WO2021217893A1 - 结合tigit抗原的抗体及其制备方法与应用 - Google Patents

结合tigit抗原的抗体及其制备方法与应用 Download PDF

Info

Publication number
WO2021217893A1
WO2021217893A1 PCT/CN2020/102091 CN2020102091W WO2021217893A1 WO 2021217893 A1 WO2021217893 A1 WO 2021217893A1 CN 2020102091 W CN2020102091 W CN 2020102091W WO 2021217893 A1 WO2021217893 A1 WO 2021217893A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
variable region
tigit
seq
region
Prior art date
Application number
PCT/CN2020/102091
Other languages
English (en)
French (fr)
Inventor
周群敏
孙薇薇
陈蕞
马晓晓
范金玲
胡红群
Original Assignee
广州昂科免疫生物技术有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 广州昂科免疫生物技术有限公司 filed Critical 广州昂科免疫生物技术有限公司
Priority to US17/915,109 priority Critical patent/US11897955B2/en
Priority to EP20933685.8A priority patent/EP4108684A4/en
Publication of WO2021217893A1 publication Critical patent/WO2021217893A1/zh

Links

Images

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
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/56—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of biotechnology-antibody.
  • the present invention relates to an antibody that antagonizes the binding of T-cell immunoglobulin and ITIM domain protein TIGIT (T-cell immunoreceptor with Ig and ITIM domains) to its ligand, and its coding sequence as well as a preparation method and application thereof.
  • TIGIT T-cell immunoreceptor with Ig and ITIM domains
  • T-lymphocyte-mediated cellular immunity plays a key role in the process of identifying, monitoring, attacking and killing tumor cells.
  • T lymphocytes transmit immune efficacy through the combination of co-stimulatory molecules or co-inhibitory molecules with ligands expressed on tumor target cells or antigen presenting cells (APC) .
  • APC antigen presenting cells
  • Inhibitory immune checkpoint molecules which mainly include CTLA-4 (cytotoxic T-lymphocyte antigen-4) and its ligands B7-1 (CD80), B7-2 (CD86); procedural Death receptor PD-1 (programmed death-1) and its ligands PD-L1 and PD-L2; LAG-3 (lymphocyte activation gene-3) and its ligands; TIM-3 (T-cell immunoglobulin domain and mucin domain 3 ) And its ligands; BTLA (B and T lymphocyte attenuator) and its ligands, etc.
  • CTLA-4 cytotoxic T-lymphocyte antigen-4
  • B7-1 CD80
  • B7-2 CD86
  • procedural Death receptor PD-1 programmed death-1 and its ligands PD-L1 and PD-L2
  • LAG-3 lymphocyte activation gene-3
  • TIM-3 T-cell immunoglobulin domain and mucin domain 3
  • BTLA B and T lymphocyte attenuator
  • a number of monoclonal antibody drugs that antagonize and inhibit CTLA-4, or PD-1/PD-L1 have been marketed, and have shown significant efficacy in the treatment of a variety of tumors.
  • Anti-PD-1 monoclonal antibody drugs Nivolumab (trade name Opdivo), Pembrolizumab (trade name Keytruda), and the anti-PD-L1 monoclonal antibody drug Atezolizumab (trade name Tecentriq) approved in 2016 for the treatment of non-small cell lung cancer.
  • TIGIT is a newly discovered immunosuppressive checkpoint molecule with similar potential drug development prospects.
  • TIGIT T cell immunoreceptor with Ig and ITIM domain
  • WUCAM Woodington University cell adhesion molecule
  • Vermi KS Eur J Immunol 2009, 39: 695-703
  • VSTM3 V-set and transmembrane domain-containing protein 3
  • Levin S Eur J Immunol, 2011, 41: 902-915
  • VSIG9 V-set and immunoglobulin domain-containing protein 9
  • the human TIGIT protein has a total length of 244 amino acids, including 141 amino acids in the extracellular region, 23 amino acids in the transmembrane region, and 80 amino acids in the cytoplasmic region.
  • the mouse TIGIT protein has a full length of 241 amino acids, and its amino acid sequence homology with human TIGIT protein is 60%.
  • An important structural feature of the TIGIT protein is the presence of a typical immunoreceptor tyrosine-based inhibition motif (ITIM) in the tail of its intracellular region, which mediates immunosuppressive signals. ITIM also exists in PD-1 domain.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • TIGIT is mainly expressed on T cells (including helper T cells, memory T cells, CD8+ effector T cells) and natural killer (NK) cells, while ligands that recognize and bind TIGIT It is mainly expressed on antigen-presenting cells such as dendritic cells (DC), macrophages, tumor cells and other targeted cells.
  • DC dendritic cells
  • CD155 is also called PVR (poliovirus receptor, poliovirus receptor); CD112 and CD113 are also called PVRL-2 and PVRL-3 (poliovirus receptor-related protein-2, -3), respectively.
  • PVR poliovirus receptor, poliovirus receptor
  • CD112 and CD113 are also called PVRL-2 and PVRL-3 (poliovirus receptor-related protein-2, -3), respectively.
  • the combination of CD155 and TIGIT is stronger than that of CD112 or CD113 and TIGIT (Yu X, Nat Immunol. 2009, 10: 48-57; Stanietsky N, Proc Natl Acad Sci USA. 2009, 106: 17858-63).
  • CD155, CD112 and CD113 are all members of the Nectin family.
  • CD155 or CD112 can also be combined with the costimulatory factor CD226 (DNAM-1), thereby activating or up-regulating the immune function of T cells.
  • CD226 costimulatory factor CD226
  • the balance system formed by the interaction of TIGIT-CD226/CD155-CD112 is similar to the CD28-CTLA4/CD80-CD86 immune network balance system.
  • CD155 is expressed at a low level on a variety of human normal cells, and is highly expressed on a variety of tumor cells. Tumor cells inhibit the immune killing effect of T cells/NK cells by up-regulating the expression of CD155. Therefore, if a monoclonal antibody that antagonizes the binding of TIGIT to its ligand such as CD155 is developed, immunosuppression can be lifted, and the attack and killing effect of T cells/NK cells on tumors can be restored.
  • the anti-TIGIT monoclonal antibody code-named "BMS-986207" by BMS is in phase II clinical research and is used as a single drug or combined with BMS's PD-1 targeting drug Nivolumab (trade name Opdivo) to treat tumors; BMS Submitted and applied for the anti-TIGIT monoclonal antibody patent and authorization (US patent application number: 2016/0176963A1; authorized US patent number: US10189902B2).
  • the anti-TIGIT monoclonal antibody codenamed "MK-7684" by MSD is in the phase I/II clinical research phase, and is used in combination with Pembrolizumab (trade name Keytruda) to treat tumors; MSD submitted an application for the anti-TIGIT monoclonal antibody patent and authorization (U.S. Patent Application No.: 2016/0355589 A1; Authorized U.S. Patent No.: US10618958B2).
  • These new monoclonal antibodies can be used as single drugs or combined with other drugs under development or on the market, such as anti-PD-1 monoclonal antibodies, anti-CTLA-4 monoclonal antibodies, anti-41BB monoclonal antibodies, anti-OX40 monoclonal antibodies, anti-CD38 monoclonal antibodies, Anti-CD47 monoclonal antibody, anti-VISTA monoclonal antibody, anti-BTLA monoclonal antibody, anti-VEGF/VEGFR and other drugs are used in combination to treat a variety of diseases including tumors.
  • One purpose of the present invention is to obtain a new monoclonal antibody that can bind to human TIGIT antigen with high affinity and antagonize blocking TIGIT antigen and its ligands such as CD155 (PVR) or CD122 (PVRL-2).
  • Such antibodies or derivatives thereof can be used as pharmaceutical ingredients, used alone or in combination with other drugs that are already on the market or under research, for the treatment of various diseases including tumors.
  • One of the technical problems to be solved by the present invention is to provide an antibody or its derivatives such as antibody Fab fragments, single chain antibodies and the like that bind to human TIGIT antigen.
  • the antibody or its derivative can bind to human TIGIT antigen with high affinity and antagonize the binding of TIGIT antigen to its ligand such as CD155 (PVR).
  • the second technical problem to be solved by the present invention is to provide DNA molecules or genes encoding the above-mentioned antibodies.
  • the third technical problem to be solved by the present invention is to provide drugs or pharmaceutical compositions containing the above-mentioned antibodies.
  • the fourth technical problem to be solved by the present invention is to provide the application of the above-mentioned antibody or its derivatives in the preparation of drugs for treating tumors.
  • the fifth technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned antibody or its derivative.
  • the present invention adopts the following technical solutions:
  • an antibody or a derivative thereof that binds human TIGIT antigen with high affinity and antagonizes the binding of TIGIT to its ligand CD155 which comprises a first variable region and a second variable region, wherein
  • the first variable region is the variable region of the antibody light chain, and its antigen complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively;
  • the second variable region is an antibody heavy chain variable region, and its antigen complementarity determining regions CDR1, CDR2 and CDR3 are the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • the antibodies include humanized monoclonal antibodies, and the derivatives include antibody Fab fragments, single-chain antibodies, bi-specific antibodies, and the like.
  • the antibody or derivative thereof has one or more of the following characteristics:
  • the light chain variable region sequence is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 17, and the heavy chain variable region sequence is the same as SEQ ID NO:
  • the amino acid sequence shown in 19 is at least 90% identical;
  • Binding affinity for binding to human TIGIT antigen is 10 nM or less than 10 nM (at 10 nM or below 10 nM), and the affinity constant can be determined by Fortebio-Octet, BIACORE or other similar surface plasmon resonance technology (surface plasmon resonance, SPR) measurement;
  • the half-maximal inhibitory concentration (IC50) of the antagonist blocking the binding of TIGIT to its ligand CD155 (PVR) is 1 nM or less than 1 nM (at 1 nM or below 1 nM) (IC50 means that the substance exerts its maximum Concentration at half the inhibitory effect).
  • the first variable region is an antibody light chain variable region, which is the amino acid sequence shown in SEQ ID NO: 17; and the second variable region is an antibody heavy chain variable region , Is the amino acid sequence shown in SEQ ID NO: 19.
  • the first variable region is an antibody light chain variable region, and its amino acid sequence is shown in SEQ ID NO: 8;
  • the second variable region is an antibody heavy chain variable region, Its amino acid sequence is shown in SEQ ID NO: 13.
  • the antibody or its derivative can antagonize and block the binding of TIGIT to its ligand CD155 (PVR) or CD112 (PVRL2).
  • the present invention comprises the antibody light chain variable region and human antibody light chain constant region, and comprises the hinge region and CH1 region of the antibody heavy chain variable region and the human antibody heavy chain constant region, CH 2 area and CH3 area.
  • the human antibody light chain constant region is derived from a human antibody kappa chain or an antibody lamda chain
  • the human antibody heavy chain constant region is derived from human IgG1, IgG2, IgG3 or IgG4 subtypes, of which the preferred For the IgG4 subtype.
  • the first variable region is the variable region of the antibody light chain, and its amino acid sequence Is shown in SEQ ID NO: 17; the second variable region is the antibody heavy chain variable region, and its amino acid sequence is shown in SEQ ID NO: 19), and the nucleotide sequence encoding the antibody light chain variable region is shown in SEQ ID NO: 18 is shown in SEQ ID NO: 20, and the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO: 20.
  • the present invention also provides another DNA molecule or gene encoding the above antibody or its derivative (corresponding to the following antibody or its derivative: the first variable region is the variable region of the antibody light chain, and its amino acid sequence is SEQ ID NO: 8; the second variable region is the antibody heavy chain variable region, and its amino acid sequence is shown in SEQ ID NO: 13), and the nucleotide sequence encoding the antibody light chain variable region is shown in SEQ ID NO :7, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO: 12.
  • the third aspect of the present invention is to provide an expression vector, which contains a DNA molecule or gene encoding the above-mentioned antibody or a derivative thereof and an expression control sequence operatively linked to the DNA molecule or gene.
  • the fourth aspect of the present invention provides a recombinant host cell, which is transformed by the above-mentioned expression vector.
  • the recombinant host cell or its progeny cells express the above-mentioned antibody or its derivative.
  • the antibodies include humanized monoclonal antibodies, and derivatives include antibody Fab fragments, single-chain antibodies, bi-specific antibodies, and the like.
  • the fifth aspect of the present invention is to provide a medicament or pharmaceutical composition, which contains a pharmaceutically effective amount of the above-mentioned antibody or derivative thereof, and a pharmaceutically acceptable carrier.
  • the components of the pharmaceutical composition include other similar antibodies and drugs such as an anti-programmed death-1 (PD-1) antibody or an anti-PDL-1 antibody.
  • the sixth aspect of the present invention is to provide the application of the above-mentioned antibody or its derivatives in the preparation of drugs for treating tumors.
  • the above antibodies can be used as single drugs or in combination with other drugs under development or on the market such as anti-PD-1 antibodies, anti-PDL1 antibodies, anti-CTLA-4 antibodies, anti-41BB antibodies, anti-OX40 antibodies, anti-CD2 antibodies, anti-CD3 antibodies, Anti-CD20 antibody, anti-CD24 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD47 antibody, anti-BTLA antibody, anti-EGFR antibody, anti-Her2 antibody, anti-VISTA antibody, anti-VEGF Antibodies, anti-VEGFR antibodies and other drugs are used in combination to treat a variety of diseases including tumors.
  • the present invention describes the application of the anti-TIGIT mAb 33D2 and anti-PD-1 mAb to inhibit the growth of colon cancer in mice.
  • the seventh aspect of the present invention provides a method for preparing the above-mentioned antibody or derivative thereof, the method comprising:
  • step b) culturing the host cell obtained in step b) under conditions suitable for the expression of the antibody:
  • monoclonal antibody refers to an immunoglobulin obtained from a pure cell line, which has the same structure and chemical properties, and is specific for a single antigenic determinant.
  • Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually with different antibodies directed against different determinants). Each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinantly engineered cell culture and will not be mixed with other immunoglobulins.
  • the modifier "monoclonal” indicates the characteristics of the antibody, which is obtained from a homogeneous antibody population, which should not be interpreted as requiring any special method to produce the antibody.
  • humanized monoclonal antibody refers to the amino acid sequence of the murine monoclonal antibody, except for the retention of complementarity-determining regions (CDR), other sequences (including the framework region sequence in the variable region) ) Replace all or most of the amino acid sequence of adult immunoglobulin to minimize the immunogenicity of murine monoclonal antibodies through genetic engineering.
  • CDR complementarity-determining regions
  • antibody and immunoglobulin are heterotetrameric glycoproteins of about 150,000 daltons with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains ( H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end. Followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in the three fragments in the light chain and heavy chain variable regions that become complementarity determining regions (CDR) or hypervariable regions.
  • CDR complementarity determining region
  • the definition of the amino acid sequence of the complementarity determining region (CDR) can refer to the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91- 3242) or (see Table 1 below):
  • CDR complementarity determining region
  • variable regions The more conserved parts of variable regions are called framework regions (FR).
  • the variable regions of the antibody heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)).
  • the constant regions of antibodies do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity. , CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the antibodies of the present invention can generally be prepared by the following methods:
  • expression control sequence generally refers to a sequence involved in controlling gene expression.
  • the expression control sequence includes a promoter and a termination signal operatively connected to the target gene.
  • the gene (DNA) sequence encoding the antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the protein sequence disclosed in the present invention or amplification by PCR. Thereafter, the DNA fragments obtained by synthesis or PCR amplification can be inserted into a suitable expression vector by methods well known in the art.
  • the expression vector used in the present invention can be a commercially available expression vector known to those skilled in the art, such as the pCDNA3.1 expression vector of Invitrogen company or a self-made vector such as the company's pQY series vector.
  • Suitable host cells for transformation with an expression vector generally include prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
  • eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the preferred host cells are mammalian cells, especially Chinese hamster ovary (CHO) cells such as CHO-S.
  • CHO Chinese hamster ovary
  • the culture supernatant is harvested, and the culture supernatant is then used for affinity with protein-A Chromatography, ion exchange chromatography, filtration sterilization and other conventional separation steps or means well known to those skilled in the art are purified to obtain the antibody of the present invention.
  • the purified antibody of the present invention can be dissolved in an appropriate solvent such as sterile physiological saline liquid, and the solubility can be between 0.01 and 100 mg/ml, and the ideal final solubility can be between 1 and 20 mg/ml.
  • Such antibodies or their derivatives can be used as pharmaceutical ingredients, used alone or in combination with other drugs under development or on the market, such as anti-PD-1 antibodies, anti-PDL1 antibodies, anti-CTLA-4 antibodies, anti-41BB antibodies, and anti-OX40 antibodies , Anti-CD2 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD24 antibody, anti-CD27 antibody, anti-CD28 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD47 antibody, anti-BTLA antibody, anti-EGFR antibody, anti Her2 antibody, anti-VISTA antibody, anti-VEGF antibody, anti-VEGFR antibody and other drugs are used in combination to treat a variety of diseases including tumors.
  • this type of antibody or its derivative is combined with anti-PD-1 monoclonal antibody for administration in TIGIT/PD-1 double-derived mice (human PD-1 and human TIGIT double gene knock-in mice) in vivo testing of its drug activity and its results (see Example 10); the test results show that the anti-TIGIT antibody of the present invention, whether administered alone or in combination with anti-PD-1 monoclonal antibodies, Both showed obvious anti-tumor efficacy.
  • the present invention selects recombinant human TIGIT extracellular protein expressed in mammals as immune antigens .
  • the polyclonal antibody secreting anti-TIGIT protein is obtained; then the mice containing the high titer antibody are selected from the mice, and the spleen cells of the mice are fused with mouse myeloma cells in vitro.
  • the present invention obtains the gene fragments encoding the variable region of the heavy chain and the variable region of the light chain of the murine antibody by means of genetic engineering and the like, and on this basis, the antibody is modified by human-mouse chimerism and humanized genetic engineering.
  • the gene encoding human-mouse chimeric antibody (c33D2) or humanized antibody (h33D2) is transfected into Chinese hamster ovary (CHO) cells to obtain recombinant engineered cells that stably secrete and express the human-mouse chimeric or humanized antibody , And separated and purified from the recombinant engineered cell culture fluid to obtain human-mouse chimeric and humanized antibodies with antagonistic TIGIT biological activity.
  • the anti-TIGIT antibody as a single drug or in combination with other drugs under development or on the market, such as anti-PD-1 antibody, anti-PDL1 antibody, anti-CTLA-4 antibody, anti-41BB antibody, anti-OX40 antibody, anti-CD2 antibody, anti-CD3 Antibodies, anti-CD20 antibodies, anti-CD24 antibodies, anti-CD27 antibodies, anti-CD28 antibodies, anti-CD33 antibodies, anti-CD38 antibodies, anti-CD40 antibodies, anti-CD47 antibodies, anti-BTLA antibodies, anti-EGFR antibodies, anti-Her2 antibodies, anti-VISTA antibodies, Anti-VEGF antibody, anti-VEGFR antibody and other drugs are used in combination to treat a variety of diseases including tumors.
  • Figure 1 is a schematic diagram of the comparison and analysis of the amino acid sequence of human TIGIT protein and mouse TIGIT protein in Example 1 of the present invention.
  • Figure 2 shows the 96-well plate coated with recombinant human TIGIT extracellular protein in Example 1 of the present invention.
  • Fig. 3A is a schematic diagram of the results of a 96-well plate coated with recombinant human TIGIT extracellular protein in Example 1 of the present invention, and the result of screening and detecting multiple strains of mouse hybridoma culture supernatant samples by indirect ELISA method, wherein the negative control
  • the sample is an unfused SP2/0 myeloma cell culture supernatant sample.
  • Figure 3B is a schematic diagram of the results of the indirect ELISA method in Example 1 of the present invention to detect the binding of the culture supernatant sample of the hybridoma cell line subcloned with the recombinant human TIGIT protein coated on the 96-well plate; wherein the positive control sample is after TIGIT immunization Of mouse serum.
  • Fig. 4 is an SDS-PAGE analysis result chart of the purified mouse 33D2 antibody protein sample after DTT-denatured reduction in Example 1 of the present invention.
  • Fig. 5 is a schematic diagram showing the results of detecting the binding of purified mouse monoclonal antibody to recombinant human TIGIT protein or other immune-related recombinant proteins coated on a 96-well plate by the indirect ELISA method in Example 2 of the present invention. .
  • Figure 6 is a schematic diagram of the binding results of the purified mouse antibody sample detected and analyzed by flow cytometry and the CHO cells (CHO/TIGIT) stably transfected with the human TIGIT gene in Example 3 of the present invention.
  • Figure 7 is a flow cytometer detection and analysis of purified mouse antibody samples and derived from wild-type C57B/L mice (wild type mice) or humanized (human TIGIT gene knock-in mice) in Example 4 of the present invention. Schematic diagram of the results of peripheral blood lymphocyte (PBL) binding in mice.
  • PBL peripheral blood lymphocyte
  • Figure 8 is a schematic diagram of the results of in vitro detection of monoclonal antibody samples antagonizing and blocking the binding of CD155 and human TIGIT protein coated on 96-well plates by competitive ELISA in Example 5 of the present invention.
  • Figure 9 shows the binding of CHO-S cell culture supernatant sample transiently transfected with recombinant humanized h33D2 antibody gene expression vector to recombinant human TIGIT protein coated on 96-well plate by ELISA in Example 9 of the present invention Schematic diagram of the results.
  • Fig. 10 is a graph showing the results of analyzing the binding and dissociation dynamics of 33D2 monoclonal antibody and TIGIT antigen with a protein interaction instrument in Example 10 of the present invention.
  • Figure 11A shows the growth trend of tumor volume in each individual animal in the normal saline negative treatment control group (group A) in the TIGIT/PD-1 double-derived mouse MC38 colon cancer model subcutaneously implanted in the TIGIT/PD-1 double-derived mouse in Example 11 of the present invention.
  • Fig. 11B shows the growth trend of tumor volume in each individual animal in the PD-1 monoclonal antibody treatment group (group B) in the TIGIT/PD-1 double-derived mouse MC38 colon cancer model subcutaneously implanted in Example 11 of the present invention.
  • Figure 11C shows the growth trend of tumor volume in each individual animal in the TIGIT monoclonal antibody treatment group (group C) in the TIGIT/PD-1 double-derived mouse MC38 colon cancer model subcutaneously implanted in the mouse in Example 11 of the present invention.
  • Figure 11D shows each individual in the TIGIT/PD-1 double-derived mouse MC38 colon cancer model subcutaneously implanted in mice in Example 11 of the present invention, in the combined administration of PD-1 monoclonal antibody and TIGIT monoclonal antibody (group D) The trend of tumor growth in animals.
  • Fig. 12A shows the trend of average growth volume of animal tumors in each experimental group in Example 11 of the present invention.
  • Fig. 12B shows the growth trend of the average body weight of the experimental animals in each group in Example 11 of the present invention.
  • Example 1 Establishment, screening and identification of mouse hybridoma cell lines secreting anti-TIGIT antibodies
  • FIG. 1 The comparison and analysis of the amino acid sequence of human TIGIT protein and that of mouse TIGIT protein are shown in Figure 1:
  • the amino acid sequence marked in italics is the signal peptide that guides extracellular secretion and expression of TIGIT protein, and the box is underlined
  • the amino acid sequence marked in bold is the transmembrane domain of the TIGIT protein.
  • Figure 1 The amino acid sequence homology between human TIGIT protein and mouse TIGIT protein is only about 60%; among them, in the extracellular region directly involved in the recognition and binding of its ligand (CD155 or CD112), human TIGIT protein There are also more than 30 amino acid differences between the protein and mouse TIGIT. Therefore, it is speculated that if the traditional antigen protein immunization of mice and hybridoma preparation technology is used, it should be possible to prepare mouse anti-human TIGIT monoclonal antibodies.
  • Step 1 Source of recombinant human TIGIT protein (immune antigen) and animal immunity
  • the antigen used for immunization is a recombinant human TIGIT extracellular protein (TIGIT-his, Sino Biologicals) expressed by mammals with a histidine-tag at the C-terminus. , Product number 10917-H08H).
  • TIGIT-his human TIGIT extracellular protein
  • Freund's complete adjuvant product of Sigma, USA
  • Balb/c mice 100 ⁇ l/mouse, 10 ⁇ g TIGIT-his protein each time
  • Two to three weeks after the first immunization the mice were given subcutaneous multiple injections of a mixture of human TIGIT-His protein and Freund's incomplete adjuvant (product of Sigma in the United States).
  • mice were taken Serum, use a 96-plate coated with recombinant human TIGIT-his protein to detect the titer of anti-TIGIT antibody in mouse serum by ELISA (see Figure 2), and take the spleen cells of the mouse with the higher titer for the next step Cell fusion.
  • Step 2 Cell fusion
  • the mouse spleen cell suspension was aseptically prepared, and the mouse SP2/0 myeloma cells (purchased from the Cell Collection Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) were aseptically prepared at a ratio of 5:1 or 10:1
  • the proportion is fused under the action of 50% PEG-1000 (product of Sigma, USA).
  • the fusion was carried out according to the conventional method (Kohler G. and Milstein C: Nature1975; 256:495-497), the dosage of PEG was 1ml, and the addition was completed slowly within 60 seconds.
  • RPMI 1640-10% FCS medium adjust the cell concentration to 1 ⁇ 10 6 /ml, add it to a 96-well flat-bottom cell culture plate (200 ⁇ l per well), at 37°C, 5% Culture in a CO2 incubator (product of Thermo Company, USA) for 2-3 weeks.
  • Step 3 Enzyme-linked immunosorbent assay (ELISA) screens mouse hybridoma cells with positive antibody secretion
  • the 96-well microtiter plate was coated with recombinant human TIGIT-his protein (2 ⁇ g/ml, pH 9.6, 0.1M NaHCO3 solution), after coating for 2 hours at 37°C, then 2% bovine serum albumin (BSA) was added Block overnight at 4°C.
  • human TIGIT-his protein 2 ⁇ g/ml, pH 9.6, 0.1M NaHCO3 solution
  • BSA bovine serum albumin
  • the culture supernatant of hybridoma cells to be tested was added (using unfused SP2/0 myeloma fine culture supernatant as a negative control) and incubated at 37°C for 2 hours; After washing with PBS-0.1% Tween20 solution, add horseradish peroxidase HRP-labeled goat anti-mouse IgG (product of Sigma, USA), incubate at 37°C for 1 hour; then fully wash with PBS-0.1% Tween20 solution , Add o-phenylenediamine (OPD)-0.1% H 2 O 2 substrate solution to develop color for 10-15 min, then add 0.1M HCl to stop the reaction.
  • HRP-labeled goat anti-mouse IgG product of Sigma, USA
  • the OD value at 492 nm was read in the MK3-Multiskan microplate reader (product of Thermo Scientific, USA).
  • the hybridoma cells with the measured OD 492 value 5-10 times higher than the negative control value were recloned and expanded and cryopreserved.
  • Figure 3A shows the retest results of the 11 strains of mouse hybridoma cell culture supernatants with positive ELISA screening results.
  • Step 4 Subcloning of positive hybridoma cells-limiting dilution method
  • Figure 3B shows the representative results of the supernatant of subclonal cell lines detected by ELISA: the supernatant samples of each subclonal of hybridoma No. 33 with code names 33-D2, 33-F3, 33-F6 and 33-F7 And the hybridoma subclone, codenamed 188-2-D7, both contain high titers of anti-TIGIT antibodies.
  • Step 5 Subclones of hybridomas are expanded in serum-free medium and the antibodies in the supernatant are separated and purified
  • the subclone of the hybridoma cell line codenamed 33D2 and the subclone of the hybridoma cell line codenamed 188-2-D7 were replaced with a serum-free medium for expansion and culture, and about 1L of hybridomas were collected respectively.
  • use Protein G Sepharose (product of GE) to purify the antibody protein (refer to the instructions for the method); the purified antibody protein is subjected to SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) analysis.
  • FIG 4 is an SDS-PAGE analysis chart of purified mouse 33D2 antibody protein sample reduced by DTT (DTT-denatured). As shown in Figure 4: The reduced antibody protein sample presents two bands: the upper end is the antibody heavy chain, and the lower end is the antibody light chain.
  • the basic steps of the ELISA method are as follows: Dilute the purified mouse 33D2 monoclonal antibody and 188-2-D7 monoclonal antibody to 1 ⁇ g/mL, and add them to the pre-coated recombinant TIGIT-his protein or other immune-related genes-Fc fusions.
  • Protein including CD28, B7-1, CTLA4, CD3, PD-1, PD1H (PD2), PD-L1, PD-L2 and other 12 antigens, each antigen coating concentration is 2 ⁇ g/mL) 96-well plate After incubating at 37°C for 1 hour and washing, add HRP-labeled goat anti-mouse IgG (product of Jackson, USA) to each well of the 96-well plate, then incubate at 37°C for 1 hour and wash, add OPD substrate to each well for color development .
  • HRP-labeled goat anti-mouse IgG product of Jackson, USA
  • Table 2 shows the representative results (OD at 492nm) of the binding of the purified 33D2 monoclonal antibody and 188-2-D7 monoclonal antibody to TIGIT and other immune-related proteins by the ELISA method:
  • Figure 5 is a bar graph based on the ELISA detection and analysis results (OD at 495nm) in Table 2: The results show that both the 33D2 mAb and the 188-2-D7 mAb only specifically bind to human TIGIT protein and are related to other immune-related proteins For example, PD-1, CD28, B7, CTLA-4, CD3, PD-L1, PD-L2, etc. have no obvious binding.
  • Example 3 Flow-cytometry detection and analysis of the binding of mouse-derived antibodies to CHO cells (CHO/TIGIT) transfected and expressing human TIGIT gene
  • mouse-derived hybridoma cell line 33D2 supernatant was used as the primary antibody, and FITC fluorescently-labeled goat anti-mouse IgG was used as the secondary antibody.
  • Flow cytometry was used to detect and analyze the mouse-derived 33D2 monoclonal antibody sample and its stable expression.
  • CHO cell (CHO/TIGIT) combination of human TIGIT gene was used as the primary antibody, and FITC fluorescently-labeled goat anti-mouse IgG was used as the secondary antibody.
  • Flow cytometry was used to detect and analyze the mouse-derived 33D2 monoclonal antibody sample and its stable expression.
  • CHO/TIGIT cells were respectively immunized with mouse IgG (negative control sample, isotype control), mouse-derived 33D2 supernatant and TIGIT antigen immunized mouse positive serum (positive control sample immu.sera, 1:200 dilution) After incubating for 30 minutes at 4°C and washing with PBS-1% BSA solution, add FITC-labeled goat anti-mouse IgG (product of Sigma); after incubating for 30 minutes at 4°C and washing with PBS-1% BSA solution, the sample Load the sample to Biosciences Accuri C6 flow cytometry (BD Company, USA).
  • Figure 6 is a schematic diagram of the representative results of the flow cytometer. As shown in Figure 6: Compared with the mouse IgG negative control sample (isotype control), the TIGIT immunized mouse positive seropositive sample (immu.sera) and the test sample 33D2 monoclonal antibody supernatant (33D2) can specifically interact with CHO/ TIGIT cell binding; the binding strength of the 33D2 monoclonal antibody sample is similar to that of the positive serum control sample.
  • Example 4 Flow cytometry to detect the binding of mouse-derived antibodies to the peripheral blood lymphocytes of TIGIT humanized mice
  • the peripheral blood of Human TIGIT gene knock-in mice (the TIGIT humanized mouse comes from Biocytogen) was used to isolate lymphocytes, and the mouse-derived 33D2 supernatant was used as the primary antibody.
  • FITC-labeled polyclonal rabbit anti-mouse IgG is the secondary antibody (PE-labeled anti-Mouse CD3e is also added to label T lymphocytes).
  • Flow cytometry is used to detect and analyze the TIGIT protein in 33D2 monoclonal antibody samples and humanized TIGIT mice The combination.
  • the specific method is as follows:
  • Figure 7 is a schematic diagram of the representative results of the flow cytometer. As shown in Figure 7: 33D2 monoclonal antibody samples can specifically bind to human TIGIT gene knock-in mouse lymphocytes, but not to C57/B6 wild-type mouse lymphocytes.
  • One of the methods to detect the biological activity of anti-TIGIT monoclonal antibody in vitro is to use competitive ELISA to detect whether it antagonizes and blocks the binding of TIGIT protein to its receptor such as CD155 (PVR).
  • CD155(PVR)-Fc protein product of ACRO Biosystems
  • 33D2 antibodies of different solubility such as anti-VEGF monoclonal antibody hPV19
  • Table 3 and Figure 8 show the representative results of the competition ELISA method for detecting the monoclonal antibody sample to antagonize and block the binding of the TIGIT protein and its receptor CD155.
  • Fig. 8 is a fitted line graph made with the mapping software (OriginPro 9.0) according to the detection results (OD at 495nm) of the competition ELISA method in Table 3.
  • RNA is first extracted from the mouse 33D2 hybridoma cells, and then the RNA is used as a template, using degenerate primers, and the reverse transcription-polymerase chain reaction (RT-PCR) method (Wang Y et al. :Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA. BMC Bioinformatics. 2006; 7 Suppl(4): S9)
  • RT-PCR reverse transcription-polymerase chain reaction
  • Step 1 Use RNA extraction reagent (RNAiso Plus, a product of Takara) to extract total RNA from mouse m33D2 hybridoma cells;
  • Step 2 Using reverse transcription PCR (RT-PCR) method to obtain cDNA template in eppendorf tube.
  • RT-PCR reverse transcription PCR
  • the sequence of the reverse transcription PCR primer (33-D2-L) for the light chain variable region of the murine 33D2 antibody is: TGT CGT TCA CTG CCA TCA AT (SEQ ID NO.:1)
  • the sequence of the reverse transcription PCR primer (33-D2-H) for the heavy chain variable region of the murine 33D2 antibody is: GCA AGG CTT ACA ACC ACA ATC (SEQ ID NO.: 2);
  • the RT-PCR reaction system is as follows:
  • the reaction was carried out at 42°C for 1 hour, and then the temperature was raised to 70°C. After 15 minutes of inactivation, the obtained cDNA was placed at -20°C and stored for later use.
  • Step 3 PCR cloning and amplification of murine 33D2 antibody light chain variable region and heavy chain variable region genes.
  • a pair of primers used to clone and amplify the 33D2 antibody light chain variable region genes are:
  • Reverse primer CTG AGG CAC CTC CAG ATG TT (SEQ ID NO.: 4)
  • the pair of primers used to clone and amplify the 33-D2 antibody heavy chain variable region gene is:
  • Reverse primer GTG CTG GAG GGG ACA GTC ACT (SEQ ID NO.: 6)
  • the DNA products amplified by PCR were analyzed by electrophoresis in 1.5% agarose gel. After the electrophoresis, the separated DNA bands are cut off and sequenced respectively to obtain the nucleotide sequences of the variable region DNA of the antibody light chain and heavy chain.
  • the measured nucleotide sequence of the murine antibody light chain variable region DNA is shown in SEQ ID NO.: 7, and the amino acid sequence of the antibody light chain variable region inferred from the DNA nucleotide sequence is shown in SEQ ID NO.: 8.
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the complementarity-determining regions (CDR) of the light chain antigen are shown in SEQ ID NO.: 9, SEQ ID NO.: 10 and SEQ ID NO.: 11, respectively.
  • the measured nucleotide sequence of the murine antibody heavy chain variable region DNA is shown in SEQ ID NO.: 12, and the amino acid sequence of the antibody heavy chain variable region inferred from the nucleotide sequence of the DNA is shown in SEQ ID NO. :13.
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain antigen complementarity determining region are shown in SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16, respectively.
  • the murine 33D2 antibody light chain variable region gene and heavy chain variable region gene obtained by cloning and amplification in Example 6 were respectively compared with human-kappa light chain constant region (C-domain) and human IgG4-heavy chain constant region gene.
  • the fragments were fused to obtain human-mouse chimeric light chain gene (c33D2L) and human-mouse chimeric heavy chain gene (c33D2H).
  • the light chain chimeric gene and the heavy chain chimeric gene were cloned into pcDNA3.1 expression plasmids, and transformed into E. coli for amplification, and a large number of expression plasmids containing human-mouse chimeric antibody genes were isolated and obtained.
  • c33D2 human-mouse chimeric antibody
  • PCR and other series of genetic engineering cloning methods are used for the light chain and heavy chain of the chimeric antibody.
  • the antigen complementarity determining region (CDR) gene fragments were respectively transplanted to the corresponding human kappa-light chain and IgG4-heavy chain variable region frameworks (framework regions, FR) to obtain humanized h33-D2 antibodies.
  • the expression product of the germline gene of the first V region of the human immunoglobulin Kappa light chain (IgKV1-9, NCBI Gene ID: 28941) has the highest homology with the 33D2 light chain variable region. Accordingly, the 33D2 light chain framework region (FR) was replaced with the homologous sequence of human IgKV1-9, and then the replaced variable region gene was spliced with the constant region coding sequence of the human immunoglobulin IgG-Kappa light chain, and finally succeeded
  • the humanized light chain coding gene (h33D2-L) was obtained.
  • the amino acid sequence of the variable region of the humanized h33D2 antibody light chain is shown in SEQ ID NO.: 17, and its nucleotide sequence is shown in SEQ ID NO.: 18.
  • the expression product of the germline gene of the third V region of the human immunoglobulin heavy chain (IgHV1-2, NCBI Gene ID: 28474) has the highest homology with the 33D2 antibody heavy chain. Accordingly, the 33D2 heavy chain framework region (FR) was replaced with the homologous sequence of human IgHV1-2, and at the same time, in order to reduce the binding of the humanized antibody to the immunoglobulin-Fc receptor (FcR) in the body and the cell mediation The killing effect of antibody-dependent cellular cytotoxicity (ADCC) on TIGIT-expressing positive immune cells (lymphocytes).
  • ADCC antibody-dependent cellular cytotoxicity
  • the humanized 33D2 antibody heavy chain variable region gene and the constant encoding human immunoglobulin-IgG4 heavy chain are deliberately combined The region sequence is spliced, and the amino acid at position 228 in the hinge region is replaced with serline (S228P) from the original proline.
  • serline serline
  • the full-length h33D2 antibody heavy chain containing the humanized heavy chain coding variable region and human IgG4-heavy chain constant region (S228P) was finally successfully obtained.
  • the amino acid sequence of the variable region of the humanized h33D2 antibody heavy chain is shown in SEQ ID NO.: 19, and its nucleotide sequence is shown in SEQ ID NO.: 20.
  • Example 9 Establishment of a cell engineering strain secreting and expressing humanized 33D2 antibody and separation and purification of antibody protein
  • Example 8 containing the humanized heavy chain gene (h33D2-H) and the humanized light chain gene (h33D2-L) was cloned step by step into the pQY-Hygro expression vector, and then transformed into E. coli and amplified and isolated to obtain the expression human source Recombinant plasmid of the 33D2 antibody.
  • the recombinant plasmids were then transiently transfected into CHO-S cells.
  • TIGIT-his protein as the coating antigen
  • HRP enzyme-labeled Goat-anti-human-IgG as the detection secondary antibody (purchased from Shanghai Xitang Biological Company)
  • OPD OPD
  • Table 6 below shows the representative test results of the ELISA.
  • Figure 9 is a fitting line diagram made with mapping software (OriginPro9.0) based on the ELISA analysis results (OD at 492nm) in Table 6.
  • mapping software OlinPro9.0
  • ELISA analysis results OD at 492nm
  • Table 6 The results show that the humanized h33D2 antibody (IgG4-kappa) can specifically interact with the human TIGIT protein It binds, and its binding activity (or affinity) is similar to that of human-mouse chimeric c33D2 antibody.
  • transfected cells were cloned and screened and cultured in suspension culture in a serum-free medium, a number of engineered CHO cell strains that stably and efficiently secreted and expressed humanized h33D2 antibody protein were successfully obtained.
  • a cell engineering strain was selected and amplified and cultured in a serum-free medium, and the culture supernatant was collected.
  • the supernatant was centrifuged and filtered with a 0.45 ⁇ m filter membrane, and the sample was loaded to include the affinity layer containing Protein A
  • the final product is obtained Humanized antibody (h33D2) with high purity (protein purity over 99%).
  • the purified humanized antibody (h33D2) was dissolved in sterile saline and stored at low temperature (below -20°C).
  • a biosensor protein interaction instrument: Fortebio-Octet RED96, product of PALL was used to monitor and determine the affinity and kinetic curve of the purified humanized 33D2 monoclonal antibody binding to the TIGIT antigen in real time.
  • the detection method and results are as follows:
  • Biosensor chip The surface is coated with anti-Human Fab-CH1 2nd Generation antibody (FAB2G, product of ForteBio, product number 1802083).
  • FAB2G anti-Human Fab-CH1 2nd Generation antibody
  • KB buffer 0.1% BSA 0.05% Tween 20 dissolved in commercial PBS with pH 7.2.
  • Antibody working solution The humanized 33D2 monoclonal antibody sample is prepared with KB buffer to 10 ⁇ g/mL.
  • Antigen working solution Recombinant human TIGIT-His protein (product of Beijing Yiqiao Shenzhou Company, product number 10917-H08H) was prepared into 3 gradients of 100nM, 25nM, and 6.25nM with KB buffer.
  • Figure 10 shows the binding and dissociation dynamic results curve of humanized 33D2 monoclonal antibody and TIGIT antigen; 0s to 400s represent binding, 400s to 1000s represent dissociation, and the three curves from top to bottom represent humanized 33D2 monoclonal antibody and 100nM, 25nM, respectively Binding curve to TIGIT-His at a concentration of 12.5 nM.
  • Table 8 shows the affinity kinetic detection constants: the results show that the humanized 33D2 monoclonal antibody has a higher affinity with TIGIT, and the binding affinity constant is 0.843 nM.
  • Table 8 shows the calculated binding and dissociation values and the affinity values in the Global mode.
  • KD represents the affinity value
  • kon represents the binding value
  • kdis represents the dissociation value
  • R 2 represents the fitting curve and the binding solution. From the correlation coefficient between the curves, the closer the value is to 1, the closer the result of the fitting calculation is to the true result
  • Example 11 TIGIT/PD-1 double gene knock-in mice (human PD-1 and human TIGIT double gene knock-in mice) in vivo testing of the anti-tumor efficacy of anti-TIGIT monoclonal antibodies
  • mouse colon cancer cells expressing the CD155 gene were specifically selected to test in vivo mouse anti-human TIGIT monoclonal antibodies in genetically engineered TIGIT/PD-1 double-derived mice 33D2, as a single drug, or a series of studies on the anti-tumor efficacy of combined administration with mouse anti-human PD-1 monoclonal antibody hAb21 (self-developed product by Stanway).
  • the animal experiment study is divided into two phases.
  • the first phase of the experiment model, administration group and experiment results are described as follows:
  • the animals were given intraperitoneal injection (ip) twice a week from the day of grouping (ie 5-6 days after tumor inoculation) (1 time every 3-4 days) for a total of 4 doses (continuous administration) Two weeks). During this period, the general clinical symptoms of the animals were observed every day, and the long diameter (mm) and short diameter (mm) of the tumor and the animal weight were measured every 3-4 days.
  • test results are summarized in Table 10 below, Tables 11A-11D, Figures 11A-11D, and Figures 12A and 12B.
  • Figures 11A-11D are graphs showing the trend of tumor growth in each individual animal in each administration group.
  • Figure 12A shows the average growth volume trend of tumors in each group of experimental animals.
  • Figure 12B shows the growth trend of the average body weight of the experimental animals in each group.
  • the normal saline negative treatment control group (group A) tumors grow rapidly, and no tumor shrinkage or complete rejection of the test animals is observed (complete rejection CR:0/6) ; While the PD-mab treatment group (group B), the TIGIT monoclonal antibody treatment group (group C), the PD-1 monoclonal antibody and TIGIT monoclonal antibody combined administration group (group D), the tumor grows 5-8 days after administration That is, it is suppressed, and the tumor shrinks or disappears completely.
  • Figure 12A shows the average growth volume trend of each group of experimental animals during the tumor experiment.
  • Figure 12B shows the average weight gain trend of each group of experimental animals during the experiment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

提供一种结合人TIGIT抗原并拮抗抑制TIGIT与其配体如CD155结合的抗体或其衍生体，包括抗体轻链和重链可变区的抗原互补决定区(CDR-L1，CDR-L2和CDR3-L3，CDR-H1，CDR-H2和CDR-H3)氨基酸序列。还公开了该抗体的人源化制备方法及该人源化抗体重链可变区与轻链可变区氨基酸序列。该抗体或其衍生体可作为药物组合物成分或制备成合适药物制剂，单独给药或与其他药物如抗PD-1单抗等或治疗手段合并使用，用于治疗肿瘤等疾病。

Description

结合TIGIT抗原的抗体及其制备方法与应用 技术领域
本发明属于生物技术-抗体领域。本发明涉及一种拮抗抑制T细胞免疫球蛋白和ITIM结构域蛋白TIGIT(T-cell immunoreceptor with Ig and ITIM domains)与其配体结合的抗体及其编码序列与其制备方法和用途。
背景技术
T淋巴细胞介导的细胞免疫在识别监控、与攻击杀灭肿瘤细胞的过程中起着非常关键的作用。T淋巴细胞通过协同刺激因子(co-stimulatory molecules)或协同抑制因子(co-inhibitory molecules)与表达在如肿瘤靶细胞或抗原呈递细胞(Antigen presenting cells，APC)上的配体结合而传导免疫功效。其中协同抑制因子也叫免疫抑制检查点(Inhibitory immune checkpoint molecules)，主要包括CTLA-4(cytotoxic T-lymphocyte antigen-4)与其配体B7-1(CD80)、B7-2(CD86)；程序性死亡受体PD-1(programmed death-1)与其配体PD-L1和PD-L2；LAG-3(lymphocyte activation gene-3)与其配体；TIM-3(T-cell immunoglobulin domain and mucin domain 3)与其配体；BTLA(B and T lymphocyte attenuator)与其配体等。这些免疫检查点蛋白在分子结构上都很相似，多数属于免疫球蛋白超家族成员。
已有多个拮抗抑制CTLA-4，或PD-1/PD-L1的单克隆抗体药物上市销售，在多种肿瘤治疗上表现出显著的疗效。如2011年美国FDA批准上市用于治疗晚期黑色素瘤(Melanoma)的抗CTLA-4单抗药物Ipilimumab(商品名YERVOY)；2014年批准用于治疗非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)的抗PD-1单抗药物Nivolumab(商品名Opdivo)、Pembrolizumab(商品名Keytruda)和2016年批准用于治疗非小细胞肺癌的抗PD-L1单抗药物Atezolizumab(商品名Tecentriq)等。
目前该领域的一大热点是寻找新型靶点以研究开发出更多类似靶向CTLA-4和PD-1/PD-L等拮抗免疫抑制检查点的单抗类药物。TIGIT则是新发现的一种具有类似潜在药物开发前景的免疫抑制检查点分子。
TIGIT(T cell immunoreceptor with Ig and ITIM domain)，最早由Yu X等发现与报道(Yu X：Nat Immunol.2009，10:48-57)。TIGIT又称为WUCAM(Washington University cell adhesion molecule；Vermi KS：Eur J Immunol 2009,39:695-703)，VSTM3(V-set and transmembrane domain-containing protein 3；Levin S：Eur J Immunol 2011,41:902-915)或VSIG9(V-set and immunoglobulin domain-containing protein 9)。
人TIGIT蛋白全长有244个氨基酸，其中胞外区有141个氨基酸，跨膜区23个氨基酸，胞质区80个氨基酸。小鼠TIGIT蛋白全长有241个氨基酸，与人TIGIT蛋 白氨基酸序列同源性为60％。TIGIT蛋白的一个重要结构特征是在其胞内区尾部存在典型的免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibition motif,ITIM)，从而介导出免疫抑制信号，ITIM同样也存在于PD-1结构域中。
与PD-1类似，TIGIT主要表达在T细胞(包括辅助性T细胞，记忆性T细胞，CD8+效应T细胞)和自然杀伤(Natural killer,NK)细胞上，而识别与结合TIGIT的配体则主要表达在抗原呈递细胞如树突状细胞(dendritic cells，DC)、巨噬细胞、肿瘤细胞等靶向细胞上。
已知的识别与结合TIGIT的配体主要有3个：分别为CD155、CD112和CD113。CD155又称为PVR(poliovirus receptor，脊髓灰质炎病毒受体)；CD112和CD113则又分别称为PVRL-2和PVRL-3(poliovirus receptor-related protein-2，-3)。CD155与TIGIT的结合强于CD112或CD113与TIGIT的结合(Yu X,Nat Immunol.2009,10:48-57；Stanietsky N,Proc Natl Acad Sci USA.2009,106:17858-63)。CD155、CD112及CD113均为Nectin家族成员。
CD155或CD112也可与协同刺激因子CD226(DNAM-1)结合，从而激活或上调T细胞免疫功效。但CD155或CD112与免疫抑制因子TIGIT结合的强度要稍高于其与CD226(DNAM-1)的结合。TIGIT-CD226/CD155-CD112这种相互作用形成的平衡系统类似于CD28-CTLA4/CD80-CD86免疫网络平衡系统。
CD155在人多种正常细胞上低水平表达，而在多种肿瘤细胞上高表达。肿瘤细胞通过上调表达CD155而抑制T细胞/NK细胞的免疫杀伤作用。因此，如开发出拮抗阻断TIGIT与其配体如CD155结合的单克隆抗体，则可解除免疫抑制，达到恢复T细胞/NK细胞对肿瘤的攻击杀伤作用。
国际上目前尚无TIGIT靶点相关药物获批上市，但已有多家制药企业如美国基因泰克公司(Genentech)、百时美施贵宝(BMS)、默沙东(MSD)、OncoMed等启动了抗TIGIT抗体新药研制并申请了相关专利保护。其中临床进展最快的为Genentech公司代号为MTIG-7192A的抗TIGIT单抗Tiragolumab,目前已进入III期临床试验阶段，用于联合靶向PD-L1单克隆抗体Atezolizumab(商品名Tecentriq)治疗非小细胞肺癌；Genentech公司递交申请了抗TIGIT单抗一系列专利(包括授权美国专利号：US9499596B2及US10017572B2)。
BMS公司代号为“BMS-986207”的抗TIGIT单抗药物处于II期临床研究阶段，用于单药或与BMS公司的PD-1靶向药Nivolumab(商品名Opdivo)联合使用治疗肿瘤；BMS公司递交申请了抗TIGIT单抗专利及授权(美国专利申请号:2016/0176963A1；授权美国专利号：US10189902B2)。
MSD公司代号为“MK-7684”的抗TIGIT单抗药物在I/II期临床研究阶段，用于 与Pembrolizumab(商品名Keytruda)联合使用治疗肿瘤；MSD公司递交申请了抗TIGIT单抗专利及授权(美国专利申请号:2016/0355589 A1；授权美国专利号:US10618958B2)。
除此之外，目前国外还有其他几家企业，分别有抗TIGIT单克隆抗体药物进入临床研究阶段，其中包括Potenza Therapeutics公司代号为“ASP8374”单抗(授权美国专利号:US9713641B2)；OncoMed公司代号为“OMP-313M32”单抗(美国专利申请号为NO：2016/0376365 A1；授权美国专利号:US10544219B2)和Arcus Biosciences公司代号为“AB-154”单抗。
鉴于小鼠TIGIT与人TIGIT蛋白氨基酸序列同源性为60％左右，因此，理论上推测，采用传统的抗原蛋白免疫小鼠及杂交瘤技术，应可以制造或开发出全新的、或针对TIGIT抗原不同表位(epitope)的单克隆抗体。这些新的单抗可作为单药或与在研发的或已上市的其他药物如抗PD-1单抗、抗CTLA-4单抗、抗41BB单抗、抗OX40单抗、抗CD38单抗、抗CD47单抗、抗VISTA单抗、抗BTLA单抗,抗VEGF/VEGFR等药物联合使用，用于治疗包括肿瘤在内的多种疾病。
本发明的一个目的在于获得新的、可高亲和力结合人TIGIT抗原并拮抗阻断TIGIT抗原与其配体如CD155(PVR)或CD122(PVRL-2)的单抗。该类抗体或其衍生体可作为药物成分，单独使用或与其他已上市或在研药物组合用于治疗包括肿瘤在内的多种疾病。
发明内容
本发明要解决的技术问题之一是提供一种结合人TIGIT抗原的抗体或其衍生体如抗体Fab片段、单链抗体等。该抗体或其衍生体可高亲和力结合人TIGIT抗原并拮抗阻断TIGIT抗原与其配体如CD155(PVR)结合。
本发明要解决的技术问题之二是提供编码上述抗体的DNA分子或基因。
本发明要解决的技术问题之三是提供含有上述抗体的药物或药物组合物。
本发明要解决的技术问题之四是提供上述抗体或其衍生体在制备治疗肿瘤的药物中的应用。
本发明要解决的技术问题之五是提供制备上述抗体或其衍生体的方法。
为解决上述技术问题，本发明采用如下技术方案：
在本发明第一方面，提供了一种高亲和力结合人TIGIT抗原并拮抗抑制TIGIT与其配体CD155结合的抗体或其衍生体，其包含第一可变区和第二可变区，其中所述第一可变区是抗体轻链可变区，其抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：9，SEQ ID NO：10及SEQ ID NO：11所示的氨基酸序列；其中所述 第二可变区是抗体重链可变区，其抗原互补决定区CDR1，CDR2和CDR3分别为SEQ ID NO：14，SEQ ID NO：15及SEQ ID NO：16所示的氨基酸序列。
所述抗体包括人源化单克隆抗体，所述衍生体包括抗体Fab片段、单链抗体、双特异抗体(bi-specific)等。
作为本发明优选的技术方案，所述抗体或其衍生体具有以下一项或多项特征：
a)包含抗体轻链可变区及抗体重链可变区，其轻链可变区序列与SEQ ID NO：17所示的氨基酸序列至少90％以上相同，其重链可变区序列与SEQ ID NO：19所示的氨基酸序列至少90％以上相同；
b)其与人TIGIT抗原结合的亲和力常数值(binding affinity，KD value)为10nM或者小于10nM(at 10nM or below 10nM)，该亲和力常数值可由Fortebio-Octet，BIACORE或其他类似的表面等离子共振技术(surface plasmon resonance，SPR)测定；
c)拮抗阻断TIGIT与其配体CD155(PVR)或CD112(PVRL2)结合。优选地，所述拮抗阻断TIGIT与其配体CD155(PVR)结合的半数抑制浓度(half maximal inhibitory concentration，IC50)为1nM或者小于1nM(at 1nM or below 1nM)(IC50是指表示物质发挥其最大抑制效果的一半时的浓度)。
作为本发明优选的技术方案，所述第一可变区是抗体轻链可变区，为SEQ ID NO：17所示的氨基酸序列；其所述第二可变区是抗体重链可变区，为SEQ ID NO：19所示的氨基酸序列。
作为本发明优选的技术方案，所述第一可变区是抗体轻链可变区，其氨基酸序列为SEQ ID NO：8所示；所述第二可变区是抗体重链可变区，其氨基酸序列为SEQ ID NO：13所示。
作为本发明优选的技术方案，所述抗体或其衍生体能拮抗阻断TIGIT与其配体CD155(PVR)或CD112(PVRL2)结合。
作为本发明优选的技术方案，其包含所述抗体轻链可变区和人抗体轻链恒定区，及包含所述抗体重链可变区和人抗体重链恒定区的铰链区，CH1区，CH 2区和CH3区。
作为本发明优选的技术方案，所述人抗体轻链恒定区来自人抗体kappa链或抗体lamda链，所述人抗体重链恒定区来自人IgG1，IgG2，IgG3或IgG4等亚型，其中优选的为IgG4亚型。
在本发明第二方面，提供了一种编码上述抗体或其衍生体的DNA分子或基因(对应以下抗体或其衍生体：所述第一可变区是抗体轻链可变区，其氨基酸序列为SEQ ID NO：17所示；所述第二可变区是抗体重链可变区，其氨基酸序列为SEQ ID NO：19所示)，编码抗体轻链可变区的核苷酸序列如SEQ ID NO：18所示，编码抗体重链可变区的核苷酸序列如SEQ ID NO：20所示。
本发明还提供了另一种编码上述抗体或其衍生体的DNA分子或基因(对应以下抗体或其衍生体：所述第一可变区是抗体轻链可变区，其氨基酸序列为SEQ ID NO：8所示；所述第二可变区是抗体重链可变区，其氨基酸序列为SEQ ID NO：13所示)，编码抗体轻链可变区的核苷酸序列如SEQ ID NO：7所示，编码抗体重链可变区的核苷酸序列如SEQ ID NO：12所示。
本发明的第三方面是提供了一种表达载体，它含有编码上述抗体或其衍生体的DNA分子或基因以及与该DNA分子或基因操作性相连的表达调控序列。
本发明的第四方面提供了一种重组宿主细胞，它由上述表达载体转化而成。该重组宿主细胞或其子代细胞表达上述抗体或其衍生体。该抗体包括人源化单克隆抗体，衍生体包括抗体Fab片段、单链抗体、双特异抗体(bi-specific)等。
本发明的第五方面是提供一种药物或药物组合物，它含有药学上有效量的上述抗体或其衍生体，以及药学上可接受的载体。所述药物组合物成分包含如抗程序性细胞死亡-1(programmed death-1,PD-1)抗体或抗PDL-1抗体等其他类似抗体与药物。
本发明的第六方面是提供上述抗体或其衍生体在制备治疗肿瘤的药物中的应用。上述抗体可作为单药或与在研发的或已上市的其他药物如抗PD-1抗体、抗PDL1抗体、抗CTLA-4抗体、抗41BB抗体、抗OX40抗体、抗CD2抗体、抗CD3抗体、抗CD20抗体、抗CD24抗体、抗CD27抗体、抗CD28抗体、抗CD33抗体、抗CD38抗体、抗CD40抗体、抗CD47抗体、抗BTLA抗体、抗EGFR抗体、抗Her2抗体、抗VISTA抗体、抗VEGF抗体、抗VEGFR抗体等药物联合使用，用于治疗包括肿瘤在内的多种疾病。
在本发明的具体实施实例中，本发明描述了该抗TIGIT单抗33D2与抗PD-1单抗在小鼠体内抑制结肠癌生长的应用。
本发明第七方面是提供制备上述抗体或其衍生体的方法，该方法包括：
a)提供上述表达载体，该表达载体含有上述DNA分子或基因以及与该分子或基因操作性相连的表达调控序列；
b)用步骤a)所述的表达载体转化宿主细胞；
c)在适合所述抗体表达的条件下培养步骤b)所得的宿主细胞：和
d)采用亲合层析从该宿主细胞培养液中分离纯化获得所述抗体或其衍生体。
本文所采用的术语“单克隆抗体(单抗)”指从一纯系细胞得到的免疫球蛋白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语“单克隆”表示了抗体的特性，是从均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本文所采用的术语“人源化单克隆抗体”系将鼠源单克隆抗体的氨基酸序列除保留互补决定区(complementarity-determining regions，CDR)外，其它序列(包括可变区中的框架区序列)全部或大部分替换成人免疫球蛋白的氨基酸序列，以达到通过基因工程手段最大限度地降低鼠源性单克隆抗体的免疫原性。
本文所用的术语“抗体”和“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)。其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊氨基酸残基在轻链和重链的可变区之间形成界面。
本文所用的术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中成为互补决定区(CDR)或超变区中的三个片段中。互补决定区(CDR)的氨基酸序列的界定可参考Kabat系统(Kabat,E.A.,et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)或(见下表1)：
其中在本发明描述中，互补决定区(CDR)的氨基酸序列的界定采用Kabat系统。
表1
Figure PCTCN2020102091-appb-000001
Figure PCTCN2020102091-appb-000002
可变区中较保守的部分称为构架区(Framework regions，FR)。抗体重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷1，647-669页(1991))。抗体恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性(antibody-dependent cellular cytotoxicity，ADCC)或补体介导毒性(complement-dependent cytotoxicity，CDC)。
本发明的抗体通常可以通过以下方法来制备：
首先，将含有编码本发明的抗体的基因插入到含有合适的表达调控序列的表达载体中。
本文所用的术语“表达调控序列”通常指参与控制基因表达的序列。表达调控序列包括与目标基因操作性相连的启动子和终止信号。编码本发明抗体的基因(DNA)序列可用本领域技术人员熟知的常规手段，如根据本发明公开的蛋白质序列人工合成或用PCR法扩增得到。其后可用本领域熟知的方法将合成或PCR扩增得到的DNA片段插入到合适的表达载体中。
本发明中所用的表达载体可以是本领域技术人员已知的市售表达载体，如Invitrogen公司的pCDNA3.1表达载体或自制载体如公司的pQY系列载体。
用于接纳表达载体转化的合适宿主细胞一般包括原核细胞和真核细胞。常用的原 核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞、哺乳动物细胞等。在本发明中，较佳的宿主细胞是哺乳动物细胞，尤其是中华仓鼠卵巢(CHO)细胞如CHO-S。
表达载体转化的宿主细胞在合适的条件下(如以无血清培养基在细胞培养瓶或生物反应器中贴壁或悬浮培养)培养后，收获培养上清液，然后用包括protein-A亲和层析、离子交换层析、过滤除菌等本领域技术人员熟知的常规分离步骤或手段纯化得到本发明的抗体。纯化得到的本发明抗体可以溶于适当的溶剂如无菌生理盐水液体中，溶度可以制备成0.01至100mg/ml之间，理想的最终溶度可以制备成1至20mg/ml之间。
该类抗体或或其衍生体可作为药物成分，单独使用或与在研发的或已上市的其他药物如抗PD-1抗体、抗PDL1抗体、抗CTLA-4抗体、抗41BB抗体、抗OX40抗体、抗CD2抗体、抗CD3抗体、抗CD20抗体、抗CD24抗体、抗CD27抗体、抗CD28抗体、抗CD33抗体、抗CD38抗体、抗CD40抗体、抗CD47抗体、抗BTLA抗体、抗EGFR抗体、抗Her2抗体、抗VISTA抗体、抗VEGF抗体、抗VEGFR抗体等药物联合使用，用于治疗包括肿瘤在内的多种疾病。
其中在本发明中，特别描述了该类抗体或其衍生体与抗PD-1单抗合联合给药，用于在TIGIT/PD-1双人源化小鼠(human PD-1 and human TIGIT double gene knock-in mice)体内测试其药物活性及其结果(见实施例10)；测试结果表明，本发明中的抗TIGIT抗体，无论是单独给药或者是抗PD-1单抗联合给药，均表现出明显的抗肿瘤疗效。
为获得可高亲和力结合人TIGIT抗原并拮抗抑制TIGIT与其配体CD155结合的鼠源单克隆抗体及分泌它的杂交瘤细胞系，本发明选取哺乳动物表达的重组人TIGIT胞膜外蛋白为免疫抗原，通过反复多次小剂量的小鼠皮下免疫，获得分泌抗TIGIT蛋白的多克隆抗体；再从中挑取含高效价抗体的小鼠，取其脾脏细胞，通过体外与小鼠骨髓瘤细胞融合、再经药物筛选及亚克隆等步骤而建立了多株稳定分泌抗人TIGIT蛋白的抗体的杂交瘤单克隆细胞。其中经ELISA、免疫印迹、免疫组化及体外与TIGIT配体竞争结合等多种方法鉴定，获得一代号为m33D2(简称33D2)的小鼠杂交瘤细胞株亚克隆，其所分泌的单克隆抗体不但能够高亲和力特异结合人TIGIT蛋白，而且可阻断抑制TIGIT蛋白与其配体(如CD155,CD112)结合。
本发明通过基因工程等手段获得了编码该鼠源抗体重链可变区及轻链可变区的基因片断，在此基础上对该抗体进行了人鼠嵌合及人源化基因工程改造。编码人鼠嵌合抗(c33D2)体或人源化抗体(h33D2)的基因经转染进入中华仓鼠卵巢(CHO)细胞，获得稳定分泌表达该人鼠嵌合或人源化抗体的重组工程细胞，并从重组工程细胞培养 液中分离纯化获得到具有拮抗TIGIT生物活性的人鼠嵌合及人源化抗体。
该抗TIGIT抗体，作为单药或与在研发的或已上市的其他药物如抗PD-1抗体、抗PDL1抗体、抗CTLA-4抗体、抗41BB抗体、抗OX40抗体、抗CD2抗体、抗CD3抗体、抗CD20抗体、抗CD24抗体、抗CD27抗体、抗CD28抗体、抗CD33抗体、抗CD38抗体、抗CD40抗体、抗CD47抗体、抗BTLA抗体、抗EGFR抗体、抗Her2抗体、抗VISTA抗体、抗VEGF抗体、抗VEGFR抗体等药物联合使用，用于治疗包括肿瘤在内的多种疾病。
附图说明
图1为本发明实施例1中人TIGIT蛋白与小鼠TIGIT蛋白的氨基酸序列比对分析示意图。
图2为本发明实施例1中以重组人TIGIT胞膜外蛋白包被96-孔板，通过间接
ELISA(in-direct ELISA)法检测免疫后小鼠血清中抗TIGIT抗体的效价结果示意图。
图3A为本发明实施例1中以重组人TIGIT胞膜外蛋白包被96-孔板，通过间接ELISA法筛选检测到的多株小鼠杂交瘤培养上清液样品的结果示意图，其中阴性对照样品为未融合的SP2/0骨髓瘤细胞培养上清液样品。
图3B为本发明实施例1中间接ELISA法检测杂交瘤细胞株亚克隆的培养上清液样品与包被在96-孔板重组人TIGIT蛋白结合的结果示意图；其中阳性对照样品为TIGIT免疫后的小鼠血清。
图4为本发明实施例1中纯化后的鼠源33D2抗体蛋白样品经DTT还原后(DTT-denatured)的SDS-PAGE分析结果图谱。
图5为本发明实施例2中间接ELISA法检测纯化的鼠源单抗与包被在96-孔板上的重组人TIGIT蛋白或免疫相关其他重组蛋白的结合的结果示意图。.
图6为本发明实施例3中以流式细胞仪检测分析纯化的小鼠抗体样品与稳定转染人TIGIT基因的CHO细胞(CHO/TIGIT)的结合结果示意图。
图7为本发明实施例4中以流式细胞仪检测分析纯化的小鼠抗体样品与来源于野生型C57B/L小鼠(wild type mice)或人源化(human TIGIT gene knock-in mice)小鼠的外周血淋巴细胞(PBL)结合的结果示意图。
图8为本发明实施例5中以竞争ELISA法体外检测单抗样品拮抗阻断CD155与包被在96-孔板上人TIGIT蛋白结合的结果示意图。
图9为本发明实施例9中ELISA检测瞬时转染了重组人源化h33D2抗体基因表达载体的CHO-S细胞培养上清液样品与包被在96-孔板上的重组人TIGIT蛋白结合的结果示意图。
图10为本发明实施例10中以蛋白质相互作用仪分析33D2单抗与TIGIT抗原结合解离动态的结果曲线图。
图11A为本发明实施例11中TIGIT/PD-1双人源化小鼠皮下种植鼠源MC38结肠癌模型中，生理盐水阴性治疗对照组(A组)每只各个体动物体内肿瘤增长体积趋势。
图11B为本发明实施例11中TIGIT/PD-1双人源化小鼠皮下种植鼠源MC38结肠癌模型中，PD-1单抗治疗组(B组)每只个体动物体内肿瘤增长体积趋势。
图11C为本发明实施例11中TIGIT/PD-1双人源化小鼠皮下种植鼠源MC38结肠癌模型中，TIGIT单抗治疗组(C组)每只个体动物体内肿瘤增长体积趋势。
图11D为本发明实施例11中TIGIT/PD-1双人源化小鼠皮下种植鼠源MC38结肠癌模型中，PD-1单抗与TIGIT单抗联合给药治疗组(D组)每只个体动物体内肿瘤增长体积趋势。
图12A为本发明实施例11中各试验组动物肿瘤的平均增长体积趋势。
图12B为本发明实施例11中各组试验动物平均体重增长趋势。
具体实施方式
下面将结合实施实例来进一步描述本发明，这些实施例只是为了起说明作用，而不是用来限制本发明。
实施例1:分泌抗TIGIT抗体的小鼠杂交瘤细胞系的建立与筛选鉴定
1)人TIGIT蛋白的氨基酸序列与小鼠TIGIT蛋白的氨基酸序列比对分析
人TIGIT蛋白的氨基酸序列与小鼠TIGIT蛋白的氨基酸序列比对分析如图1:其中的以方框斜体标注的氨基酸序列为引导细胞外分泌表达TIGIT蛋白的信号肽(signal peptide)，以方框下划线粗体标注的氨基酸序列为TIGIT蛋白的跨细胞膜区域(transmembrane domain)。如图1所示：人TIGIT蛋白与小鼠TIGIT蛋白的氨基酸序列同源性仅有60％左右；其中在直接参与识别及结合其配体(CD155或CD112)的胞膜外区域,人TIGIT蛋白和小鼠TIGIT蛋白的氨基酸差异位点数也有30多个。因此推测，如采用传统的抗原蛋白免疫小鼠及杂交瘤制备技术，应可以制备出鼠抗人TIGIT单克隆抗体。
2)分泌抗TIGIT抗体的小鼠杂交瘤细胞系的建立与筛选鉴定
步骤1.重组人TIGIT蛋白(免疫抗原)的来源与动物免疫
在本实施例中，用于免疫的抗原为由哺乳动物表达的C-端带组氨酸(histidine)-标签的重组人TIGIT胞膜外蛋白(TIGIT-his,Sino Biologicals北京义翘神州公司产品，产品编号10917-H08H)。该重组人TIGIT-His蛋白与弗氏完全佐剂(美国Sigma公司 产品)混合后，于皮下多点注射Balb/c小鼠(100μl/只，每次10μg TIGIT-his蛋白)。首次免疫2-3周后，小鼠再给予皮下多点注射含人TIGIT-His蛋白与弗氏不完全佐剂(美国Sigma公司产品)的混合物，加强免疫3-4次后，取少量小鼠血清，用包被重组人TIGIT-his蛋白的96-板以ELISA法检测小鼠血清中抗TIGIT抗体的效价(见图2)，取效价高者小鼠的脾细胞用于下一步的细胞融合。
步骤2、细胞融合
在末次免疫后3-4天，无菌制备小鼠脾细胞悬液，与小鼠SP2/0骨髓瘤细胞(购自中国科学院上海生命科学院细胞保藏中心)，以5：1或10：1的比例在50％PEG-1000(美国Sigma公司产品)作用下融合。融合按常规法(Kohler G.and Milstein C:Nature1975；256:495-497),PEG用量1ml，60秒内缓慢加完。反应90秒后，以无血清的RPMI-1640培养基终止反应，1000rpm离心10min,去除上清液，再将离心沉淀下的细胞以含10％HAT(H为次黄嘌呤、A氨基碟呤、T胸腺嘧啶核苷，为美国Sigma公司产品)的RPMI 1640-10％FCS培养基将细胞浓度调节至1Х10 6/ml，加入96孔平底细胞培养板(每孔200μl)，于37℃，5％CO2培养箱中(美国Thermo公司产品)培养2-3周。
步骤3、酶联免疫吸附试验(ELISA)筛选抗体分泌阳性的小鼠杂交瘤细胞
同样，以重组人TIGIT-his蛋白(2μg/ml，pH 9.6，0.1M NaHCO3液)包被96-孔酶标板，37℃包被2小时后，再加入2％牛血清白蛋白(BSA)4℃封闭过夜。次日，包被板经PBS-0.1％Tween20液洗涤后，加入待检杂交瘤细胞培养上清液(以未融合的SP2/0骨髓瘤细培养上清为阴性对照)37℃孵育2小时；经PBS-0.1％Tween20液洗涤后，加入辣根过氧化物酶HRP-标记的羊抗小鼠IgG(美国Sigma公司产品)，37℃孵育1小时；再经PBS-0.1％Tween20液充分洗涤后，加入邻苯二胺(OPD)-0.1％H 2O 2底物液显色10-15min后再加入0.1M HCl终止反应。其后在MK3-Multiskan酶标仪(美国Thermo Scientific公司产品)中读取492nm处OD值。测得的OD 492值比阴性对照值高5-10倍的杂交瘤细胞再克隆化，并进行扩增冻存。
其中图3A为ELISA筛选结果阳性的11株小鼠杂交瘤细胞培养上清液复测结果。
步骤4、阳性杂交瘤细胞的亚克隆-有限稀释法
将上述初筛得到的阳性杂交瘤细胞以RPMI-1640-10％FCS培养基稀释至每孔1-10个细胞，铺于96-孔细胞培养板，于37℃，5％CO2培养箱中培养2-3周。待克隆长成，取上清液以ELISA再次检测鉴定抗TIGIT抗体。
其中图3B为以ELISA法检测亚克隆细胞株上清的代表性结果：其中代号为33-D2，33-F3，33-F6及33-F7的33号杂交瘤的各亚克隆上清液样品和代号为188-2-D7的杂交瘤亚克隆清均含高滴度的抗TIGIT抗体。
步骤5、杂交瘤亚克隆在无血清培养基下扩大培养及上清中抗体的分离纯化
在本实施例中，将上述代号为33D2的杂交瘤细胞株亚克隆及代号为188-2-D7的杂交瘤细胞株亚克隆改用无血清培养基进行扩大培养，各收集约1L杂交瘤上清后用Protein G Sepharose(GE公司产品)进行抗体蛋白纯化(方法参考说明书)；纯化后的抗体蛋白进行SDS-PAGE(Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)分析。
图4为纯化获得的鼠源33D2抗体蛋白样品经DTT还原(DTT-denatured)的SDS-PAGE分析图谱。如图4所示：还原后的抗体蛋白样品呈现两个条带：其中位于上端的为抗体重链，位于下端的为抗体轻链。
实施例2 ELISA法检测分析鼠源单抗与TIGIT及免疫相关其他蛋白的结合
采用ELISA法比较分析了纯化的鼠源33D2单抗、188-2-D7单抗与TIGIT及其他相关蛋白的结合活性。
该ELISA法检测基本步骤如下：将纯化的鼠源33D2单抗、188-2-D7单抗稀释至1μg/mL，分别加入到预包被了重组TIGIT-his蛋白或免疫相关其他基因-Fc融合蛋白(包括CD28、B7-1、CTLA4、CD3、PD-1、PD1H(PD2)、PD-L1、PD-L2等12种抗原，各抗原包被浓度均为2μg/mL)的96孔板中，经37℃孵育1h及洗涤后，96孔板各孔再加入HRP-标记的山羊抗鼠IgG(美国Jackson公司产品)，再经37℃孵育1h及洗涤后，各孔加入OPD底物显色。
表2为该ELISA法检测分析纯化的33D2单抗、188-2-D7单抗与TIGIT及免疫相关其他蛋白结合的代表性结果(OD at 492nm)：
表2鼠源33D2单抗与TIGIT及免疫相关其他蛋白的结合(OD at 495nm)
Figure PCTCN2020102091-appb-000003
图5为根据表2的ELISA法检测分析结果(OD at 495nm)所作的直条图：结果显示，33D2单抗及188-2-D7单抗均仅特异结合人TIGIT蛋白，与免疫相关其他蛋白如PD-1、CD28、B7、CTLA-4、CD3、PD-L1、PD-L2等均无明显结合。
实施例3流式细胞仪(Flow-cytometry)检测分析鼠源抗体与转染表达人TIGIT基因的CHO细胞(CHO/TIGIT)结合
在本实施例中，以鼠源杂交瘤细胞株33D2上清为一抗，以FITC荧光标记的羊抗小鼠IgG为二抗，采用流式细胞仪检测分析鼠源33D2单抗样品与稳定表达人TIGIT基因的CHO细胞(CHO/TIGIT)结合。为此,将CHO/TIGIT细胞分别与含小鼠IgG(阴性对照样品,isotype control)、鼠源33D2上清液及TIGIT抗原免疫小鼠阳性血清(阳性对照样品immu.sera，1:200稀释)；4℃孵育30min及经PBS-1％BSA液洗涤后，加入FITC-标记的羊抗小鼠IgG(Sigma公司产品)；4℃孵育30min及再经PBS-1％BSA液洗涤后，将样品上样至Biosciences Accuri C6流式细胞仪检测(美国BD公司)。
图6为该流式细胞仪检测代表性结果示意图。如图6所示：与小鼠IgG阴性对照样品相比(isotype control)，TIGIT免疫小鼠阳性血清阳性样品(immu.sera)及测试样品33D2单抗上清(33D2)均可特异与CHO/TIGIT细胞结合；33D2单抗样品的结合强度与阳性血清对照样品相近。
实施例4流式细胞仪检测鼠源抗体与TIGIT人源化小鼠的外周血淋巴细胞的结合
在本实施例中，采取Human TIGIT gene knock-in小鼠外周血(该TIGIT人源化小鼠来自于百奥赛图生物公司)，分离获得淋巴细胞，再以鼠源33D2上清为一抗，FITC标记的polyclonal rabbit anti-mouse IgG为二抗(同时加入PE标记的anti-Mouse CD3e以标记T淋巴细胞)，采用流式细胞仪检测分析33D2单抗样品与人源化TIGIT小鼠中TIGIT蛋白的结合。具体方法如下：
1)预先在EP管内加入100μl的抗凝剂，分别取C57/B6野生型小鼠和human TIGIT gene knock-in小鼠尾静脉采血约150μl，混匀；加入溶血素(BD公司)，室温避光作用10min；离心弃上清，将TIGIT人源化小鼠样本平均分为三份，离心；其中两管分别做FITC单阳管和PE单阳管，一管为检测管。
2)弃上清，PE单阳管加稀释液；FITC单阳管和其他检测管加抗TIGIT mAb33-D2杂交瘤上清，500μl/孔，4℃作用30min。1500rpm，离心5min；弃上清，用1％BSA洗涤一次；其中FITC单阳管加入FITC标记的Polyclonal Rabbit Anti-Mouse IgG，PE单阳管加入PE标记的Anti-Mouse CD3e(1:25),检测管加入PE标记的Anti-Mouse CD3e和FITC标记的polyclonal rabbit anti-mouse IgG，100μl/孔，4℃作用30min。1500rpm，离心5min，用1％BSA洗涤一次，用150uL 1％BSA重悬细胞，将样品上样至Biosciences AccuriC6流式细胞仪检测(美国BD公司)。
图7为该流式细胞仪检测代表性结果示意图。如图7所示：33D2单抗样品能够与human TIGIT gene knock-in小鼠淋巴细胞特异结合，而不与C57/B6野生型小 鼠淋巴细胞结合。
实施例5竞争性ELISA法检测单抗拮抗阻断TIGIT蛋白与其受体的结合
体外检测抗TIGIT单抗的生物活性方法之一是采用竞争性ELISA法检测其是否拮抗阻断TIGIT蛋白与其受体如CD155(PVR)的结合。
其中，该竞争性ELISA法检测的具体步骤如下：
1)用重组人TIGIT-his胞膜外蛋白(北京义翘神州公司产品)包被96-孔板(包被溶度：2μg/mL，50μL/孔)，4℃过夜；
2)经PBS液漂洗及5％牛奶(稀释在PBS-0.1％tween20液中)室温封闭后，分别加入含固定溶度的生物素标记的CD155(PVR)-Fc蛋白(ACRO Biosystems公司产品)与不同溶度的33D2抗体，或非相关的抗体(如抗VEGF单抗hPV19)，37℃孵育1h；
3)经PBS-T洗脱后，加入辣根过氧化物酶标记的Avidin(1：5000)，37℃孵育1h；
4)经PBS-T洗脱后，加入显色液(邻苯二胺)-3％双氧水，室温5-10min至显色；
5)加入1M HCL终止反应，以酶联免疫仪测定492nm波长处各孔的吸光值。
表3及图8为该竞争ELISA法检测单抗样品拮抗阻断TIGIT蛋白与其受体CD155的结的代表性结果。
表3体外竞争ELISA法检测结果
Figure PCTCN2020102091-appb-000004
Figure PCTCN2020102091-appb-000005
图8为根据表3的竞争ELISA法检测结果(OD at 495nm)使用作图软件(OriginPro 9.0)所作的拟合线图。
如图8所示:在加入不同浓度的33D2单抗(33D2-sample #2,33D2-sample #4)与固定浓度的生物素标记的CD155-Fc蛋白的样品中，各孔显色反应OD值与加入的抗体蛋白量成反比关系：即加入的33D2单抗量越高，其显色OD值越低；而非相关单抗样品(hPV19，抗VEGF单抗)的加入量多少对各孔OD值影响不大。此结果表明，33D2单抗体外可拮抗阻断TIGIT与其受体(CD155)结合，根据拟合线图测得其平均半数抑制浓度(IC 50值)在0.13μg/ml(换算成摩尔浓度,其IC 50均值为0.867nM),具体结果见表4。
表4半数抑制浓度(IC 50值)
Sample ID IC50(μg/mL) IC50(nM)
33D2-sample#2 0.135 0.900
33D2-sample#4 0.125 0.833
Average 0.130 0.867
实施例6.鼠源33D2抗体可变区编码基因的克隆
在此，先从小鼠33D2杂交瘤细胞中提取出总RNA中，再以该RNA为模板，采用简并引物(degenerate primers)，以Reverse transcription-polymerase chain reaction(RT-PCR)法(Wang Y等：Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA。BMC Bioinformatics.2006；7 Suppl(4):S9)分别克隆扩增获得m33-D2抗体重链可变区及轻链可变区的cDNA基因片段。其中cDNA基因克隆步骤如下：
步骤1、采用RNA提取试剂(RNAiso Plus，Takara公司产品)从小鼠m33D2杂交瘤细胞中提取出总RNA；
步骤2、采用逆转录PCR(RT-PCR)方法在eppendorf管获得cDNA模板。
其中用于鼠源33D2抗体轻链可变区逆转录PCR引物(33-D2-L)序列为：TGT CGT TCA CTG CCA TCA AT(SEQ ID NO.:1)
用于鼠源33D2抗体重链可变区逆转录PCR引物(33-D2-H)序列为：GCA AGG CTT ACA ACC ACA ATC(SEQ ID NO.:2)；
RT-PCR反应体系如下：
Figure PCTCN2020102091-appb-000006
随后加入：
Figure PCTCN2020102091-appb-000007
于42℃温度下反应1小时，随后温度升至70℃，15分钟灭活后将获得的cDNA置于-20℃，保存备用。
步骤3、鼠源33D2抗体轻链可变区及重链可变区基因的PCR克隆扩增用于克隆扩增该33D2抗体轻链可变区基因的一对引物为：
正向引物：GAC ATC CAG ATG A(SEQ ID NO.:3)
反向引物：CTG AGG CAC CTC CAG ATG TT(SEQ ID NO.:4)
而用于克隆扩增33-D2抗体重链可变区基因的一对引物为：
正向引物：GTG CAG TCT GGA CCT GA(SEQ ID NO.:5)
反向引物：GTG CTG GAG GGG ACA GTC ACT(SEQ ID NO.:6)
PCR扩增得到的DNA产物在1.5％agarose胶中电泳分析。电泳结束后，将分离的DNA条带切下并分别进行测序获得抗体轻链及重链可变区DNA的核苷酸序列。测得的该鼠源抗体轻链可变区DNA的核苷酸序列见SEQ ID NO.:7，由该DNA核苷酸序列推测得到的抗体轻链可变区氨基酸序列见SEQ ID NO.:8。该轻链抗原互补决定区(complementarity-determining regions，CDR)的CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:9、SEQ ID NO.:10和SEQ ID NO.:11。
测得的该鼠源抗体重链可变区DNA的核苷酸序列见SEQ ID NO.:12，由该DNA的核苷酸序列推测得到的抗体重链可变区氨基酸序列见SEQ ID NO.:13。该重链抗原互补决定区的CDR1、CDR2及CDR3的氨基酸序列分别见SEQ ID NO.:14、SEQ ID NO.:15和SEQ ID NO.:16。
实施例7鼠源33D2抗体的人-鼠嵌合抗体(c33D2)的构建
将实施例6中克隆扩增获得的鼠源33D2抗体轻链可变区基因和重链可变区基因分别与人-kappa轻链恒定区(C-domain)和人IgG4-重链恒定区基因片段融合，获得人-鼠嵌合轻链基因(c33D2L)及人-鼠嵌合重链基因(c33D2H)。其后将轻链嵌合基因与重链嵌合基因分别克隆至pcDNA3.1表达质粒，转入大肠杆菌扩增，分离获得大量含人-鼠嵌合抗体基因的表达质粒。
含人-鼠嵌合抗体基因轻链基因的表达质粒样品及人-鼠嵌合抗体重链基因的表达质粒样品组合，再与Fugen-6脂质体(Roche)混合后共转染入CHO细胞。细胞转染后2天，取培养上清液，用包被人TIGIT蛋白的96-孔板，用HRP酶标记的Goat-anti-human-IgG(Fab specific)(购自Sigma-Aldrich)为检测二抗，以ELISA法检测上清中的嵌合抗体(c33D2)与人TIGIT蛋白结合。该ELISA代表性检测结果如下表：
表5 ELISA法检测嵌合抗体(c33D2)基因瞬时转染细胞培养上清与人TIGIT蛋白的结合
Figure PCTCN2020102091-appb-000008
表5结果表明，人-鼠嵌合抗体基因c33D2表达质粒转染的CHO细胞培养上清可与人TIGIT的蛋白特异结合。
上述转染细胞上清经离心及0.45μm滤膜过滤后，上样至Protein A亲合层析柱(protein A-Sepharose Fast Flow，美国通用电气GE公司产品)，经分离纯化，获得人-鼠嵌合抗体(c33D2)抗体蛋白。
实施例8鼠源33D2抗体的人源化基因工程改造
在ELISA法检测初步证明人-鼠嵌合抗体(c33D2)保持有与人TIGIT蛋白高亲合力结合活性的基础上，采用PCR等系列基因工程克隆手段将该嵌合抗体轻链和重链中的抗原互补决定区(CDR)基因片段分别移植到对应人kappa-轻链和IgG4-重链可变区骨架(framework regions，FR)上，获得人源化h33-D2抗体。
1)33D2抗体轻链的人源化
通过氨基酸序列分析，确定人免疫球蛋白Kappa轻链第一V区胚系基因的表达产物(IgKV1-9，NCBI Gene ID:28941)与33D2轻链可变区具有最高同源性。据此，将33D2轻链骨架区(FR)用人IgKV1-9的同源序列替换，然后将替换后的可变区基因与 人免疫球蛋白IgG-Kappa轻链的恒定区编码序列拼接，最后成功获得人源化的轻链编码基因(h33D2-L)。其中人源化h33D2抗体轻链可变区的氨基酸序列见SEQ ID NO.:17,其核苷酸序列为SEQ ID NO.：18所示。
2)33-D2抗体重链的人源化
通过氨基酸序列分析，确定人免疫球蛋白重链第三V区胚系基因的表达产物(IgHV1-2，NCBI Gene ID:28474)与33D2抗体重链具有最高同源性。据此，将33D2重链骨架区(FR)用人IgHV1-2的同源序列替换，同时为了降低人源化抗体与机体内的免疫球蛋白-Fc受体(FcR)结合及其介导的细胞毒性(antibody-dependent cellular cytotoxicity，ADCC)对TIGIT表达阳性免疫细胞(淋巴细胞)的杀伤作用，特意将人源化的33D2抗体重链可变区基因与编码人免疫球蛋白-IgG4重链的恒定区序列拼接，并将其中铰链区(hinge region)内处于第228点位的氨基酸由原有的proline替换成serline(S228P)。经此基因工程系列改造，最后成功获得含人源化重链编码可变区、人IgG4-重链恒定区(S228P)的全长h33D2抗体重链。其中人源化h33D2抗体重链可变区的氨基酸序列见SEQ ID NO.:19,其核苷酸序列为SEQ ID NO.：20所示。
实施例9分泌表达人源化33D2抗体的细胞工程株的建立及抗体蛋白的分离纯化
将实施例8含人源化重链基因(h33D2-H)、人源化轻链基因(h33D2-L)分步克隆到pQY-Hygro表达载体，转入大肠杆菌后扩增分离获得表达人源化33D2抗体的重组质粒。其后将重组质粒分别瞬时转染CHO-S细胞。转染48小时后，吸取孔内细胞培养上清，以TIGIT-his蛋白为包被抗原，HRP酶标记的Goat-anti-human-IgG为检测二抗(购自上海西塘生物公司)，OPD为显色底物，以直接ELISA法检测转染细胞上清中抗体与人TIGIT抗原结合的活性。
下表6为该ELISA代表性检测结果。
表6 ELISA分析瞬时转染CHO细胞培养上清与人TIGIT蛋白的结合
Figure PCTCN2020102091-appb-000009
Figure PCTCN2020102091-appb-000010
图9为根据表6的ELISA法分析结果(OD at 492nm)使用作图软件(OriginPro9.0)所作的拟合线图，结果显示人源化h33D2抗体(IgG4-kappa)可特异与人TIGIT蛋白结合，且其结合活性(或亲和力)与人-鼠嵌合型c33D2抗体相近似。
上述转染细胞经克隆筛选及无血清培养基悬浮培养驯化后，成功获得多个稳定高效分泌表达人源化h33D2抗体蛋白的CHO细胞工程株。
其后，从中选取一细胞工程株再经无血清培养基放大扩增培养后，收集培养上清液，上清液经离心及0.45μm滤膜过滤后，上样至包括含Protein A亲合层析柱(proteinA-Sepharose Fast Flow，美国通用电气GE公司)、离子交换析柱、病毒去除/灭活、及过滤除菌(0.22μm滤膜过滤)在内的多个分离纯化步骤后，最终获得高纯度(蛋白纯度达99％以上)的人源化抗体(h33D2)。纯化的人源化抗体(h33D2)再溶于无菌生理盐水中低温(-20℃以下)保存。
实施例10抗体亲和动力学检测
采用生物传感器(蛋白质相互作用仪：Fortebio-Octet RED96,PALL公司产品)实时监测与测定了纯化的人源化33D2单抗与TIGIT抗原结合的亲和力与动力学曲线。该检测方法及结果如下：
1.生物传感器及实验材料溶液配制
生物传感器芯片：表面用anti-Human Fab-CH1 2nd Generation抗体(FAB2G，ForteBio公司产品，产品编号1802083)包被。
其中实验材料溶液配制：
1.1.KB buffer：0.1％BSA 0.05％Tween 20以pH7.2的商品化PBS溶解。
1.2.抗体工作溶液：人源化33D2单抗样品用KB buffer配制成10μg/mL。
1.3.抗原工作溶液：重组人TIGIT-His蛋白(北京义翘神州公司产品，产品编号10917-H08H)用KB buffer配制成100nM、25nM、6.25nM 3个梯度。
2.检测方法及步骤
2.1.打开Fortebio仪器及相关软件，选择Advance Kientics实验模式；
2.2.分析程序按下表7进行
表7
Assay step Assay time(s) Sample
Baseline 60 KB buffer
Loading 400 mAb 33-D2
Baseline 60 KB buffer
Association 400 TIGIT-His
Dissociation 600 KB buffer
3.实验结果
图10为人源化33D2单抗与TIGIT抗原结合结合解离动态结果曲线；0s到400s表示结合，400s到1000s表示解离，三条曲线从上到下分别表示人源化33D2单抗与100nM，25nM和12.5nM浓度的TIGIT-His结合的曲线。表8为亲和动力学检测常数：结果显示人源化33D2单抗与TIGIT具有较高的亲和力，结合亲和力(binding affinity)常数为0.843nM。
表8单抗亲和动力学检测结果(monovalent binding)
Figure PCTCN2020102091-appb-000011
(表8表示在Global的模式下计算出来的结合解离的数值和亲和力的数值，KD表示亲和力的数值，kon表示结合的数值，kdis表示解离的数值，R 2表示拟合曲线与结合解离曲线之间的相关系数,该数值越接近1表示拟合计算出来的结果越接近真实的结果)
实施例11 TIGIT/PD-1双人源化小鼠(human PD-1and human TIGIT double gene knock-in mice)体内测试抗TIGIT单抗的抗肿瘤疗效
由于33D2单抗不识别小鼠TIGIT，故无法在一般的普通小鼠体内直接测试该单抗的疗效。为此，在本实施例中，特别选用表达CD155基因的小鼠结肠癌细胞(MC38-CD155)在基因工程改造过的TIGIT/PD-1双人源化小鼠进行体内测试鼠抗人TIGIT单抗33D2，作为单药，或者与鼠抗人PD-1单抗hAb21(思坦维公司自研产品)联合给药的抗肿瘤疗效的系列研究。
该动物试验研究分两阶段，其中第一阶段的试验模型、给药分组及试验结果描述 如下：
第一阶段研究：
动物试验模型及给药分组：
将数量为1x10 6的表达CD155基因的MC38-CD155结肠癌细胞(MC38结肠癌细胞由上海南方模式生物科技股份有限公司提供，来源于C57BL/6小鼠，)接种于基因背景同样为C57BL/6的TIGIT/PD-1双人源化纯合子小鼠右侧背部皮下(该小鼠品系简称hPDCD1/hTIGIT，由南京大学模式动物研究所提供，其通过Cas9方式利用基因同源重组，分别将人的PD-1基因及TIGIT基因相应替换到小鼠内源PD-1基因及内源TIGIT基因位置，从而表达人PD-1基因及人TIGIT基因而非小鼠PD-1基因及小鼠TIGIT基因)；待接种的肿瘤体积长至约米粒大小(约50-70mm 3，肿瘤细胞接种后第5-6天左右)时将动物随机分为4实验组(如下表9)：
表9实验分组
Figure PCTCN2020102091-appb-000012
动物自分组当天起(即接种肿瘤后第5-6天)，每周腹腔注射(i.p.)给药2次(每隔3-4天给药1次)，共给药4次(连续给药2周)。期间每天观察动物一般临床症状，每隔3-4天测量肿瘤长径(mm)和短径(mm)及动物体重。
肿瘤体积计算公式为：体积(mm 3)＝长径(mm)X短径(mm)X短径(mm)X0.5。如测量时肿瘤体积超过4000mm 3则对测试动物实行安乐死(euthanized)。
动物试验结果：
该试验结果汇总见下表10，表11A-11D，图11A-11D及图12A和图12B。
图11A-11D则分别为各给药组每只个体动物体内肿瘤增长体积趋势图。
图12A为各组试验动物肿瘤的平均增长体积趋势。
图12B为各组试验动物平均体重增长趋势。
表10实验结果
Figure PCTCN2020102091-appb-000013
Figure PCTCN2020102091-appb-000014
表11A实验A组(生理盐水阴性对照组)结果
Figure PCTCN2020102091-appb-000015
*:Numbers in()are days after received 1 st treatment
表11B实验B组(anti-PD-1单抗治疗组)结果
Figure PCTCN2020102091-appb-000016
*:Numbers in()are days after received 1 st treatment
表11C实验C组(anti-TIGIT单抗治疗组)结果
Figure PCTCN2020102091-appb-000017
*:Numbers in()are days after received 1 st treatment
#:Mean±SE(without C01 animal,n＝5)
表11D实验D组(anti-PD-1单抗与anti-TIGIT单抗联合给药组)结果
Group D:Tumor volume(mm 3)in TIGIT&PD1 double gene knock-in mice treated with anti-PD1 mAb(1mg/kg)and anti-TIGIT mAb(1mg/kg),n＝6)
Figure PCTCN2020102091-appb-000018
*:Numbers in()are days after received 1 st treatment
如上表11A-11D，图11A-11D实验结果所示：生理盐水阴性治疗对照组(A组)肿瘤快速增长，没有观察到测试动物有肿瘤萎缩或被彻底排斥(complete rejection CR:0/6)；而PD-单抗治疗组(B组)、TIGIT单抗治疗组(C组)、PD-1单抗与TIGIT单抗联合给药组(D组)在给药后5-8天肿瘤增长即受到抑制，肿瘤出现萎缩或彻底消 失。
PD-单抗治疗组的结果与预期的一致，所有测试动物在给药2-3次后(即首次给药后第11-13天)肿瘤全部出现萎缩或彻底被排斥(complete rejection:6/6)，停止给药后整个实验期间未再见肿瘤复发(最长已观察到接种肿瘤后的第76天)；而更惊奇的及令人鼓舞是在TIGIT单抗治疗组，除1只小鼠(动物编号C01)肿瘤增长未受到抑制，其余5只小鼠(6/6)在仅给药2-3次后(即首次给药后第11-13天)肿瘤也全部出现萎缩或被彻底排斥(complete rejection:5/6)，且停止给药后整个实验期间也未再见肿瘤复发(最长已观察到接种肿瘤后的第76天)；PD-1单抗与TIGIT单抗联合治疗组在各自剂量减半(1mg/kg)的情况下，所有测试动物的仅给药2-3次后(即首次给药后第11-13天)肿瘤也全部萎缩或被彻底排斥(complete rejection:6/6),停止给药后整个实验期间未再见肿瘤复发(最长已观察到接种肿瘤后的第76天)；。
图12A为各组试验动物肿瘤实验期间的平均增长体积趋势。
图12B则为各组试验动物实验期间平均体重增长趋势。
如图12B所示，与对照组相比，TIGIT单抗无论是单独给药还是与PD-单抗联合给药后，实验期间对测试动物体重增均长无明显影响。

Claims (18)

  1. 一种结合人TIGIT抗原的抗体或其衍生体，其特征在于，其包含第一可变区和第二可变区，其中所述第一可变区是抗体轻链可变区，其抗原互补决定区CDR1，CDR2和CDR3的氨基酸序列分别为SEQ ID NO：9，SEQ ID NO：10及SEQ ID NO：11所示；其中所述第二可变区是抗体重链可变区，其抗原互补决定区CDR1，CDR2和CDR3的氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：15及SEQ ID NO：16所示。
  2. 根据权利要求1所述的抗体或其衍生体，其特征在于，所述抗体或其衍生体具有以下一项或多项特征：
    a)包含抗体轻链可变区及抗体重链可变区，其轻链可变区序列与SEQ ID NO：17所示的氨基酸序列至少90％以上相同，其重链可变区序列与SEQ ID NO：19所示的氨基酸序列至少90％以上相同；
    b)其与人TIGIT抗原结合的亲和力常数值为10nM或小于10nM；
    c)拮抗阻断TIGIT与其配体CD155(PVR)或CD112(PVRL-2)结合。
  3. 根据权利要求2所述的抗体或其衍生体，其特征在于，所述拮抗阻断TIGIT与其配体CD155(PVR)结合的IC50值为1nM或小于1nM。
  4. 根据权利要求2所述的抗体或其衍生体，其特征在于，所述第一可变区是抗体轻链可变区，其氨基酸序列为SEQ ID NO：17所示；所述第二可变区是抗体重链可变区，其氨基酸序列为SEQ ID NO：19所示。
  5. 根据权利要求1所述的抗体或其衍生体，其特征在于，所述第一可变区是抗体轻链可变区，其氨基酸序列为SEQ ID NO：8所示；所述第二可变区是抗体重链可变区，其氨基酸序列为SEQ ID NO：13所示。
  6. 根据权利要求1-5任一项所述的抗体或其衍生体，其特征在于，其能拮抗阻断TIGIT与其配体CD155(PVR)或CD112(PVRL-2)结合。
  7. 根据权利要求1-5任一项所述的抗体或其衍生体，其特征在于，其包含所述抗体轻链可变区和人抗体轻链恒定区，及包含所述抗体重链可变区和人抗体重链恒定区的铰链区，CH1区，CH2区和CH3区。
  8. 根据权利要求7所述的抗体或其衍生体，其特征在于，所述人抗体轻链恒定区来自人抗体kappa链或抗体lamda链，所述人抗体重链恒定区来自人IgG1，IgG2，IgG3或IgG4亚型。
  9. 一种编码权利要求4所述抗体或其衍生体的DNA分子或基因，其特征在于，编码抗体轻链可变区的核苷酸序列如SEQ ID NO：18所示，编码抗体重链可变区的核苷酸序列如SEQ ID NO：20所示。
  10. 一种编码权利要求5所述抗体或其衍生体的DNA分子或基因，其特征在于，编码抗体轻链可变区的核苷酸序列如SEQ ID NO：7所示，编码抗体重链可变区的核苷酸序列如SEQ ID NO：12所示。
  11. 一种表达载体，它含有编码权利要求1-8任一项所述抗体或其衍生体的DNA分子或基因以及与该DNA分子或基因操作性相连的表达调控序列。
  12. 根据权利要求11所述的表达载体，其特征在于，它含有权利要求9或10所述的DNA分子或基因以及与该DNA分子或基因操作性相连的表达调控序列。
  13. 一种重组宿主细胞，其特征在于，它由权利要求11或权利要求12所述的表达载体转化而成。
  14. 根据权利要求13所述的重组宿主细胞，其中所述重组宿主细胞表达权利要求1-8任一项所述的抗体或其衍生体。
  15. 一种药物或药物组合物，其特征在于，它含有药学上有效量如权利要求1-8任一项所述的抗体或其衍生体，以及药学上可接受的载体。
  16. 根据权利要求15所述的药物组合物，其特征在于，所述药物组合物成分包含如抗PD-1抗体或抗PDL-1抗体等其他类似抗体与药物。
  17. 根据权利要求1-8任一项所述的抗体或其衍生体在制备治疗肿瘤的药物中的应用。
  18. 一种制备权利要求1，2，3，或7所述的抗体或其衍生体的方法，其特征在于，该方法包括如下步骤：
    a)提供权利要求10所述表达载体；
    b)用步骤a)所述的表达载体转化宿主细胞；
    c)在适合所述抗体表达的条件下培养步骤b)所得的宿主细胞：和
    d)采用亲合层析从宿主细胞培养液中分离纯化获得所述抗体或其衍生体。
PCT/CN2020/102091 2020-04-29 2020-07-15 结合tigit抗原的抗体及其制备方法与应用 WO2021217893A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/915,109 US11897955B2 (en) 2020-04-29 2020-07-15 Monoclonal antibody binding to TIGIT antigen, preparation method and use thereof
EP20933685.8A EP4108684A4 (en) 2020-04-29 2020-07-15 ANTIBODY BINDING TO TIGIT ANTIGEN, PREPARATION METHOD AND USE THEREOF

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010353714.6A CN113563470B (zh) 2020-04-29 2020-04-29 结合tigit抗原的抗体及其制备方法与应用
CN202010353714.6 2020-04-29

Publications (1)

Publication Number Publication Date
WO2021217893A1 true WO2021217893A1 (zh) 2021-11-04

Family

ID=78158346

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/102091 WO2021217893A1 (zh) 2020-04-29 2020-07-15 结合tigit抗原的抗体及其制备方法与应用

Country Status (4)

Country Link
US (1) US11897955B2 (zh)
EP (1) EP4108684A4 (zh)
CN (1) CN113563470B (zh)
WO (1) WO2021217893A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115043910A (zh) * 2022-03-25 2022-09-13 湖南中晟全肽生化有限公司 抑制tigit与cd155结合的多肽及其应用
CN116003606A (zh) * 2023-01-03 2023-04-25 上海百英生物科技股份有限公司 一种tigit纳米抗体及其制备方法与应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116355095A (zh) * 2021-12-27 2023-06-30 上海健信生物医药科技有限公司 靶向tigit的抗体和双特异性抗体及其应用
CN117285637B (zh) * 2023-11-22 2024-03-22 江苏迈威康新药研发有限公司 一种抗独特型抗体及其应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016028656A1 (en) * 2014-08-19 2016-02-25 Merck Sharp & Dohme Corp. Anti-tigit antibodies
US20160176963A1 (en) 2014-12-23 2016-06-23 Bristol-Myers Squibb Company Antibodies to tigit
US9499596B2 (en) 2008-04-09 2016-11-22 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
US20160376365A1 (en) 2015-05-28 2016-12-29 Oncomed Pharmaceuticals, Inc. Tigit-binding agents and uses thereof
US9713641B2 (en) 2015-10-01 2017-07-25 Potenza Therapeutics, Inc. Anti-TIGIT antigen-binding proteins and methods of use thereof
US10017572B2 (en) 2015-09-25 2018-07-10 Genentech, Inc. Anti-tigit antibodies and methods of use
CN109384846A (zh) * 2018-09-25 2019-02-26 合肥瑞达免疫药物研究所有限公司 能够结合tigit的抗体或其抗原结合片段及用途
CN109734806A (zh) * 2019-03-15 2019-05-10 安徽安科生物工程（集团）股份有限公司 一种全人源抗huTIGIT单克隆抗体及其应用
CN110997720A (zh) * 2017-07-27 2020-04-10 Iteos治疗公司 抗tigit抗体
CN111050788A (zh) * 2017-02-28 2020-04-21 西雅图基因公司 抗tigit抗体

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MY183503A (en) * 2013-07-16 2021-02-23 Genentech Inc Method of treating cancer using pd-1 axis binding antagonists and tigit inhibitors
PT3347379T (pt) * 2016-08-17 2020-02-18 Compugen Ltd Anticorpos anti-tígitos, anticorpos anti-pvrig e as suas combinações
CN106939049B (zh) * 2017-04-20 2019-10-01 苏州思坦维生物技术股份有限公司 拮抗抑制人pd-1抗原与其配体结合的单克隆抗体及其制备方法与应用
JP7348072B2 (ja) * 2017-06-01 2023-09-20 コンピュジェン リミテッド 三重併用抗体療法
EP3689909A4 (en) * 2017-09-29 2021-12-29 Jiangsu Hengrui Medicine Co., Ltd. Tigit antibody, antigen-binding fragment thereof, and medical use thereof
BR112020008127A2 (pt) * 2017-10-27 2020-10-13 Pfizer Inc. anticorpos e conjugados de anticorpo-fármaco específicos para cd123 e seus usos

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9499596B2 (en) 2008-04-09 2016-11-22 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
US20160355589A1 (en) 2014-08-19 2016-12-08 Merck Sharp & Dohme Corp. Anti-tigit antibodies
WO2016028656A1 (en) * 2014-08-19 2016-02-25 Merck Sharp & Dohme Corp. Anti-tigit antibodies
US10618958B2 (en) 2014-08-19 2020-04-14 Merck Sharp & Dohme Corp. Anti-TIGIT antibodies
US20160176963A1 (en) 2014-12-23 2016-06-23 Bristol-Myers Squibb Company Antibodies to tigit
WO2016106302A1 (en) * 2014-12-23 2016-06-30 Bristol-Myers Squibb Company Antibodies to tigit
US10189902B2 (en) 2014-12-23 2019-01-29 Bristol-Myers Squibb Company Antibodies to TIGIT
US10544219B2 (en) 2015-05-28 2020-01-28 Oncomed Pharmaceuticals, Inc. TIGIT-binding agents and uses thereof
US20160376365A1 (en) 2015-05-28 2016-12-29 Oncomed Pharmaceuticals, Inc. Tigit-binding agents and uses thereof
US10017572B2 (en) 2015-09-25 2018-07-10 Genentech, Inc. Anti-tigit antibodies and methods of use
US9713641B2 (en) 2015-10-01 2017-07-25 Potenza Therapeutics, Inc. Anti-TIGIT antigen-binding proteins and methods of use thereof
CN111050788A (zh) * 2017-02-28 2020-04-21 西雅图基因公司 抗tigit抗体
CN110997720A (zh) * 2017-07-27 2020-04-10 Iteos治疗公司 抗tigit抗体
CN109384846A (zh) * 2018-09-25 2019-02-26 合肥瑞达免疫药物研究所有限公司 能够结合tigit的抗体或其抗原结合片段及用途
CN109734806A (zh) * 2019-03-15 2019-05-10 安徽安科生物工程（集团）股份有限公司 一种全人源抗huTIGIT单克隆抗体及其应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
KABAT ET AL., NIH PUBL., vol. 1, 1991, pages 647 - 669
KOHLER GMILSTEIN C, NATURE, vol. 256, 1975, pages 495 - 497
LEVIN S, EUR J IMMUNOL, vol. 41, 2011, pages 902 - 915
See also references of EP4108684A4
STANIETSKY N, PROC NATL ACAD SCI USA., vol. 106, 2009, pages 17858 - 63
VERMI KS, EUR J IMMUNOL, vol. 39, 2009, pages 695 - 703
WANG Y ET AL.: "Degenerated primer design to amplify the heavy chain variable region from immunoglobulin cDNA", BMC BIOINFORMATICS, 2006
YU X, NAT IMMUNOL., vol. 10, 2009, pages 48 - 57

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115043910A (zh) * 2022-03-25 2022-09-13 湖南中晟全肽生化有限公司 抑制tigit与cd155结合的多肽及其应用
CN116003606A (zh) * 2023-01-03 2023-04-25 上海百英生物科技股份有限公司 一种tigit纳米抗体及其制备方法与应用
CN116003606B (zh) * 2023-01-03 2023-06-20 上海百英生物科技股份有限公司 一种tigit纳米抗体及其制备方法与应用

Also Published As

Publication number Publication date
CN113563470A (zh) 2021-10-29
CN113563470B (zh) 2023-02-10
EP4108684A4 (en) 2023-10-25
US11897955B2 (en) 2024-02-13
EP4108684A1 (en) 2022-12-28
US20230272064A1 (en) 2023-08-31

Similar Documents

Publication Publication Date Title
JP6908722B2 (ja) 抗ｐｄ−１抗体及びその使用
US20230348602A1 (en) Novel anti-pd-1 antibodies
US10696745B2 (en) Anti-PD-L1 antibodies
US11345754B2 (en) Monoclonal antibody antagonizing and inhibiting the binding of human PD-1 antigen to its ligand, preparation method therefor and application thereof
JP6993992B2 (ja) 抗ｐｄ－１抗体、その産生方法及びその使用方法
US8735553B1 (en) Anti-PD1 antibodies and their use as therapeutics and diagnostics
WO2021217893A1 (zh) 结合tigit抗原的抗体及其制备方法与应用
WO2016000619A1 (en) Anti-pd-l1 antibodies and their use as therapeutics and diagnostics
CN111196852A (zh) 抗tigit抗体及其用途
US20220267438A1 (en) Trispecific antibody targeting bcma, gprc5d, and cd3
WO2020056790A1 (zh) 特异结合人及猴cd38抗原的单克隆抗体及其制备方法与应用
WO2023088337A1 (zh) 抗tigit-抗pd-l1双特异性抗体、其药物组合物及用途
WO2022100694A1 (zh) 抗体及其制备方法
EP4154910A1 (en) Binding protein having h2l2 and hcab structures
WO2023186111A1 (zh) 一种靶向cd40的抗原结合蛋白及其制备和应用
TWI833227B (zh) 靶向pd-l1和cd73的特異性結合蛋白及其應用
WO2022068891A1 (zh) Pd-1抗体及其制备方法与应用
WO2023241629A1 (zh) 抗cldn18.2抗体、其药物组合物及用途
US20230365714A1 (en) Antibodies capable of binding to ror2 and bispecific antibodies binding to ror2 and cd3
RU2745560C2 (ru) 5-бром-2,6-ди-(1н-пиразол-1-ил)пиримидин-4-амин для применения в лечении рака
KR20230012559A (ko) 암 치료를 위한 결합 분자
KR20240103034A (ko) Tigit 및 pd-l1에 대한 이중특이적 항체, 그의 의약 조성물, 및 그의 용도
CN116554325A (zh) 抗b7h3抗体及其应用
CN112079924A (zh) Pd-l1靶向结合剂及其用途

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: 20933685

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020933685

Country of ref document: EP

Effective date: 20220923

NENP Non-entry into the national phase

Ref country code: DE