cells can be diagnosed by detecting or measuring cells expressing CD3 by using the antibodies or antibody fragments thereof of the present invention.
- a known immunodetection can be used, and preferably immunoprecipitation, fluorescent cell staining or immunohistochemical staining etc. can be used. Furthermore, a fluorescent antibody staining method etc. using FMAT8100HTS system (Applied Biosystem) can be used.
- the room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30°C.
- CD3 antigen A combination of Jurkat E6.1, DNA and recombinant protein antigens were used to immunize mice.
- the recombinant ectodomain of human CD3E and CD3D were expressed and purified as heterodimer and were commercially available at AcroBiosystems (catalog numbers CDD-H52Wa, CDD-H52W1, CDD-C52W4, CDD-C52W9) .
- Either Fc, His Tag&Fc, Flag Tag or His Tag&Tag Free were used.
- Anti-CD3 antibodies were obtained by immunizing genetically modified mouse encoding human immunoglobulin heavy and kappa light chain variable regions with a combination of human CD3 antigen, cynomolgus CD3 antigen, human CD3 DNA, and/or Jurkat E6.1 cells (Sigma-Aldrich, Catalog No. 88042803) which naturally express human CD3. The antibody immune response was monitored by a CD3-specific immunoassay.
- splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for CD3 specificity, or (2) B-cell sorted using a human CD3 with a C-terminal 6-His tag as the sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells) .
- Figure 1 shows the results of the antibody titer in serum detected by Flow cytometry after immunization with CD3 protein.
- mice after 3 times of immunization with the immunogens, the selected mice had an antigen-selective cell binding titer of more than 1: 50, indicating that mice had a better humoral immune response to the immunogen, and their spleen cells can be used for Hybridoma cell preparation.
- the spleen lymphocytes and myeloma cells Sp2/0 were fused to obtain hybridoma cells by electrofusion or PEG fusion.
- PEG fusion was performed using Clonacell TM HY technology (STEMCELL technologies) , following manufacturer’s instructions.
- the primary cell: mouse myeloma cell line ratio was 1: 1 for electrofusion, and 10: 1 for PEG fusion.
- ELISA was performed using the DuoSet ELISA Ancillary Kit (R&D System, DY008) . ELISA plates were coated with 5ug/ml of human CD3DE (AcroBio CDD-H52W1) and cyno CD3DE (AcroBio, CDD-C52W4) overnight. Excess unbound proteins were washed off by washing the plates three times with the wash buffer before blocking for 1 hour at room temperature. 50ul CD3 hybridoma supernatant was added in duplicate wells and incubated for 1 hour.
- Hybridoma supernatants was subjected to binding tests on human T cell lines (CD3 + Jurkat cell line clone E6.1 and CD3 - clone J. RT-T3.5 (ATCC) using flow cytometry analysis.
- the primary staining was performed using undiluted hybridoma supernatants, followed by fluorescence labeled secondary antibody (PE Goat anti-mouse IgG, Biolegend Cat#405307) .
- Figure 2 shows examples of selected flow cytometry histograms. The clone 13A1 was identified with enhanced binding profile to Jurkat E6.1 compared to J. RT-T3.5 cells.
- T cell activation activity of hybridoma supernatants or recombinant anti-CD3 antibodies were measured using NFAT reporter assay, using Jurkat-Lucia TM NFAT cells (InvivoGen jktl-nfat) .
- U-bottom 96-well plates were dry coated with capturing antibody (goat anti-mouse IgG Fc ⁇ fragment specific, Jackson ImmunoResearch 115-005-071) , at 4 ⁇ g/ml in PBS and 50 ⁇ l/well.
- Hybridoma supernatants or recombinant anti-CD3 antibodies diluted in culture medium were added to the pre-coated plate at 50 ⁇ l/well for overnight.
- the Jurkat-Lucia TM NFAT cells were added to each well at 400,000 cells per well in 200ul culture medium. The cells were maintained at 37°C with 5%CO 2 for 24 hours, then the culture supernatants were collected. Luciferase activity in culture supernatant was measured using QUANTI-Luc assay solution (InvivoGen) according to manufacturer’s instruction. Luminescence signal was recorded by VICTOR Nivo Multimode Microplate Reader (PerkinElmer) . Purified anti-CD3 antibodies F2B and OKT3 were used as positive controls. Supernatant from another hybridoma clone was used as a negative control. Clone 13A1 was selected for their ability to active human T cells indicated by the in vitro reporter assay as shown in Figure 4.
- the process of cloning sequences from positive hybridomas are as follows.
- the logarithmic growth phase hybridoma cells were collected, RNA was extracted, and reverse transcription was performed then followed by VDJ region amplification.
- Amplified cDNA library from each clones were subjected to next-generation sequencing.
- the amino acid sequences of the heavy and light chain variable region DNA sequences corresponding to the antibodies 13A1 were obtained (the amino acid residues of the CDRs in VH/VL are underlined according to Kabat & Wu annotation:
- VH and VL regions of selected clones were directly synthesized as DNA fragments with 5’-end in-frame leader sequence (MGWSCIILFLVATATGVHS) . These DNA fragments were cloned into selected vectors using NEBuilder DNA Assembly Cloning Kit (New England Biolabs) .
- VH region was cloned into pFUSE-CHIg_hG1 vector (InvivoGen #pfuse-hchg1) , which in-frame with constant region of hIgG1 heavy chain in the vector.
- VL region was cloned into pFUSE2-CLIg_hk vector (InvivoGen, #pfuse2-hclk) , which in-frame with constant region of hIg kappa light chain in the vector.
- the heavy chain expression plasmid and light chain plasmids were co-transfected into Expi293F cells (ThermoFisher, #A14527) using ExpiFectamine 293 Transfection Kit (ThermoFisher, A14524) , or into ExpiCHO-Scells (ThermoFisher #A29127) using ExpiFectamine CHO Transfection Kit (ThermoFisher, A29129) . Based on the manufacturer’s instructions, plasmid DNA concentration reached 1.0 ug per ml of suspended cells, with LC: HC vector ratio 1: 1. The transfected cells were cultured 5 to 7 days on an orbital shaker at 37 C, 8%CO 2 .
- the purpose of the mutation is to obtain a group of anti-CD3 antibodies with varied affinities and potentially lower immunogenicity by converting certain residues to the germline sequence.
- I14T indicates a mutation from I to T at position 14 according to Kabat numbering system.
- WT indicates that the sequence does not contain amino acid mutations.
- 13A1_mut01 indicates that two kinds of mutation (13A1_VL1. A and 13A1_VH1) are present on the 13A1_mut01, and so on.
- TEST EXAMPLE 1 Enzyme-linked immunosorbent assay (ELISA) detection of the binding of antibodies to hCD3 ⁇ and cynoCD3 ⁇
- ELISA plates were coated with 5ug/ml human or cyno CD3 ⁇ -His (AcroBiosystems) overnight. Serial dilutions of recombinant antiCD3 antibodies 13A1 wereperformed, starting at 100nM and added to CD3 coated plates. The excess non-bound antibodies were washed away before addition of the mouse anti-human IgG Fc-HRP (Abcam 99774) . The results were measured by O. D. at 450nm on a microplate reader. Data were analyzed with GraphPad Prism 9 ( Figure 5) . EC50 values were calculated for the binding of the CD3 antibody to human and cyno CD3 ⁇ protein (Table 5) .
- the binding curves to cell surface CD3 were determined for 13A1_VH1_VL1, 13A1_VH1_VL2 ( Figure 6) recombinant antibodies by flow cytometry analysis.
- Jurkat E6.1 cells were aliquoted into 0.3 million/well and 60ul of serially diluted antibodies were added to each well. The staining was performed for 1 hour at 4 degrees in the dark.
- the PE goat anti human IgG Fc Jackson ImmunoResearch was used as the secondary antibody at 1: 200 dilution.
- 13A1_VH1_VL1 binding to primary T cell was determined by using the freshly isolated PBMCs from one healthy donor. 0.3 million PBMCs were aliquoted to each well and stained with anti-human TCR ⁇ / ⁇ -FITC (Biolegend Cat 306706) . 13A1 antibody was serially diluted and 60ul was added to each well as primary staining. The PE goat anti human IgG was used as secondary antibody as Figure 7A.
- TEST EXAMPLE 3 Detection of the effect of CD3 antibody on T cell activation by NFAT reporter assay or human PBMCs activation.
- 96 well U-bottom plates were dry coated with 10 ug/ml capturing antibody (Jackson ImmunoResearch’s AffiniPure goat anti-human IgG, Fc ⁇ specific, #109-005-170) , and then after PBS wash, were wet coated with 10 ug/ml anti-CD3 antibodies.
- NFAT reporter cells InvivoGen #jktl-nfat
- RPMI 1640 RPMI 1640
- Reporter activity was measured using QUANTI-Luc assay (InvivoGen) as manufacture’s instruction.
- PBMCs from single healthy donor were plated in RPMI1640 with GlutaMax 10%FBS medium at a density of 1x10 6 cells/ml and incubated overnight at 37°C.
- Anti-CD3 antibodies OKT3, 13A1 and F2B, as well as control human IgG were serially diluted.
- PBMCs were then added to the antibody solutions at 50,000 cells per well, and final volume of 200 ⁇ l per well. Cells were incubated at 37°C 5%CO2 for 2 days, then stained with anti-human CD69 antibody (Biolegend Catalog No. 310938) and analyzed by BD Fortessa X20. Data were ananlyzed by FlowJo and GraphPad Prism 9 software.
- PBMCs activation by anti-CD3 antibodies indicated as the ⁇ MFI of CD69 over control were shown in Figure 9, EC 50 values were shown in Table 10.
- CD4 + CD25 + represents the percentage of CD25 positive CD4 cells in a given group
- T cell activation was determined as percent of CD4 T cells expressing CD25 (FIG. 10A) or CD69 (FIG. 10B) , respectively CD8 T cells expressing CD25 (FIG. 10C) and CD69 (FIG. 10D) .
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
L'invention concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci, et en particulier de tels anticorps et fragments de liaison à l'antigène qui ont une affinité et une activité de stimulation de l'activation des cellules T. Des utilisations de tels anticorps et fragments de liaison à l'antigène dans le traitement du cancer, de maladies immunitaires et infectieuses et d'autres affections sont présentées.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263388825P | 2022-07-13 | 2022-07-13 | |
US63/388,825 | 2022-07-13 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024012513A1 true WO2024012513A1 (fr) | 2024-01-18 |
Family
ID=89535619
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2023/107132 WO2024012513A1 (fr) | 2022-07-13 | 2023-07-13 | Anticorps, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée |
Country Status (2)
Country | Link |
---|---|
TW (1) | TW202413405A (fr) |
WO (1) | WO2024012513A1 (fr) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014047231A1 (fr) * | 2012-09-21 | 2014-03-27 | Regeneron Pharmaceuticals, Inc. | Anticorps anti-cd3, molécules de liaison à un antigène bispécifiques qui se lient à cd3 et cd20, et leurs utilisations |
WO2014163684A1 (fr) * | 2013-04-03 | 2014-10-09 | Ibc Pharmaceuticals, Inc. | Polytherapie pour induire une reponse immunitaire a une maladie |
WO2017053856A1 (fr) * | 2015-09-23 | 2017-03-30 | Regeneron Pharmaceuticals, Inc. | Anticorps bispécifiques anti-cd3 optimisés et leurs utilisations |
WO2019045856A1 (fr) * | 2017-08-28 | 2019-03-07 | Systimmune, Inc. | Anticorps anti-cd et leurs procédés de fabrication et d'utilisation |
WO2021104371A1 (fr) * | 2019-11-26 | 2021-06-03 | Shanghai Epimab Biotherapeutics Co., Ltd. | Anticorps anti-cd3 et anti-bcma, et protéines de liaison bispécifiques fabriquées à partir de ceux-ci |
-
2023
- 2023-07-13 WO PCT/CN2023/107132 patent/WO2024012513A1/fr unknown
- 2023-07-13 TW TW112126218A patent/TW202413405A/zh unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014047231A1 (fr) * | 2012-09-21 | 2014-03-27 | Regeneron Pharmaceuticals, Inc. | Anticorps anti-cd3, molécules de liaison à un antigène bispécifiques qui se lient à cd3 et cd20, et leurs utilisations |
WO2014163684A1 (fr) * | 2013-04-03 | 2014-10-09 | Ibc Pharmaceuticals, Inc. | Polytherapie pour induire une reponse immunitaire a une maladie |
WO2017053856A1 (fr) * | 2015-09-23 | 2017-03-30 | Regeneron Pharmaceuticals, Inc. | Anticorps bispécifiques anti-cd3 optimisés et leurs utilisations |
WO2019045856A1 (fr) * | 2017-08-28 | 2019-03-07 | Systimmune, Inc. | Anticorps anti-cd et leurs procédés de fabrication et d'utilisation |
WO2021104371A1 (fr) * | 2019-11-26 | 2021-06-03 | Shanghai Epimab Biotherapeutics Co., Ltd. | Anticorps anti-cd3 et anti-bcma, et protéines de liaison bispécifiques fabriquées à partir de ceux-ci |
Non-Patent Citations (1)
Title |
---|
ROOT, A.R. ET AL.: "Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors", MABS, vol. 13, no. 1, 31 December 2021 (2021-12-31), XP055875731, DOI: 10.1080/19420862.2020.1850395 * |
Also Published As
Publication number | Publication date |
---|---|
TW202413405A (zh) | 2024-04-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR102522693B1 (ko) | 세포독성 t-림프구-관련 단백질 4 (ctla-4)에 대한 신규의 단일클론 항체 | |
EP3689909A1 (fr) | Anticorps tigit, fragment de liaison à l'antigène de celui-ci, et son utilisation médicale | |
CN109843927B (zh) | 抗b7-h3抗体、其抗原结合片段及其医药用途 | |
JP2023503180A (ja) | 抗ヒトクローディン18.2抗体及びその適用 | |
KR20210142638A (ko) | Cd3 항원 결합 단편 및 이의 응용 | |
CA3019740A1 (fr) | Administration d'une construction bispecifique se liant a cd33 et cd3 destinee a une utilisation dans un procede de traitement de la leucemie myeloide | |
WO2021098822A1 (fr) | Anticorps bispécifiques | |
WO2023125888A1 (fr) | Anticorps gprc5d et son utilisation | |
JP7538131B2 (ja) | 抗cd79b抗体、その抗原結合フラグメントおよびそれらの医薬用途 | |
WO2022105811A1 (fr) | Anticorps cd19 humanisé et son utilisation | |
CN110461874A (zh) | 抗gitr抗体、其抗原结合片段及其医药用途 | |
EP4403572A1 (fr) | Anticorps anti-cd3 humain et son utilisation | |
CN117616048A (zh) | Cd19抗体及其应用 | |
WO2024012513A1 (fr) | Anticorps, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée | |
CN116761889A (zh) | 抗原结合蛋白及其用途 | |
CN114957468A (zh) | 一种抗Siglec15抗体及其用途 | |
WO2024012434A1 (fr) | Anticorps, fragment de liaison à l'antigène de celui-ci, et leur utilisation pharmaceutique | |
CN109593135A (zh) | 抗人pd-l1单克隆抗体及其应用 | |
CN115947855B (zh) | 抗cd24抗体的制备及其用途 | |
WO2024094151A1 (fr) | Anticorps multi-spécifique et son utilisation médicale | |
WO2024088386A1 (fr) | Anticorps, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique de celui-ci | |
WO2023036215A1 (fr) | Molécule de liaison à un antigène bispécifique et son utilisation | |
WO2024175093A1 (fr) | Anticorps, fragments de liaison à l'antigène et procédés d'utilisation | |
RU2817143C2 (ru) | Антитело против cd79b, его антигенсвязывающий фрагмент и его фармацевтическое применение | |
WO2024131846A1 (fr) | Anticorps, fragment de liaison à l'antigène de celui-ci et utilisation pharmaceutique associée |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23839003 Country of ref document: EP Kind code of ref document: A1 |