[go: up one dir, main page]

WO2023125561A1 - 靶向tigit的抗体和双特异性抗体及其应用 - Google Patents

靶向tigit的抗体和双特异性抗体及其应用 Download PDF

Info

Publication number
WO2023125561A1
WO2023125561A1 PCT/CN2022/142471 CN2022142471W WO2023125561A1 WO 2023125561 A1 WO2023125561 A1 WO 2023125561A1 CN 2022142471 W CN2022142471 W CN 2022142471W WO 2023125561 A1 WO2023125561 A1 WO 2023125561A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
tigit
heavy chain
amino acid
Prior art date
Application number
PCT/CN2022/142471
Other languages
English (en)
French (fr)
Inventor
刘佳建
王凤坡
邓洪渊
栾彦
王翠辉
张振
Original Assignee
上海健信生物医药科技有限公司
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 上海健信生物医药科技有限公司 filed Critical 上海健信生物医药科技有限公司
Publication of WO2023125561A1 publication Critical patent/WO2023125561A1/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
    • 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
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/90—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • 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 invention belongs to the field of biomedicine, and in particular relates to an antibody targeting TIGIT, a bispecific antibody and applications thereof.
  • TIGIT T cell immune receptor containing Ig and ITIM domains
  • WUCAM WUCAM
  • Vstm3 or VSIG9 WUCAM
  • CD155 family CD155 family
  • TIGIT is a type I transmembrane protein, expressed on T cells (including activated T cells, memory T cells, regulatory T cells, and follicular T helper cells) and NK cells, and plays a role in suppressing T cells and NK cell-mediated Plays an important role in the activity of anti-tumor immune function.
  • the amino acid sequence of mature human TIGIT contains 223 amino acid (aa) residues (NCBI accession number: NM_173799).
  • the extracellular domain (ECD) of mature human TIGIT consists of 120 amino acid residues with a type V Ig-like domain, followed by a 21 amino acid transmembrane sequence, and an immunoreceptor tyrosine-based inhibitory
  • the 82 amino acid cytoplasmic domain of the sex motif (ITIM) Within the ECD, human TIGIT shares 59% and 87% sequence homology with mouse and cynomolgus monkey, respectively.
  • TIGIT maintains a "many-to-many" interaction relationship with poliovirus receptor (PVR; CD155), nectin2 (CD112), CD96, and CD226.
  • PVR poliovirus receptor
  • CD155 a competitive co-stimulatory receptor
  • CD226 multiple ligands
  • ligands are mainly expressed on APCs (such as dendritic cells and macrophages) and tumor cells.
  • APCs such as dendritic cells and macrophages
  • TIGIT initiates inhibitory signaling in immune cells when bound by its ligands CD155 and CD112.
  • the binding affinity of TIGIT to CD155 (KD: about 1 nM) is much higher than that of CD112, and whether the TIGIT:CD112 interaction is functionally relevant in mediating inhibitory signals remains to be determined.
  • TIGIT potentially inhibits innate and adaptive immunity through multiple mechanisms: 1. After TIGIT-CD155 binding, CD155-expressing dendritic cells (DC) may be tolerogenic, reducing the production of IL-12 and the production of IL-10 Increase; 2. TIGIT inhibits NK cell degranulation, cytokine production and NK cell-mediated tumor cytotoxicity, TIGIT is expressed on Treg, and enhances immunosuppressive function and stability. 3. TIGIT can destroy the cis-dimerization of DNAX accessory molecule 1 (DNAM-1) on the cell surface, thereby preventing the interaction between DNAM-1 and CD155. TIGIT blocks CD155-mediated activation of CD226. 4. Compared with DNAM-1, TIGIT binds CD155 with higher affinity, so it may compete with DNAM-1 to interact with CD155. 5. TIGIT can also directly transmit inhibitory signals to T cells and NK cells through its cytoplasmic tail.
  • DC dendritic cells
  • TIGIT and other such co-inhibitory molecules play a role in tumor cell escape from immune surveillance. Similar to other co-inhibitory receptors such as CTLA-4, PD-1 and BTLA, TIGIT can function to "turn off" the immune response.
  • CTLA-4, PD-1 and BTLA co-inhibitory receptors
  • TIGIT can function to "turn off" the immune response.
  • antibody blockade of both PD-L1 and TIGIT resulted in a synergistic increase in CD8+ T cell-mediated tumor rejection. Grogan et al. (2014) J. Immunol. 192(1) Suppl. 203.15; Johnston et al. (2014) Cancer Cell 26:1-15. Similar results were obtained in animal models of melanoma.
  • TIGIT is upregulated in a variety of tumor infiltrating lymphocytes, such as melanoma, breast cancer, non-small cell lung cancer, colon cancer, acute myeloid leukemia (AML) and multiple myeloma.
  • High expression of TIGIT is associated with tumor progression and poor prognosis.
  • TIGIT is a new target with high hopes, and combining with PD-L1 is a promising anticancer treatment strategy in the future.
  • Most of the existing antibodies are monoclonal antibodies targeting the TIGIT target or anti-TIGIT monoclonal antibodies are used in combination with other target monoclonal antibodies, which have problems such as high cost, large side effects, and limited clinical effects, and the only bispecific Antibody BMS/Agenus' AGEN1777 and Innovent/Eli Lilly's IBI321 bispecific antibody are only in the early stages of clinical trials.
  • TIGIT target or anti-TIGIT monoclonal antibodies are used in combination with other target monoclonal antibodies, which have the disadvantages of high cost, large side effects, and limited clinical effect, and are not yet available.
  • No TIGIT bispecific antibody has entered late-stage clinical trials.
  • the invention provides an antibody targeting TIGIT, a bispecific antibody and applications thereof.
  • the antibody targeting TIGIT can well bind human TIGIT protein and hTIGIT+ cells, has strong affinity with the two target antigens, can effectively block the combination of human TIGIT and human PVR, can synergistically enhance T cell activity, and promote IL-2 is released, and the synergistic effect is better than that of the combined administration group; it can effectively inhibit the growth of tumor cells; it can also be combined with non-human primate TIGIT, especially providing a new way for the combination of PD-1 antibody to treat tumors, Even better options.
  • an antibody or antigen-binding fragment targeting TIGIT which includes a light chain variable region and/or a heavy chain variable region;
  • the light chain variable region comprises: CDR1 of the amino acid sequence shown in SEQ ID NO:5, CDR2 of the amino acid sequence shown in SEQ ID NO:6 and CDR3 of the amino acid sequence shown in SEQ ID NO:7;
  • the heavy chain can be
  • the variable region comprises: CDR1 of the amino acid sequence shown in SEQ ID NO:8, CDR2 of the amino acid sequence shown in SEQ ID NO:9 and CDR3 of the amino acid sequence shown in SEQ ID NO:10, and the above CDR is The CDR sequence defined by CCG, and the CDR sequence information determined by CCG and other definitions are shown in the following tables a-e.
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 GFTFSSYTMS (SEQ ID NO: 8) heavy chain CDR2 EISSSGGSTYYPDTVKG (SEQ ID NO:9) heavy chain CDR3 PGLGAWFAY (SEQ ID NO: 10)
  • the anti-TIGIT antibody mab22 of the present invention defines the CDR sequence according to Kabat
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 SYTMS (SEQ ID NO: 11) heavy chain CDR2 EISSSGGSTYYPDTVKG (SEQ ID NO:9) heavy chain CDR3 PGLGAWFAY (SEQ ID NO: 10)
  • the anti-TIGIT antibody mab22 of the present invention defines the CDR sequence according to AbM
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 GFTFSSYTMS (SEQ ID NO: 8) heavy chain CDR2 EISSSGGSTY (SEQ ID NO: 12) heavy chain CDR3 PGLGAWFAY (SEQ ID NO: 10)
  • the anti-TIGIT antibody mab22 of the present invention defines the CDR sequence according to Chothia
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6)
  • the anti-TIGIT antibody mab22 of the present invention defines the CDR sequence according to Contact
  • Antibody mab22 CDRs light chain CDR1 VHNSGNTYLEWY (SEQ ID NO: 15) light chain CDR2 LLIYKVSNRF (SEQ ID NO: 16) light chain CDR3 FQFSHVPR (SEQ ID NO: 17) heavy chain CDR1 SSYTMS (SEQ ID NO: 18) heavy chain CDR2 LVAEISSSGGSTY (SEQ ID NO: 19) heavy chain CDR3 ARPGLGAWFA (SEQ ID NO: 20)
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 23-25 or a mutation thereof; the mutation has a or multiple amino acid residue substitutions, deletions or additions, preferably having at least 99% sequence identity with the original amino acid sequence, and the mutations maintain or improve the binding of the binding protein to TIGIT.
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 26-27 or a mutation thereof; the mutation has a or multiple amino acid residue substitutions, deletions or additions, preferably having at least 99% sequence identity with the original amino acid sequence, and the mutations maintain or improve the binding of the binding protein to TIGIT.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or, the light chain can be The variable region comprises the amino acid sequence shown in SEQ ID NO:23, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 24, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 26; or, the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 25, the The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26; or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23, and the heavy chain variable region comprises the amino acid sequence shown in The amino acid sequence shown in SEQ ID NO:27; Or, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:
  • the antibody or antigen-binding fragment targeting TIGIT is an antibody, Fab, Fab', F(ab') 2 , Fv, scFv, bispecific antibody, multispecific antibody, Single domain antibody or single domain antibody, or monoclonal antibody or polyclonal antibody prepared from the above-mentioned antibodies.
  • the TIGIT-targeting antibody or antigen-binding fragment includes a human antibody light chain constant region and a human antibody heavy chain constant region.
  • the light chain constant region of the human antibody is a ⁇ or ⁇ type light chain constant region
  • the heavy chain constant region of the human antibody is a heavy chain constant region of hIgG1, hIgG2, hIgG3, hIgG4 or its mutation.
  • the light chain of the TIGIT-targeting antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 32 or its mutation
  • the heavy chain of the TIGIT-targeting antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31 or a mutation thereof.
  • the amino acid sequence of the light chain is shown in SEQ ID NO:28; the amino acid sequence of the heavy chain is shown in SEQ ID NO:29; or, the amino acid sequence of the light chain is shown in SEQ ID NO:30 shown; the amino acid sequence of the heavy chain is shown in SEQ ID NO:29; or, the amino acid sequence of the light chain is shown in SEQ ID NO:30; the amino acid sequence of the heavy chain is shown in SEQ ID NO:31 or, the amino acid sequence of the light chain is shown in SEQ ID NO:32; the amino acid sequence of the heavy chain is shown in SEQ ID NO:31.
  • the second technical solution provided by the present invention is: a bispecific antibody or multispecific antibody targeting TIGIT, which includes a first protein functional region and a second protein functional region, wherein the The first protein functional region is the TIGIT-targeting antibody or antigen-binding fragment as described in one of the technical solutions; the second protein functional region is a non-TIGIT-targeting antibody.
  • the first protein functional region and the second protein functional region are respectively selected from immunoglobulin, scFv, Fab, Fab' or F(ab') 2 , and the first protein functional region and the second protein functional region At most one protein domain in the domain is an immunoglobulin.
  • the constant region of the immunoglobulin includes a human antibody light chain constant region and a human antibody heavy chain constant region.
  • the human antibody light chain constant region is ⁇ chain or ⁇ chain
  • the human antibody heavy chain constant region is hIgG1, hIgG2, hIgG3, hIgG4 or mutations thereof.
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is one or more scFv
  • the scFv includes a heavy chain variable region and a light chain variable region. region, the heavy chain variable region and the light chain variable region are connected by a linker.
  • the scFv is connected to the immunoglobulin through a linker, the linker is preferably (G 4 S) w or (G 2 S) W , and the w is preferably an integer between 0 and 10, More preferably 1, 2, 3 or 4.
  • the scFv is a light chain variable region-linker-heavy chain variable region, the N-terminal of the light chain variable region or the C-terminal of the heavy chain variable region are connected correspondingly in the immunological
  • the C-terminal is correspondingly connected to the C-terminal or N-terminal of the immunoglobulin light chain and/or heavy chain through a linker.
  • the linker in the scFv is VE(G 2 S) W GGVD or (G 4 S) w , and the w is preferably an integer between 0 and 10, more preferably 1, 2, 3 or 4.
  • the scFv is connected to the immunoglobulin through a linker, the linker is (G 4 S) 3 or (G 2 S) 4 , and/or, the scFv
  • the quantity is two, and two scFvs are connected symmetrically at the C-terminal or N-terminal of the light chain and/or heavy chain of the immunoglobulin; the scFv is connected with the heavy chain of the immunoglobulin.
  • the scFv has a light chain variable region-linker-heavy chain variable region structure, the C-terminus of the light chain variable region of the scFv is connected to a linker, and the linker is connected to a heavy chain variable region.
  • the C-terminal of the heavy chain variable region of the scFv is connected to the N-terminal of the immunoglobulin heavy chain through the linker; or, the scFv is a heavy chain variable region-linker - light chain variable region structure, the N-terminal of the light chain variable region of the scFv is connected with the linker, and the linker is connected with the C-terminal of the heavy chain variable region, the heavy chain of the scFv can be The N-terminus of the variable region is linked to the C-terminus of the immunoglobulin heavy chain.
  • the linker in the scFv is VE(G 2 S) 4 GGVD or (G 4 S) 3 .
  • the second protein functional region targets PD-1/PD-L1, Claudin18.2, TIM-3 or LAG-3.
  • the second protein functional region is an antibody targeting PD-1/PD-L1.
  • the antibody targeting PD-1/PD-L1 is Nivolumab, Atezolizumab, Pembrolizumab, Durvalumab or Avelumab.
  • the C-terminals of the heavy chains are mutated from K to A; and/or, the second protein functional region It is two identical scFv or its mutation, wherein, the light chain variable region of the scFv is the light chain variable region of Nivolumab, Atezolizumab, Pembrolizumab, Durvalumab or Avelumab, and the heavy chain variable region of the scFv is Nivolumab, In the heavy chain variable region of Atezolizumab, Pembrolizumab, Durvalumab or Avelumab, the mutation preferably has at least 99% sequence identity with the original amino acid sequence and maintains or improves the function of the antibody.
  • the bispecific antibody targeting TIGIT is selected from the following groups:
  • the first protein functional region is an immunoglobulin, the amino acid sequence of its light chain is shown in SEQ ID NO: 32, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 31;
  • the second protein The functional region is two identical scFvs, and the linker is (G 4 S) 3 or VE(G 2 S) 4 GGVD;
  • the number of the scFv is two; the scFv is a light chain variable region-linker-heavy chain variable region structure, and the C-terminals of the heavy chain variable regions of the two scFvs pass through (G 4 S) 3 or (G 2 S) 4 is symmetrically connected to the N-terminals of the two heavy chains of the immunoglobulin, and the C-terminals are mutated from K to A; or,
  • the first protein functional region is an immunoglobulin, the amino acid sequence of its light chain is shown in SEQ ID NO: 32, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 31;
  • the second protein The functional region is two identical scFvs, and the linker is (G 4 S) 3 or VE(G 2 S) 4 GGVD;
  • the number of the scFv is two; the scFv is a heavy chain variable region-linker-light chain variable region structure, and the N-terminals of the heavy chain variable regions of the two scFv are respectively passed through (G 4 S) 3 or (G 2 S) 4 are symmetrically linked at the C-termini of the two heavy chains of the immunoglobulin.
  • the bispecific antibody targeting TIGIT comprises the following amino acid sequence:
  • Amino acid sequence such as the light chain shown in SEQ ID NO:32 such as the amino acid sequence of the heavy chain shown in SEQ ID NO:33; or, the amino acid sequence such as the light chain shown in SEQ ID NO:32, such as SEQ ID NO The amino acid sequence containing the heavy chain shown in: 34; Or, the amino acid sequence such as the light chain shown in SEQ ID NO: 32, the amino acid sequence containing the heavy chain shown in SEQ ID NO: 35; Or, the amino acid sequence is such as SEQ ID NO: For the light chain shown in ID NO:32, the amino acid sequence containing the heavy chain as shown in SEQ ID NO:36; or, for the light chain shown in SEQ ID NO:32, as shown in SEQ ID NO:37 The amino acid sequence containing the heavy chain; or, the amino acid sequence such as the light chain shown in SEQ ID NO:32, the amino acid sequence containing the heavy chain as shown in SEQ ID NO:38; or, the amino acid sequence such as SEQ ID NO:32 The light chain shown, the amino acid
  • the third technical solution provided by the present invention is: an isolated nucleic acid encoding the TIGIT-targeting antibody or antigen-binding fragment as described in the first technical solution, or as described in the second technical solution Bispecific antibody or multispecific antibody targeting TIGIT.
  • the fourth technical solution provided by the present invention is: an expression vector comprising the isolated nucleic acid according to the third technical solution.
  • the fifth technical solution provided by the present invention is: a host cell comprising the expression vector according to the fourth technical solution.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the sixth technical solution provided by the present invention is: a method for preparing a TIGIT-targeting antibody or antigen-binding fragment, or a TIGIT-targeting bispecific antibody or multispecific antibody, which comprises culturing such as In the host cell of the fifth technical solution, the TIGIT-targeting antibody or antigen-binding fragment, or the TIGIT-targeting bispecific antibody or multispecific antibody is obtained from culture.
  • the seventh technical solution provided by the present invention is: an antibody-drug conjugate comprising a cytotoxic agent, and an antibody or antigen-binding fragment targeting TIGIT as described in one of the technical solutions, or The bispecific antibody or multispecific antibody targeting TIGIT as described in the second technical solution.
  • the eighth technical solution provided by the present invention is: a pharmaceutical composition comprising the TIGIT-targeting antibody or antigen-binding fragment as described in the technical solution one, and the TIGIT-targeting antibody or antigen-binding fragment described in the second technical solution.
  • the pharmaceutical composition further comprises an antibody targeting PD-1/PD-L1, and the antibody targeting PD-1/PD-L1 is Nivolumab, Atezolizumab, Pembrolizumab, Durvalumab and/or Avelumab.
  • the pharmaceutical composition comprises an antibody or antigen-binding fragment targeting TIGIT and an antibody targeting PD-1/PD-L1, and the light chain amino acid sequence of the antibody or antigen-binding fragment targeting TIGIT is as follows: As shown in SEQ ID NO: 32, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 34, and the antibody targeting PD-1/PD-L1 is Atezolizumab.
  • the ninth technical solution provided by the present invention is: a set of medicine boxes, which includes medicine box A and medicine box B, wherein:
  • the kit A comprises the TIGIT-targeting antibody or antigen-binding fragment as described in one of the technical solutions, the TIGIT-targeting bispecific antibody or multispecific antibody as described in the second technical solution, and/or the TIGIT-targeting antibody or antigen-binding fragment as described in the second technical solution.
  • Kit B contains an antibody targeting PD-1/PD-L1, and the antibody targeting PD-1/PD-L1 is Nivolumab, Atezolizumab, Pembrolizumab, Durvalumab and/or Avelumab.
  • the kit A comprises an antibody targeting TIGIT or an antigen-binding fragment
  • the light chain amino acid sequence of the TIGIT-targeting antibody or antigen-binding fragment is shown in SEQ ID NO:32
  • the heavy chain amino acid sequence is shown in SEQ ID NO:32.
  • the kit B includes an antibody targeting PD-1/PD-L1, and the antibody targeting PD-1/PD-L1 is Atezolizumab.
  • the tenth technical solution provided by the present invention is: the TIGIT-targeting antibody or antigen-binding fragment described in the first technical solution, the TIGIT-targeted bispecific antibody or the The multispecific antibody, the antibody drug conjugate described in the seventh technical scheme, the pharmaceutical composition described in the eighth technical scheme and/or the drug combination described in the ninth technical scheme are used in diagnosis, treatment and/or prevention Applications in cancer medicine.
  • the cancer is a solid tumor or a liquid tumor, such as colon cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, liver cancer, epithelial squamous cell carcinoma, esophageal cancer, rectal cancer, kidney cancer, ovarian cancer , bladder cancer, pancreatic cancer, melanoma and glioma
  • the liquid tumors such as acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma and diffuse large B-cell lymphoma.
  • the eleventh technical solution provided by the present invention is: the TIGIT-targeting antibody or antigen-binding fragment as described in the first technical solution, and the TIGIT-targeted bispecific antibody described in the second technical solution or multispecific antibody, the antibody drug conjugate described in the seventh technical scheme, the pharmaceutical composition described in the eighth technical scheme and/or the drug combination as described in the ninth technical scheme in diagnosis, treatment and/or Application in cancer prevention.
  • the cancer comprises solid tumors and/or liquid tumors, such as colon cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, liver cancer, epithelial squamous cell carcinoma, esophageal cancer, rectal cancer, kidney cancer, Ovarian cancer, bladder cancer, pancreatic cancer, melanoma and glioma, the liquid tumors such as acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma and Diffuse large B-cell lymphoma.
  • liquid tumors such as acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma and Diffuse large B-cell lymphoma.
  • the twelfth technical solution provided by the present invention is: the TIGIT-targeting antibody or antigen-binding fragment as described in the first technical solution for diagnosis, treatment and/or prevention of cancer, the second technical solution
  • the bispecific antibody or multispecific antibody targeting TIGIT, the antibody drug conjugate described in the seventh technical scheme, the pharmaceutical composition described in the eighth technical scheme, and/or the pharmaceutical composition described in the ninth technical scheme The above-mentioned drug combination.
  • the cancer comprises solid tumors and/or liquid tumors, such as colon cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, liver cancer, epithelial squamous cell carcinoma, esophageal cancer, rectal cancer, kidney cancer, Ovarian cancer, bladder cancer, pancreatic cancer, melanoma and glioma, the liquid tumors such as acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma and Diffuse large B-cell lymphoma.
  • liquid tumors such as acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma and Diffuse large B-cell lymphoma.
  • the thirteenth technical solution provided by the present invention is: an antibody or antigen-binding fragment targeting TIGIT as described in the first technical solution, or a TIGIT-targeting antibody as described in the second technical solution Preparations of bispecific antibodies or multispecific antibodies,
  • the formulation comprises citric acid-sodium citrate, Tween 80 and the TIGIT-targeting antibody or antigen-binding fragment, or the TIGIT-targeting bispecific antibody or multispecific antibody.
  • the formulation further includes one or more of arginine, glutamic acid and trehalose.
  • the pH of the formulation is 5.5-6.5.
  • the preparation comprises 20 mM citric acid-sodium citrate, 50 mM arginine, 50 mM glutamic acid, 200 mM trehalose, 0.02% Tween 80 and 14.2 mg/mL
  • the antibody or antigen-binding fragment targeting TIGIT, or the bispecific antibody or multispecific antibody targeting TIGIT, the pH of the preparation is 6.0.
  • the preparation includes 20 mM His-HCl, 50 mM arginine or glutamic acid, 200 mM trehalose, 0.02% PS80 and 11.4 mg/mL of the TIGIT-targeting antibody Or the antigen-binding fragment, or the bispecific antibody or multispecific antibody targeting TIGIT, the pH of the preparation is 6.0.
  • the preparation includes 20 mM His-HCl, 125 mM glycine, 125 mM trehalose, 0.02% PS80 and 11 mg/mL of the antibody or antigen-binding fragment targeting TIGIT, or the The bispecific antibody or multispecific antibody targeting TIGIT, the pH of the preparation is 6.0.
  • the preparation includes 20 mM His-HCl, 50 mM arginine hydrochloride, 150 mM trehalose, 0.02% Tween 80 and 11.6 mg/mL of the TIGIT-targeted
  • the pH of the preparation is 6.0.
  • targeting TIGIT and “anti-TIGIT” both refer to binding specificity to TIGIT, and can be used interchangeably.
  • first and “second” in the present invention have no practical meaning, and are only used to distinguish the same terms.
  • “One pair” and “two”, “two pairs” and “four” have the same meaning when referring to the number of scFv or cytokine or cytokine receptor or Fab' or F(ab') 2 .
  • “One” and “one”, “two” and “two” have the same meaning when referring to the number of light or heavy chains or light or heavy chain variable regions.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • antibody generally refers to an immunoglobulin composed of two pairs of polypeptide chains, each pair having a light (L) chain and a heavy (H) chain.
  • a heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody
  • a light chain refers to a polypeptide chain with a smaller molecular weight in an antibody.
  • Light chains can be classified as kappa and lambda light chains.
  • Heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • CL light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs)] interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino terminus to carboxyl terminus.
  • the variable regions (VH and VL) corresponding to each heavy chain/light chain form the antibody binding site, respectively.
  • the heavy chain may also comprise more than 3 CDRs, such as 6, 9 or 12.
  • the heavy chain in the bispecific antibody of the present invention, can be the ScFv of another antibody linked to the N-terminal of the heavy chain of the IgG antibody, in this case the heavy chain contains 9 CDRs.
  • Antibody when the term “antibody” is referred to, it includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • Antigen-binding fragment refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to an antigen, which is also referred to as Referred to as "antigen-binding portion”. See generally Fundamental Immunology, Ch. 7, Paul, W., ed., 2nd Ed., Raven Press, NY (1989), which is hereby incorporated by reference in its entirety.
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain binding fragments (e.g., scFv), chimeric antibodies, bis Antibodies (diabodies) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide.
  • Fv means an antibody fragment consisting of VL and VH domains of a single arm of an antibody
  • Fab means an antibody fragment consisting of VL, VH, CL and CH1 (or CH) domains
  • F( ab′) 2 ′′ means an antibody fragment comprising two Fab fragments linked by a disulfide bond at the hinge region.
  • the antigen-binding fragment of an antibody is a single-chain binding fragment (e.g., a scFv) in which VL and VH are paired to form a monovalent molecule via a linker that enables production as a single polypeptide chain (see, e.g., Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
  • scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeating G4S amino acid sequence or variants thereof. For example, linkers having the amino acid sequence ( G4S ) 4 or ( G4S ) 3 may be used, although variants thereof may also be used.
  • Antigen-binding fragments such as the above-mentioned antibody fragments
  • Those skilled in the art can use conventional techniques (such as recombinant DNA technology or enzymatic or chemical cleavage methods) to obtain its antigen-binding fragments (such as the above-mentioned antibody fragments) from a given antibody, and use the same method as screening intact antibodies. Antigen-binding fragments were screened for specificity.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide is isolated with high purity from this natural state, which is called isolated. .
  • isolated does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • the term "host cell” refers to cells that can be used to introduce vectors, including but not limited to, prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, such as S2 Drosophila cells or Sf9 Insect cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells and HEK 293 cells etc. human or animal cells.
  • KD refers to the dissociation equilibrium constant (KD) of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • KD dissociation equilibrium constant
  • the antibody binds the antigen with a dissociation equilibrium constant of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less, e.g. , as determined in a BIACORE instrument using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the reagents and raw materials used in the present invention are all commercially available.
  • the TIGIT-targeting antibody and bispecific antibody of the present invention have better affinity and better function than the prior art.
  • the TIGIT-targeting antibody of the present invention has high degree of humanization, low immunogenicity, is combined with non-human primate antigen, and is convenient for preclinical safety evaluation.
  • the structure of the bispecific antibody described in the present invention is simple, and the bispecific antibody similar to the structure of conventional antibody IgG makes the purification simple and easy; the molecule is stable, which provides great convenience for later development; the expression is high and the cost is low; the double target is retained Point binding and blocking activity; enhanced T cell functional activity and better drug efficacy in mice, better than combined administration.
  • Figure 1 shows the activity of some preferred humanized antibodies of the present invention to block the binding of hTIGIT to PVR.
  • Fig. 2 is an SDS-PAGE diagram of some preferred bispecific antibodies of the present invention.
  • Fig. 3 is the SDS-PAGE detection result after the preferred bispecific antibody of the present invention is treated at 40°C for 28 days.
  • Fig. 4 is the activity of the preferred bispecific antibody of the present invention to induce activated T cells to release IL-2.
  • Figure 5 shows the anti-tumor activity of preferred humanized antibodies and bispecific antibodies of the present invention.
  • the antigen used in the present invention may be purchased from the following companies: Beijing Yiqiao Shenzhou Technology Co., Ltd. cyno TIGIT-hFc (Cat#MB12OC2203), mouse TIGIT-his (Cat#MB09AP0804), or Taizhou Baiying Biotechnology Co., Ltd. PD-L1 - mFc (Cat#1576), or obtained by expression and purification of the present invention.
  • the expressed human TIGIT (-his, -Fc Tag or -mFc tag) sequence is NCBI Reference Sequence: NP_776160.2, with a total length of 244 amino acids, of which amino acids 1-21 are signal peptides; the extracellular (ECD) region is 22 -141 amino acids; 32-42 amino acids in the extracellular (ECD) region are homodimer formation regions, 39-127 amino acids are Ig regions, and 32 and 101 are glycosylation sites.
  • the expressed rhesus monkey TIGIT (RhTIGIT-mFc tag) sequence is NCBI Reference Sequence: XP_014985302.2, with a total length of 245 amino acids, of which amino acids 1-21 are the signal peptide; amino acids 39-128 are the Ig region.
  • the human CD155 (PVR, hFc tag) protein sequence is GenBank: AAA36462.1, with a total length of 392 amino acids, of which amino acids 1-20 are signal peptides; the extracellular (ECD) region is amino acids 21-343. Among them, amino acids 43-142 are the Ig1_PVR_like region, and amino acids 145-240 are the Ig2_Nectin-2_like region.
  • the human PD-1 (hFc/his tag) protein sequence is NCBI Reference Sequence: NP_005009.2, with a total length of 288 amino acids, of which the 1-20th is the signal peptide; the ECD is the 21-167th amino acid.
  • the human PD-L1 (hFc/his tag) protein sequence is NCBI Reference Sequence: NP_054862.1, with a total length of 290 amino acids, of which the 1-18th is the signal peptide; the ECD is the 19th-239th amino acid.
  • the hFc tag antigens used in the present invention are all connected to the IgG1 Fc region at the C-terminal, and the his tag antigen is connected to 6 ⁇ his at the C-terminal.
  • the mFc tag antigens are all connected to the mIgG2a Fc region at the C-terminus.
  • Antibodies used in the present invention include positive control antibody Ref1 (ie Roche RG6058, sequence from WO2015009856A2, #19 light chain, #17 heavy chain) and positive control antibody Ref2 (ie 22G2 of BMS, sequence from WO2016/106302A1, #9 light chain , #7 heavy chain) were expressed and purified by the present invention.
  • positive control antibody Ref1 ie Roche RG6058, sequence from WO2015009856A2, #19 light chain, #17 heavy chain
  • positive control antibody Ref2 ie 22G2 of BMS, sequence from WO2016/106302A1, #9 light chain , #7 heavy chain
  • the pTT5 vector (Bio Vector, Cat#102762) was used for expression.
  • the expressed recombinant protein, antibody light and heavy chain sequences were cloned into pTT5 vector, expressed by transient transfection into HEK293E cells (Life Technologies, Cat#11625019), and then purified.
  • 293 cells were expanded in SMM 293-TII Expression Medium (Beijing Sino Biological Technology Co., Ltd., Cat#M293TII) medium. 48 hours before the start of the transient, the cell concentration was adjusted to 1 ⁇ 10 6 cells/ml, and cultured in a shaker for 48 hours. The culture conditions were 36.5° C., 6% CO 2 , and 120 rpm. Before transfection, the survival rate was checked again under the microscope, and the cell concentration was 4 ⁇ 10 6 cells/mL.
  • SMM 293-TII Expression Medium Beijing Sino Biological Technology Co., Ltd., Cat#M293TII
  • Purification of antibody or -Fc fusion protein Centrifuge the sample at high speed to remove impurities, and equilibrate the gravity column containing Protein A (Mabselect, GE Healthcare Life Science, Cat#71-5020-91AE) with PBS pH7.4 (Shenggong Biotechnology, Cat# F506606-0001), wash with 2-5 column volumes. The sample was passed through the column, the flow rate was controlled, and the retention time was 5 min. Wash the column with 5-10 column volumes of PBS (Sangon, Cat#B548117-0500). The target protein was then eluted with pH 3.5 0.1M acetic acid, adjusted to neutrality with pH 8.0 Tris-HCl, and the concentration was measured with a microplate reader.
  • Purification of His Tag protein Centrifuge the sample at high speed to remove impurities; equilibrate nickel column (Ni smart beads 6FF, Changzhou Tiandi Renhe Biotechnology Co., Ltd., Cat#SA036010), use PBS (pH7.4) solution containing 10mM imidazole 0.5M NaCl Equilibrate nickel column, wash with 2-5 column volumes. Pass the supernatant to be purified through the column. Control the flow rate so that the retention time is 5 min.
  • Rinse impurity proteins Use PBS (pH7.4) solution containing 10mM imidazole and 0.5M NaCl to wash the chromatography column to remove non-specific binding impurity proteins, and collect the effluent. The target protein was eluted with PBS (pH7.4) containing 250mM imidazole 0.5M NaCl.
  • Buffer replacement pass the eluted and neutralized hFc/mFc tag or eluted his tag protein through an ultrafiltration tube, centrifuge at 12000g for 10min, (ultrafiltration tube, Merck Millipore, Cat#UFC500308), if the volume is large, you can Centrifuge repeatedly until all proteins are concentrated, add PBS, centrifuge twice to remove residual buffer as much as possible, invert the ultrafiltration tube into a new collection tube, centrifuge at low speed for 1 min, then add 1 mL of PBS, measure the concentration, aliquot, Store for later use.
  • Example 2 Overexpression cell line construction and cell activity (ELISA) detection
  • the overexpression cell line CHO-K1-T1001 used in the present invention is constructed by the inventors through our company's stable cell line construction platform. Preservation Committee cell bank, Cat#GNHu17) were seeded in two 6cm culture dishes, 7.5 ⁇ 10 5 cells/dish.
  • the 293T cell culture medium was replaced with 4 mL DMEM high-glucose medium (Shanghai Yuanpei Biotechnology Co., Ltd., Cat#L130KJ).
  • 5 ⁇ 10 5 CHO-K1 cells (Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences, Cat#SCSP-507) were inoculated on a 10 cm culture dish.
  • the cells were subcultured on the 7th day, and from the 8th day, 10 ⁇ g/mL puromycin was added to the cells for selection (Shanghai Yuanpei Biotechnology Co., Ltd., Cat#S250J0). After 2-3 days, a large number of cells died, and the medium was replaced to continue culturing until the cells no longer died, and the cells were amplified in large numbers, screened for monoclonal cell lines, expanded, and frozen for preservation.
  • the amino acid sequence NP_776160.2 of the human TIGIT (pTBE1001) used in this embodiment example has a total length of 244 amino acids, wherein the 1-21 position is a signal peptide; the 22-244 position is expressed by the CHO-K1 TIGIT+ cell line constructed in the present invention protein sequence.
  • TIGIT-hFc Human TIGIT-hFc, TIGIT-his, rhesus monkey TIGIT-mFc (RhTIGIT-mFc), cynomolgus monkey TIGIT-hFc (cyno TIGIT-hFc), mouse TIGIT-his ( muTIGIT-his) or recombinant proteins such as PD1 and PD-L1 were diluted to 5 ⁇ g/mL or 2 ⁇ g/mL, added to a 96-well microtiter plate (Corning, Cat#CLS3590-100EA) at a volume of 50 ⁇ L/well, and incubated at 37°C in the box for 2 hours.
  • PD1 and PD-L1 recombinant proteins
  • skimmed milk (bright skimmed milk powder) blocking solution diluted with PBS, and incubate in an incubator at 37°C for 3 hours or place at 4°C overnight (16-18 hours) for blocking.
  • PBST buffer (1 ⁇ PBS, pH7.4, containing 0.05% Tweeen20)
  • 50 ⁇ L supernatant containing detection antibody
  • 10 ⁇ g/mL initial, 5-fold gradient to each well Dilute the antibody to be tested, incubate at 37°C for 1 hour, wash the plate 5 times with PBST, add 1:2500 diluted Anti-mouse Fc-HRP or Anti-human Fc-HRP secondary antibody (Jackson Immuno Research, Cat#115-035- 003 or 109-035-003), 50 ⁇ L/well, and incubated at 37°C for 1 hour.
  • TMB chromogenic substrate KPL, Cat#52-00-03
  • KPL chromogenic substrate
  • 1M H 2 SO 4 50 ⁇ L 1M H 2 SO 4
  • MULTISKAN Go microplate reader ThermoFisher, model: 51119300
  • Example 6 Double-sandwich ELISA method to detect bispecific antibody binding to PD-L1 and TIGIT activity simultaneously
  • Dilute LT023 (TIGIT-his, produced by Shanghai Jianxin Biomedical Technology Co., Ltd.) with PBS at pH 7.4 to 7 ⁇ g/mL, add to 96-well ELISA plate, 100 ⁇ L/well, and incubate overnight at 4°C. After the liquid was discarded, the plate was washed once with PBST (1 ⁇ PBS, pH7.4, containing 0.05% Tweeen20), 250 ⁇ L/well. Block with 230 ⁇ L of 5% skim milk at 37° C. for 3 h. Discard the blocking solution, wash the plate three times with PBST, 250 ⁇ L/well.
  • the 96-well ELISA plate is blocked, dilute the antibody to be tested, the initial concentration is 10 ⁇ g/mL, 5-fold serial dilution, 7 points of dilution, the 8th point uses the diluted solution as a blank control, and the diluted antibody to be tested is pressed Add 100 ⁇ L/well to the ELISA plate, and incubate at 37°C for 1.5h in 1 duplicate well. After washing the plate 4 times with PBST, dilute PD-L1-mFc with antibody diluent to 0.1 ⁇ g/mL, add to the ELISA plate, 100 ⁇ L/well, and incubate at 37°C for 1 h.
  • human TIGIT-his (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Cat#10917-H08H) is used as antigen
  • Freund's adjuvant is used to immunize A/J mice for 4 times, and three days later
  • electrofusion and fusion hybridization are screened Tumors were screened from thousands of hybridoma clones, and it was unexpectedly found that as many as 467 hybridoma clones in this fusion had better binding activity to TIGIT.
  • the present invention uses the method in Example 4 to further screen to obtain a monoclonal cell line that can well block the combination of TIGIT and PVR, from which the murine antibody is obtained.
  • mice used in the experiment female, 4 weeks old, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., animal strain number: N000018. After the mice were purchased, they were raised in a laboratory environment for 1 week, with daytime light/night dark cycle adjustment, temperature 20-25°C, and humidity 40-60%. Mice were divided into 3/group/cage. Immunization was carried out with purchased TIGIT-his antigen.
  • the adjuvant for the first immunization was Freund's complete adjuvant (Sigma-Aldrich, Cat#SIGMA F5506-10M), and the adjuvant from the second immunization was Freund's incomplete adjuvant (Sigma-Aldrich, Cat#SIGMA F5881-10M).
  • the ratio of antigen to adjuvant was 1:1. 100 ⁇ L/25 ⁇ g/monkey for the first immunization, 100 ⁇ L/12.5 ⁇ g/rattle for the second, third, and fourth immunizations, and calf intramuscular injection. 3 days before fusion, 100 ⁇ L/25 ⁇ g/only booster immunization.
  • the time of immunization was 0, 14, 28, 42 and 56 days (booster immunization).
  • the ELISA method of the above-mentioned embodiment 3 was used to detect the antibody titer of mouse serum, and the mice with high antibody titer in the serum and whose titer was in the plateau stage were selected for spleen cell fusion, and spleen lymphocytes were combined with bone marrow Tumor cell Sp2/0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells spread on a 96-well plate, screened, and preferably cloned.
  • the hybridoma cell line was initially screened, and the binding activity of the antibody and human TIGIT in the secreted supernatant of the hybridoma cell line was detected by the ELISA method of Example 3, and a clone with good activity was selected, and the supernatant was obtained as described in Example 4.
  • Methods The secreted antibodies were detected to block the binding activity (Blocking activity) of hTIGIT and hPVR, and 56 clones were found to have good blocking effects.
  • 14 hybridoma cell lines are preferably subjected to 3 rounds of limited dilution, and each round of limited dilution is optimized using the methods in Example 2, Example 3 and Example 4, and finally 6
  • Table 2 The results are shown in Table 2.
  • Table 1 Some initial screening data are listed in Table 1. The data shows that many hybridoma fusion cells showed high binding activity and good blocking effect during the initial screening (close to ref or even better, the higher the ELISA binding value, the better the affinity, and the lower the blocking value, indicating The better the blocking activity), such as 3A10, 6H4, 7H4, 11A4, 17A10 and 23H3 clones. Multiple limited dilutions were performed on the preferred 14 clones (including the above 6 strains). After each round of dilution, 7-10 days after the clones multiplied, the ELISA method was used to re-test the binding activity and concentration of the antibodies (supernatants) secreted by each clone.
  • mice were expanded and counted, and 1 ⁇ 10 7 cells/200 ⁇ L were injected into the peritoneal cavity of mice. After the mice were fed for 6 days, the ascites was collected, and purified by the method in Example 1 to obtain IF071, IF081, and IF091 respectively. , IF101, IF112 and IF122 6 murine antibodies. Then, using the methods in Examples 2, 3 and 4, the binding activities of the 6 murine antibodies to human TIGIT, cynomolgus monkey TIGIT, mouse TIGIT and CHO-K1 cells (CHO-K1-T1001) stably expressing TIGIT were evaluated and Blocking activity, the results are shown in Table 3.
  • IF071, IF081, IF091, IF101, IF112 and IF122 did not bind to mouse TIGIT
  • IF081 did not bind to cynoTIGIT
  • the binding activity of IF091 to hTIGIT, cyno TIGIT and CHO-K1-T1001 blocked the binding of hTIGIT to hPVR
  • the activity of IF091 and CHO-K1-T1001 is 1.73 times better than that of Ref1
  • the activity of IF091 blocking hTIGIT binding to hPVR is 1.83 times better than that of Ref1.
  • the sequence of IF091 is extracted, which is the preferred antibody sequence of murine mab22 in the present invention.
  • Example 8 Sequence extraction, analysis and identification of the murine anti-human TIGIT antibody mab22 antibody of the present invention
  • the process of extracting antibody sequences from monoclonal cell lines preferably obtained from hybridomas is a common method used by those skilled in the art. Specifically, collect the above-mentioned monoclonal cell line, after expansion and culture, take 1 ⁇ 10 6 cells, use Trizol (Invitrogen, Cat#15596-018) to extract RNA (according to the kit instructions), and extract the RNA with reverse The transcription kit (Sangon Biotech, Cat#B532435) was used to reverse transcribe into cDNA, and the cDNA obtained by reverse transcription was used as a template for PCR amplification. The amplified products were sequenced to obtain the base/coding sequences of the light and heavy chain variable regions of the mab22 antibody (see below). For the primers used, refer to the manual TB326 Rev. C0308 published by Novagen.
  • the murine monoclonal antibody mab22 light chain variable region base sequence obtained in the preferred hybridoma cell line of the present invention (the underlined part is the coding sequence):
  • the murine monoclonal antibody mab22 heavy chain variable region base sequence obtained in the preferred hybridoma cell line of the present invention (the underlined part is the coding sequence):
  • amino acid sequences encoded by the base sequences of the light and heavy chain variable regions of the above-mentioned murine monoclonal antibody mab22 obtained in the present invention are the following SEQ ID NO: 3 and SEQ ID NO: 4.
  • Amino acid sequence of the heavy chain variable region of the murine monoclonal antibody mab22 obtained in the preferred hybridoma monoclonal strain of the present invention is amino acid sequence of the heavy chain variable region of the murine monoclonal antibody mab22 obtained in the preferred hybridoma monoclonal strain of the present invention:
  • the light and heavy chain variable region sequences of the above-mentioned antibodies of the present invention and constant regions of IgG different types such as human hIgG1, hIgG2, hIgG3, hIgG4, human light chain ⁇ , ⁇ type; mouse mIgG1, mIgG2, mIgG3, mouse light chain ⁇ , Recombinant expression and purification of ⁇ type etc. to obtain complete human-mouse chimeric antibody or mouse antibody.
  • the heavy chain constant region is hIgG1
  • the light chain ⁇ type is an example
  • the chimeric antibody mab22c is obtained according to the expression and purification method of Example 1, and the mab22c and hTIGIT, hTIGIT, and The binding activity of RhTIGIT and hTIGIT+ cells and the activity of blocking the binding of hTIGIT and hPVR were compared with the control antibody. The results are shown in Table 4.
  • the results in Table 4 show that the antibody mab22c of the present invention has better activity than Ref1 and Ref2.
  • the binding activity of mab22c to hTIGIT is better than that of Ref1 and Ref2 (0.270nM vs 0.462nM vs 0.396nM), its binding EC50 is 0.58 times and 0.68 times that of Ref1 and Ref2, respectively, and the binding EC50 of mab22c to hTIGIT+ cells is 0.47 of Ref2 EC50 times, close to the EC50 of Ref1 (0.101nM vs 0.0.216nM vs 0.138nM); the IC50 of mab22c blocking hTIGIT binding to hPVR is 0.51 times of Ref2 IC50, 0.46 times of Ref1 IC50 (0.541nM vs 1.07nM vs 1.19nM) ;
  • the binding activity of mab22c to RhTIGIT is strong, and the binding EC50
  • mab22c of the present invention is a new antibody superior to Ref1 and Ref2.
  • mab22c has better binding hTIGIT activity and blocking hTIGIT binding hPVR activity than Ref1 and Ref2, and can be used in the development of tumor therapy products, including monoclonal antibodies, bispecific antibodies, multispecific antibodies, CAR, ADC, etc.
  • mab22c can bind to TIGIT of rhesus monkeys (the Ig-like region of the TIGIT sequence of rhesus monkeys is completely consistent with that of cynomolgus monkeys), and the binding EC50 is about 2 times different from that of Ref1 and Ref2. To facilitate safety evaluation in primates.
  • the murine antibody mab22 of the present invention was humanized, designed, screened, and sequence optimized. The specific process is described as follows.
  • variable regions of the murine antibody mab22 targeting the TIGIT target described above are defined according to various methods in Table 5, and their CDR sequence marks/annotations are shown in Table 6-Table 10 below.
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 GFTFSSYTMS (SEQ ID NO: 8) heavy chain CDR2 EISSSGGSTYYPDTVKG (SEQ ID NO:9)
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 SYTMS (SEQ ID NO: 11) heavy chain CDR2 EISSSGGSTYYPDTVKG (SEQ ID NO:9) heavy chain CDR3 PGLGAWFAY (SEQ ID NO: 10)
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 GFTFSSYTMS (SEQ ID NO: 8) heavy chain CDR2 EISSSGGSTY (SEQ ID NO: 12) heavy chain CDR3 PGLGAWFAY (SEQ ID NO: 10)
  • Antibody mab22 CDRs light chain CDR1 RSSQSIVHNSGNTYLE (SEQ ID NO:5) light chain CDR2 KVSNRFS (SEQ ID NO: 6) light chain CDR3 FQFSHVPRT (SEQ ID NO: 7) heavy chain CDR1 GFTFSSY (SEQ ID NO: 13) heavy chain CDR2 SSSGGS (SEQ ID NO: 14) heavy chain CDR3 PGLGAWFAY (SEQ ID NO: 10)
  • Antibody mab22 CDRs light chain CDR1 VHNSGNTYLEWY (SEQ ID NO: 15) light chain CDR2 LLIYKVSNRF (SEQ ID NO: 16) light chain CDR3 FQFSHVPR (SEQ ID NO: 17) heavy chain CDR1 SSYTMS (SEQ ID NO: 18) heavy chain CDR2 LVAEISSSGGSTY (SEQ ID NO: 19) heavy chain CDR3 ARPGLGAWFA (SEQ ID NO: 20)
  • the human antibody germlines with relatively good homology to mab22 light chain include IGKV2-40*01, IGKV2D-40*01, IGKV2-28*01, IGKV2-29*02 , IGKV2-29*03, IGKV2/OR22-4*01, IGKV2D-28*01, IGKV2D-29*01, IGKV2-29*01, IGKV2D-29*02, etc.
  • the human antibody germline light chain IGKV2-28*01 is preferred.
  • hJk4.1 is preferably used for the J region of the mab22 light chain humanized antibody for humanized design, screening and sequence optimization.
  • the human antibody germlines with relatively good homology to mab22 heavy chain include IGHV3-7*01, IGHV3-7*02, IGHV3-7*03, IGHV3-48*01, IGHV3 -48*02, IGHV3-48*03, IGHV3-48*04, IGHV3-66*01, IGHV3-66*02, IGHV3-66*04, etc.
  • the human germline heavy chain IGHV3-66*01 sequence is preferably used for humanization of the antibody of the present invention.
  • the CDR region of the antibody mab22 of the present invention (see the definition of the above CDR) is grafted onto the selected humanized light and heavy chain human antibody germline template, and then recombined with IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the murine antibody, back-mutate the buried residues in the humanized antibody, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH , Screen these mutations and mutation combinations to see the impact on antibody activity, and optimize the chemically unstable amino acid residues in the CDR region to obtain an antibody molecular sequence optimized for structure and activity, that is, to complete the humanization of the murine antibody of the present invention.
  • mab22 The specific sequence of mab22 will be described below by taking hIgG1 heavy chain and kappa light chain (sequence as follows) as examples.
  • the number of back mutations in the humanized sequence of the light chain of the murine antibody of the present invention is relatively small, and the number of back mutation sites is only 0, 1 or 2, such as the sequences listed above, so the light chain of the antibody of the present invention is variable
  • the degree of humanization of the region sequence is very high.
  • These sequences are combined with the constant region sequence of the human antibody light chain constant region ⁇ chain or ⁇ chain to obtain the light chain sequence of the antibody of the present invention, such as the light chain of the present invention using the ⁇ type light chain constant region, such as SEQ ID NO: 21.
  • the number of back mutation sites in the heavy chain variable region used for humanization is also small, and the number of back mutation sites is only 2 or 0, such as the heavy chain variable region sequence listed above.
  • the sequence humanization of the heavy chain variable region of the humanized antibody of the present invention is also highly humanized.
  • These heavy chain variable region sequences containing different numbers of back mutations are recombined with optional human IgG1, IgG2, IgG3, IgG4 chain constant region sequences to obtain the heavy chain sequence of the present invention, for example, the heavy chain of the present invention uses hIgG1 as the constant region sequence as Example to illustrate.
  • each light chain sequence is combined with each heavy chain sequence to obtain each humanized antibody.
  • the human-mouse chimeric antibody mab22c of the present invention has better binding activity to hTIGIT than the reference molecule Ref1, and its binding EC50 is only 0.517 times that of Ref1 EC50 (0.585nM vs 1.132nM).
  • the above-mentioned humanized antibody molecules obtained by combining the light and heavy chain sequences of the antibody murine sequence mab22 of the present invention with different degrees of humanization retain almost the same binding activity as the chimeric antibody.
  • the combined antibodies with different degrees of humanization basically retain the expression level of the chimeric antibody, which is 4.4-5.4 times the expression level of Ref1.
  • the level is around 100mg/mL.
  • the humanized antibody of the present invention basically retains the chimeric
  • the combined antibody mab22c can well block the binding properties of hTIGIT and hPVR, and the activity of humanized antibody in blocking the binding of hTIGIT and hPVR is better than that of Ref1.
  • its blocking IC50 is only 0.44 of Ref1IC50 times (1.443nM vs 3.30nM).
  • Example 2 using the method in Example 2 to detect the binding activity of each humanized antibody to hTIGIT+ cells, it was found that the binding activity of the humanized antibody of the present invention to hTIGIT+ cells was basically the same (mab22-h3 was slightly weaker), and the binding activity of both was higher than that of Ref1 Well, taking mab22-h6 as an example, its binding EC50 is only 0.50 times that of Ref1 (0.0133nM vs 0.0268nM).
  • the antibody, humanized antibody, and preferably humanized antibody molecule of the present invention not only bind to hTIGIT protein and hTIGIT+ cells, but also can effectively block the binding of hTIGIT to hPVR.
  • Table 12 Humanized antibody of the present invention binds to hTIGIT+ cell activity and blocking activity
  • amino acid sequences of the light and heavy chains (including the constant regions) of some preferred humanized antibodies described in Table 12 are as follows.
  • the heavy chain sequence of the humanized antibody mab22-h2 is the same as SEQ ID NO: 29.
  • the light chain sequence of the humanized antibody mab22-h5 is the same as SEQ ID NO:30.
  • the heavy chain sequence of the humanized antibody mab22-h6 is the same as SEQ ID NO: 31.
  • the inventors unexpectedly discovered an anti-human TIGIT antibody through innovative screening. They have good binding activity, can bind human TIGIT protein and hTIGIT+ cells well, and can effectively block human TIGIT and human PVR Compared with the current clinical antibody (control antibody Ref1), it has better activity; it can combine with non-human primate TIGIT, which provides convenience for non-clinical safety evaluation; the degree of humanization is high, which can reduce the risk for later drug development. The risk of immunogenicity is eliminated; after humanization, the antibody expression level is high, which provides convenience for downstream production and processing, and saves costs.
  • the unique characteristics of the antibody of the present invention make it more suitable for the development of antibody drugs targeting human TIGIT, and as a candidate drug, it can be used alone or in combination, especially providing a new and even better anti-PD-1 antibody for the treatment of tumors. s Choice.
  • the present invention designs various bispecific antibodies.
  • the general formula of the designed bispecific antibody is as follows.
  • the sequence containing the light chain means that the sequence can also include the scFv linked to the light chain sequence in addition to the light chain sequence; the sequence containing the heavy chain means that the sequence can also include the Chain sequence linked scFv.
  • T1 represents the first protein functional region for target 1 (such as TIGIT), and T2 represents the second protein functional region for target 2 (not TIGIT).
  • T1 (scFv) represents the scFv sequence of the antibody against target 1;
  • T2 (scFv) represents the scFv sequence of the antibody against target 2.
  • (scFv) n1 , (scFv) n2 , (scFv) n3 , n1, n2, n3, and n4 in (scFv) n4 are natural numbers respectively, which can be 0, 1, 2, 3, etc., in specific embodiments of the present invention Among them, at least one value among n1, n2, n3, and n4 is 1, and the rest are 0.
  • VL represents the antibody light chain variable region sequence against target 1 or 2;
  • VH represents the antibody heavy chain variable region sequence against target 1 or 2.
  • LC represents the constant region sequence of the light chain ( ⁇ or ⁇ ), preferably a human light chain constant region sequence
  • HC represents the constant region sequence of the heavy chain, including IgG1, IgG2, IgG3, IgG4, etc. (abbreviated as HC- IgG1, HC-IgG2, HC-IgG3, HC-IgG4), preferably the human heavy chain constant region sequence (HC-hIgG).
  • the C-terminus of the heavy chain constant region is linked to scFv or other protein sequences, the last amino acid K at the C-terminus can be mutated, preferably mutated to A.
  • T1 is immunoglobulin
  • T2 is scFv
  • T1 is scFv
  • scFv targets the same target
  • scFv targets the same target
  • the scFv is a light chain variable region-linker-heavy chain variable region, and the N-terminal of the light chain variable region or the C-terminal of the heavy chain variable region is connected to the immune
  • the C-terminal is correspondingly connected to the C-terminal or N-terminal of the immunoglobulin light chain and/or heavy chain through a linker.
  • connection method is that the C-terminus of the light chain variable region is connected with the linker, and the linker is then connected with the heavy chain
  • the N-terminus of the variable region is connected, thereby exposing the N-terminus of the variable region of the scFv light chain and the C-terminus of the variable region of the heavy chain, so that it can be connected to the light chain and/or heavy chain of the immunoglobulin through a linker.
  • the C-terminal of the heavy chain variable region of scFv when it is connected to the light chain of immunoglobulin, in some specific embodiments, it is preferred to use the C-terminal of the heavy chain variable region of scFv to connect with the N-terminal of the light chain of immunoglobulin through a linker; When it is linked to the heavy chain of the immunoglobulin, it is preferred in some specific embodiments to use the N-terminus of the light chain variable region of the scFv linked to the C-terminus of the heavy chain of the immunoglobulin.
  • the connection method is that the N-terminal of the light chain variable region is connected with the linker, and the linker is then connected with the heavy chain variable region.
  • the C-terminus is linked, thereby exposing the C-terminus of the variable region of the scFv light chain and the N-terminus of the variable region of the heavy chain, so that it can be connected to the light chain and/or heavy chain of the immunoglobulin through a linker.
  • the C-terminal of the light chain variable region of the scFv in some specific embodiments linked to the N-terminal of the light chain of the immunoglobulin;
  • the heavy chain of immunoglobulin in some specific embodiments, it is preferred to use the N-terminal of the heavy chain variable region of scFv linked to the C-terminal of the heavy chain of immunoglobulin.
  • the linker connecting the scFv to the immunoglobulin is preferably (G 4 S) m or (G 2 S) m , and the m is preferably an integer between 0 and 10. Further preferably, the linker is (Gly-Gly-Gly-Gly-Ser) 3 or (Gly-Gly-Ser) 4 .
  • the quantity of said scFv is a pair, symmetrically connected at the C-terminal and/or N-terminal of said immunoglobulin light chain and/or heavy chain.
  • the linker in the scFv is VE(G 2 S) m GGVD or (G 4 S) m, and the m is preferably an integer between 0 and 10. Further preferably, the linker is VE(G 2 S) 4 GGVD or (G 4 S) 3 .
  • target antibody sequences are from published antibody sequences. Including, anti-PD-1 antibodies Nivolumab/Opidivo (referred to as Nivo) and Pembrolizumab/Keytruda (referred to as Pem); anti-PD-L1 antibodies Atezolizumab/Tecentriq (referred to as Atezo), Durvalumab/Imfinzi (referred to as Durv) and Avelumab/Bavencio (referred to as )wait. Sequences of Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, and Avelumab can all be found from public resources such as www.drugbank.ca.
  • Example 11 Design and activity evaluation of bispecific antibodies targeting TIGIT and PD-1/PD-L1 dual targets
  • the present invention designs bispecific antibodies with different sequence structures for the two targets of TIGIT and PD-1/PD-L1, as shown in Table 14 below.
  • ⁇ chain indicates that the light chain is the constant region of the ⁇ -type light chain of human IgG.
  • the following designs for introducing scFv at the C-terminus of the heavy chain all mutate the K at the end to A.
  • bispecific antibodies were respectively cloned, expressed, and purified, and each bispecific antibody molecule, human TIGIT, and PD-1 or PD- Binding activity of L1. It was found that the expression, quality and activity of bispecific antibodies with different designs were significantly different. The quality, expression and activity data of some preferred bispecific antibody molecules are shown in Figure 2 and Table 15.
  • NA not applicable, means no expression of the antibody.
  • ND not detected due to poor quality or poor binding activity to another antigen.
  • the values in brackets are the binding activity EC 50 of the monoclonal antibody corresponding to the same target under the same experimental conditions.
  • * The ratio of the binding activity EC 50 of the bispecific antibody to the corresponding monoclonal antibody under the same experimental conditions. The larger the ratio, the more weakened the binding ability of the designed bispecific antibody to the single target. For example, if the ratio is 2, it means that the designed bispecific antibody has weaker binding activity to the target than the corresponding monoclonal antibody. 1 times. The ratio is within 2 (experimental error range), indicating that the binding activity is not affected.
  • the scFv of the anti-TIGIT antibody mab22-h6 of the present invention is designed at the heavy chain N-terminal and C-terminal of the PD-1 antibody Pem and Nivo, anti-PD-L1 antibody Atezo, Durv or Avel; the light chain N-terminal and C-terminal
  • the bispecific molecule formed, or the scFv of PD-1 antibody Pem or Nivo, the scFv of anti-PD-L1 antibody Atezo, Durv or Avel and the anti-TIGIT antibody mab22-h6 of the present invention are designed to express the amount of bispecific molecule, and PD -1 or the binding activity of PD-L1 and TIGIT.
  • the bispecific antibody designed at different positions has different expression and quality, such as LB601-LB603 no expression vs LB604 expression is 3.22 mg/L, LB609 no expression vs LB606 expression was 7.68mg/L, LB612 10.54mg/L vs LB613 1.59mg/L, and the quality of LB612 was better, and there were more LB613-mers;
  • the same TIGIT antibody and anti-PD-L1 Antibody scFv bispecific antibodies designed at different positions, have different expression and quality, such as LB622 vs LB623, LB615 (12.4mg/L) vs LB621 (40mg/L) (as shown in Figure 2, the purity of LB615 SEC is much lower LB621); the same anti-TIGIT antibody and anti-PD-L1 antibody,
  • the anti-PD-L1 antibody When the anti-PD-L1 antibody is connected to the complete antibody molecule of mab22-h6 by scFv, it can basically retain the activity of the two targets, such as LB621, the expression level is 40mg/L , the binding activity to the two targets is close to that of the corresponding monoclonal antibody; the same TIGIT antibody, the same connection position, and different PD-1 or PD-L1 antibody scFv have different effects on the activity, such as LB605 vs LB610 vs LB621, indicating Pem scFv linking at the N-terminus of the mab22-h6 heavy chain affected the activity of mab22-h6, while the bispecific antibody molecules (LB610 and LB615) formed by scFv forms of Nivo and Atezo linking at the N-terminus of the mab22-h6 heavy chain basically retained activity of the two targets.
  • the two targets such as LB621
  • the expression level is 40mg/
  • the above data show that the same TIGIT antibody (the present invention) is linked to a non-TIGIT antibody in the form of scFv, and the activity of the designed bispecific antibody molecule against the TIGIT antigen is lost.
  • the scFv of different PD-1 antibody or PD-L1 antibody is connected, or the position of scFv is different, and the activity of the designed bispecific antibody molecule is also very different.
  • bispecific antibodies designed based on the TIGIT antibody sequence of the present invention have different sequences, scFv and antibody positions, and different activities. Appropriate positions and appropriate sequences are designed to obtain bispecific antibodies with good activity against dual targets.
  • the structure of these bispecific antibodies is similar to conventional IgG and has a complete Fc.
  • the present invention is called Sequence-based IgG like bispecific antibody format. That is, a bispecific antibody (SBody) with a similar sequence-specific IgG structure.
  • SBody bispecific antibody
  • These bispecific antibody molecules have the same complete Fc as normal antibodies, so that their purification process can be carried out in accordance with normal antibodies, so the process is simple and has the advantages of low production costs.
  • the values in the brackets are the IC 50 of the blocking antigen and ligand binding activity of the monoclonal antibody corresponding to the same target under the same experimental conditions.
  • the fold change of IC 50 that is, the ratio of the IC 50 of the bispecific antibody and the corresponding monoclonal antibody (control antibody). The larger the ratio, the more the functional activity of the designed bispecific antibody on the single target is weakened. For example, if the ratio is 2, it means that the functional activity of the designed bispecific antibody on the target is weakened compared with the corresponding monoclonal antibody. 1 times. A ratio within 2 is the range of experimental error, that is, the activity is not affected.
  • the affinity of the preferred bispecific antibody of the present invention was determined with a Biacore T200 (manufacturer: Cytiva) instrument.
  • the sample to be tested is 1 ⁇ g/mL.
  • Antigens were purchased from Beijing Yiqiao Shenzhou TIGIT-his, article number: 10917-H08H, PD-L1-his article number: 10084-H08H.
  • Capture ProteinA Chip Cat#29-1275-56, Cytiva.
  • Diluted analytes (TIGIT-his or PD-L1-his) flow sequentially through the Fc1 and Fc2 channels on the chip surface.
  • the flow rate was 30 ⁇ L/min
  • the binding time was 180 seconds
  • the dissociation time was 800 seconds.
  • wash the chip with 10mM Glycine-HCl, pH 1.5, 30 ⁇ L/min, 30s.
  • the experimental data was fitted with a 1:1 Langmuir model using Biacore T200 evaluation version 3.1 (GE) software to obtain the affinity value KD. The results are shown in Table 16b.
  • the bispecific antibody (SBody) designed in the present invention retains the binding and affinity activities for the dual targets.
  • its activity of blocking antigen binding to corresponding ligands is consistent with the change of binding activity, such as LB605, its binding activity to human TIGIT is slightly weakened, and the binding activity of blocking human TIGIT and human PVR is also slightly weakened (corresponding to Compared with the monoclonal antibody, the multiples of change were 3.548 and 3.729).
  • the comprehensive data show that the anti-TIGIT antibody mab22-h6 and PD1, PD-L1 antibody designed bispecific antibody SBody of the present invention not only has activity and function, but also has sequence-specific expression and quality.
  • the preferred sequence of the SBody part of the bispecific antibody designed by the anti-TIGIT antibody mab22-h6 and PD1, PD-L1 antibody of the present invention is as follows:
  • LB610 light chain sequence is the same as SEQ ID NO:32.
  • LB621 light chain sequence is the same as SEQ ID NO:32.
  • LB605 light chain sequence is the same as SEQ ID NO: 32.
  • LB615 light chain sequence is the same as SEQ ID NO:32.
  • LB623 light chain sequence is the same as SEQ ID NO: 32.
  • LB624 light chain sequence is the same as SEQ ID NO:32.
  • LB625 light chain sequence is the same as SEQ ID NO:32.
  • Example 12 Stability evaluation of TIGIT and PD-1/PD-L1 bispecific antibody of the present invention in different formulations
  • the preferred bispecific antibody LB621 of the present invention was expressed and purified by the method in the Examples and replaced with a desalted spin column (Thermo, Cat#89890) into each formulation formulation.
  • the formulation formulations are shown in Table 17.
  • the buffer replacement process of each preparation is as follows.
  • Pretreat the desalting spin column first, centrifuge at 1000g for 2min, remove the storage solution, add 1mL of each preparation buffer to the desalting spin column, centrifuge at 1000g for 2min, repeat 3 times, discard The buffer in the collection tube; put the desalting spin column in a new collection tube, slowly add an appropriate amount of LB621 to the column, and add 20 ⁇ L of the preparation buffer hydraulic layer, centrifuge at 1000g for 2min, collect the centrifuged sample, After mixing, filter with a 0.2 ⁇ m filter membrane; divide the filtered LB621 preparation samples into 80 ⁇ L/tubes, and place 4 tubes in a 40°C water bath for SEC- HPLC and SDS-PAGE detection, that is, samples treated at 40°C for 7 days, 14 days, 21 days, and 28 days; take another tube of sterile aliquots for SEC-HPLC detection, that is, samples treated at 40°C for 0 days .
  • the SEC-HPLC detection results of different formulations
  • the bispecific antibody LB621 of the present invention is at a higher concentration (14.2mg/mL), in the preparation buffer (20mM citric acid-sodium citrate, 50mM arginine, 50mM glutamic acid, 200mM trehalose, 0.02% After being treated at 40°C for 28 days in Tween 80, pH6.0), the SEC-HPLC purity only decreased to 86.22%, indicating that LB621 had better stability.
  • Example 13 Evaluation of T cell activation by TIGIT and PD-L1 bispecific antibody of the present invention
  • Human PBMCs were activated by SEB and incubated with the preferred bispecific antibody LB621 of the present invention, the corresponding monoclonal antibodies Atezo (anti-PD-L1 antibody) and Ref1 (anti-TIGIT antibody) alone or in combination, and compared the increase in the release of IL-2 induced by each antibody to evaluate its in vitro activity.
  • Human PBMC cells (Miaoshun Biotechnology Co., Ltd., Cat. No.: PB010C, Lot No.: P121040901C) were taken out from liquid nitrogen, recovered and counted, and cultured with RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Cat. No.: 10270-106) (Containing 1 ⁇ g/mL SEB) (Hyclone, product number: SH30809.01B) was adjusted to a density of 2 ⁇ 10 6 /mL and added to a 96-well culture plate (Corning, product number: 3599) at 100 ⁇ L per well.
  • the negative control is human IgG1 isotype, which is added to a 96-well culture plate at 100 ⁇ L per well. After culturing in a carbon dioxide constant temperature incubator for 3 days, the supernatant was collected by centrifugation. The IL-2 level in the supernatant was measured by human IL-2 precoated ELISA kit (Dayou, catalog number: 1110203).
  • Example 14 The optimal design of the TIGIT and PD-L1 bispecific antibody of the present invention Molecular efficacy evaluation in vivo in animals
  • mice Use BALB/cJGpt-Tigit em1Cin (hTIGIT) /Gpt female mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Zhejiang) 2019-0001) to establish an animal drug effect model, which is used in the present invention.
  • the bispecific antibody LB621 was evaluated for its efficacy in vivo.
  • CT26 cells purchased from Shanghai Chinese Academy of Sciences were cultured in RPMI1640 medium (Hyclone, product number: SH30809.01B) containing 10% fetal bovine serum (Gibco, product number: 10270-106) at 37°C with 5% CO2 .
  • RPMI1640 medium Hyclone, product number: SH30809.01B
  • 10% fetal bovine serum Gibco, product number: 10270-106
  • CT26 cells grow to logarithmic growth phase (confluence rate is 80%-90%), digest with 0.25% trypsin, collect cells, wash cells twice with RPMI1640 medium, and carry out resuspension counting with RPMI1640 medium, adjust A cell density of 10 ⁇ 10 6 /mL was used for inoculation. Inoculate 100 ⁇ L of CT26 cell suspension (1 ⁇ 10 6 ) subcutaneously in the left flank of the mouse, select the tumor cells to grow to a size of about 120-130 mm 3 , and divide them into random groups, with 7 mice in each group.
  • the samples to be tested and the positive control were aseptically prepared with PBS.
  • Blank group was PBS
  • Atezo (PD-L1 antibody) was the positive control group of monoclonal antibody
  • Atezo+Ref1 was the positive control group of combination drug
  • LB621 was the test group of bispecific antibody drug.
  • the way of administration is intraperitoneal injection.
  • the dosage of Atezo is 10 mg/kg, and the injection volume is 200 ⁇ L/piece
  • the dosage of Atezo+Ref1 is (10+10) mg/kg, and the injection volume is (100+100) ⁇ L/piece
  • the dosage of LB621 is 13.3 mg/kg (equal molar to the drug in the combination group)
  • the injection volume is 200 ⁇ L/rat.
  • the administration frequency of each group was 2 times/week, a total of 3 administrations.
  • the day of administration of each injected sample was defined as day 0. Body weight and tumor volume were measured before each administration, and the data were recorded. The test was terminated on day 18 after the first dose. The analysis and statistics of the drug efficacy data are up to the 18th day.
  • * represents p ⁇ 0.05; ** represents p ⁇ 0.01; *** represents p ⁇ 0.01.
  • Example 15 PK evaluation of TIGIT and PD-L1 bispecific antibody of the present invention
  • mice The same BALB/cJGpt-Tigit em1Cin(hTIGIT) /Gpt female mice as in Example 11 were used to evaluate the PK of the bispecific antibody of the present invention under the same feeding conditions. Three mice were randomly selected to form a group. Mice were injected with mab22-h6 and LB621 through the tail vein, the injection doses were 10mg/kg and 13.3mg/kg respectively, and the injection volume was 200 ⁇ L/mouse. 0 hours before injection, 0.25, 0.5, 2, 8, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384 hours after injection Take blood.
  • the collected blood samples were centrifuged, and the supernatant was collected and stored at -20°C.
  • the concentration of mab22-h6 in serum was used to evaluate the PK characteristics of LB621 and mab22-h6.
  • the PK data was analyzed by EXCEL software, and the T 1/2 of LB621 and mab22-h6 were calculated. The results are shown in Table 20.
  • the inventors unexpectedly discovered an anti-human TIGIT antibody through innovative screening, which has good binding activity to TIGIT and has good binding activity to non-human primate cynomolgus monkey TIGIT protein. Can effectively block the combination of human TIGIT and human PVR. It has better activity than the current clinical antibody (control antibody Ref1), and in vivo experiments show that it has better anti-tumor activity than Ref2; and the degree of humanization is high, which reduces the risk of immunogenicity for later drug development. After humanization, the antibody expression level is high, which provides convenience for downstream production and process, and saves costs.
  • the bispecific antibody designed based on the TIGIT antibody sequence of the present invention can retain the functional activity of the dual-target antibody, and the binding activity and affinity for the two targets are close to those of the corresponding monoclonal antibody, and block the interaction between the antigen and the corresponding antibody.
  • the ligand binding activity is also consistent with the corresponding monoclonal antibody binding activity, good stability, can synergistically enhance T cell activity, and has good anti-tumor activity.
  • These bispecific antibodies (referred to as SBody in the present invention) are similar in structure to conventional IgG and have the same complete Fc as normal antibodies, so that the purification process can be carried out according to normal antibodies, so the process is simple and has the advantages of low production cost.
  • the unique characteristics of the antibody of the present invention make it more suitable for the development of antibody drugs against human TIGIT targets, and as candidate drugs can be administered alone or in combination, especially providing a new combination for the treatment of tumors with PD-1 and PD-L1 antibodies It is even a better choice, and the preferred bispecific antibody of the present invention provides another choice for the multi-target therapy of tumors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Oncology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Hospice & Palliative Care (AREA)
  • Plant Pathology (AREA)

Abstract

提供一种靶向TIGIT的抗体或抗原结合片段，其包括轻链可变区和重链可变区；所述抗体或其抗原结合片段结合人TIGIT，并具有阻断 PVR和TIGIT结合的功能;所述轻链可变区包含:如 SEQIDNO: 5所示的氨基酸序列的 CDR1、如 SEQID NO:6所示的氨基酸序列的 CDR2和如 SEQ ID NO: 7所示的氨基酸序列的CDR3；所述重链可变区包含:如SEQIDNO:8所示的氨基酸序列的CDR1、如 SEQID NO:9所示的氨基酸序列的CDR2 和如 SEQ ID NO: 10 所示的氨基酸序列的 CDR3。

Description

靶向TIGIT的抗体和双特异性抗体及其应用
本申请要求申请日为2021/12/27的中国专利申请2021116173509的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，具体涉及一种靶向TIGIT的抗体和双特异性抗体及其应用。
背景技术
TIGIT(含Ig和ITIM结构域的T细胞免疫受体)是一种抑制性受体蛋白，也称为WUCAM、Vstm3或VSIG9，与CD155等蛋白结构相似，统称为CD155家族。TIGIT是Ⅰ型跨膜蛋白，在T细胞(包括活化的T细胞、记忆T细胞、调节性T细胞和滤泡性T辅助细胞)和NK细胞上表达，在抑制T细胞和NK细胞介导的抗肿瘤免疫功能活性方面起着重要作用。成熟人TIGIT的氨基酸序列含有223个氨基酸(aa)残基(NCBI登录号：NM_173799)。成熟人TIGIT的细胞外结构域(ECD)由以下组成：120个氨基酸残基，其具有V型Ig样结构域、随后是21个氨基酸跨膜序列、和具有基于免疫受体酪氨酸的抑制性基序(ITIM)的82个氨基酸细胞质结构域。在ECD内，人TIGIT分别与小鼠和食蟹猴具有59％和87％的序列同源性。
已知TIGIT与脊髓灰质炎病毒受体(PVR；CD155)、nectin2(CD112)、CD96、CD226之间保持着一种“多对多”的相互作用关系。TIGIT参与了一个复杂的调控网络，涉及多个受体、一个竞争性共刺激受体(CD226)和多个配体(如CD155、CD112)。这些配体主要在APC(如树突细胞和巨噬细胞)和肿瘤细胞上表达。作为免疫“检查点”分子，TIGIT在被其配体CD155和CD112结合时启动免疫细胞中的抑制性信号传导。TIGIT与CD155的结合亲和力(KD：约1nM)远远高于CD112，并且TIGIT:CD112相互作用在介导抑制性信号时是否在功能上相关仍有待确定。
TIGIT通过多种机制潜在地抑制固有和适应性免疫：1.TIGIT-CD155结合后，表达CD155的树突状细胞(DC)可能具有致耐受性，降低IL-12的产生和IL-10的增加；2.TIGIT抑制NK细胞脱颗粒、细胞因子生成和NK细胞介导的肿瘤细胞毒性，TIGIT在Treg上表达，增强免疫抑制功能和稳定性。3.TIGIT可以破坏细胞表面的DNAX辅助分子1(DNAM-1)顺式二聚化，从而阻止DNAM-1与CD155的相互作用。TIGIT阻碍 CD155介导的CD226活化。4.与DNAM-1相比，TIGIT以更高的亲和力结合CD155，因此可能会与DNAM-1竞争而与CD155相互作用。5.TIGIT还可以通过其胞质尾部直接将抑制信号传递给T细胞和NK细胞。
TIGIT和其它这样的共-抑制性分子(例如，CTLA-4、PD-1、Lag3和BTLA)在肿瘤细胞逃脱免疫监视中起作用。类似于其它共-抑制性受体例如CTLA-4、PD-1和BTLA，TIGIT可起“关闭”免疫反应的作用。在小鼠模型中，PD-L1和TIGIT二者的抗体阻断导致协同提高CD8+T细胞介导的肿瘤排斥。Grogan et al.(2014)J.Immunol.192(1)Suppl.203.15；Johnston et al.(2014)Cancer Cell 26:1-15。在黑素瘤的动物模型中得到类似结果。
TIGIT在多种肿瘤浸润淋巴细胞中表达上调，例如黑色素瘤、乳腺癌、非小细胞肺癌、结肠癌、急性髓细胞性白血病(AML)和多发性骨髓瘤等。TIGIT的高表达与肿瘤进展、预后不良相关。有研究发现在结直肠癌组织中TIGIT和CD155的阳性表达率与肿瘤分化程度、病理分期及淋巴结转移相关。Wang等2018，ClinImmunol,190:64-73在AML患者中发现PD-1+和TIGIT+CD8+T细胞的比例增加，CD226+CD8+T细胞降低。进一步分析显示，PD-1+和TIGIT+和CD226 low CD8+T细胞亚群与诱导化疗失败和FLT3-ITD突变相关，而后者与预后不良相关。Hutten等2018，Biol Blood Marrow Transplant,24(4):666-677在接受同种异体干细胞移植患者中检测T细胞亚群免疫检查点的表达谱，与处在缓解期患者相比，术后复发患者PD-1、TIGIT和KLRG-1在MiHA反应性CD8+T细胞上共表达更高。Liu等2019，Cancer Immunol Immunother,68(12):2041-2054研究发现乙型肝炎病毒致肝细胞癌(HBV-HCC)患者PD-1和TIGIT在CD4+、CD8+T细胞的表达显著上调，晚期和进展期患者PD-1+TIGIT+CD8+T细胞群升高，并且与总生存率和无进展生存率呈负相关。这些研究显示出TIGIT对患者预后评估的参考价值，为TIGIT作为免疫治疗靶标提供了重要依据。
因此，TIGIT是被寄予厚望的新的靶点，与PD-L1联合是未来有前途的抗癌治疗策略。已有的抗体绝大部分为靶向TIGIT靶点的单克隆抗体或者抗TIGIT单抗与其他靶点单抗联合使用，存在成本高，副作用大，临床效果有限等问题，而仅有的双特异性抗体BMS/Agenus的AGEN1777和信达/礼来的IBI321双特异抗体仅处于临床试验早期阶段。
发明内容
针对现有技术中已有抗体绝大部分为靶向TIGIT靶点的单克隆抗体或者抗TIGIT单抗与其他靶点单抗联合使用，存在成本高，副作用大，临床效果有限的缺陷，且尚无进入后期临床试验的TIGIT双特异性抗体。本发明提供了一种靶向TIGIT的抗体和双特异性 抗体及其应用。所述靶向TIGIT的抗体能很好地结合人TIGIT蛋白和hTIGIT+细胞，与两个靶点抗原的亲和力强，能有效的阻断人TIGIT和人PVR的结合，能协同增强T细胞活性，促进IL-2释放，且协同作用优于联合给药组；可有效地抑制肿瘤细胞的生长；亦能和非人灵长类TIGIT结合，特别是为联合PD-1抗体治疗肿瘤提供了新的，甚至是更好的选择。
为解决上述技术问题，本发明提供的技术方案之一为：一种靶向TIGIT的抗体或抗原结合片段，其包括轻链可变区和/或重链可变区；所述轻链可变区包含：如SEQ ID NO:5所示的氨基酸序列的CDR1、如SEQ ID NO:6所示的氨基酸序列的CDR2和如SEQ ID NO:7所示的氨基酸序列的CDR3；所述重链可变区包含：如SEQ ID NO:8所示的氨基酸序列的CDR1、如SEQ ID NO:9所示的氨基酸序列的CDR2和如SEQ ID NO:10所示的氨基酸序列的CDR3，上述CDR是以CCG定义的CDR序列，以CCG和其他定义确定的CDR序列信息见下表a-e。
表a本发明抗TIGIT抗体mab22按CCG定义的CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 GFTFSSYTMS(SEQ ID NO:8)
重链CDR2 EISSSGGSTYYPDTVKG(SEQ ID NO:9)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表b本发明抗TIGIT抗体mab22按Kabat定义CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 SYTMS(SEQ ID NO:11)
重链CDR2 EISSSGGSTYYPDTVKG(SEQ ID NO:9)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表c本发明抗TIGIT抗体mab22按AbM定义CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 GFTFSSYTMS(SEQ ID NO:8)
重链CDR2 EISSSGGSTY(SEQ ID NO:12)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表d本发明抗TIGIT抗体mab22按Chothia定义CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 GFTFSSY(SEQ ID NO:13)
重链CDR2 SSSGGS(SEQ ID NO:14)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表e本发明抗TIGIT抗体mab22按Contact定义CDR序列
抗体 mab22 CDRs
轻链CDR1 VHNSGNTYLEWY(SEQ ID NO:15)
轻链CDR2 LLIYKVSNRF(SEQ ID NO:16)
轻链CDR3 FQFSHVPR(SEQ ID NO:17)
重链CDR1 SSYTMS(SEQ ID NO:18)
重链CDR2 LVAEISSSGGSTY(SEQ ID NO:19)
重链CDR3 ARPGLGAWFA(SEQ ID NO:20)
在本发明一较佳实施方案中，所述轻链可变区包含如SEQ ID NO:3、SEQ ID NO:23-25或其突变所示的氨基酸序列；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少99％的序列同一性，并且所述突变保持或改善了所述结合蛋白对TIGIT的结合。
在本发明一较佳实施方案中，所述重链可变区包含如SEQ ID NO:4、SEQ ID NO:26-27或其突变所示的氨基酸序列；所述突变在原氨基酸序列上有一个或多个氨基酸残基的取代、缺失或添加，优选与原氨基酸序列具有至少99％的序列同一性，并且所述突变保持或改善了所述结合蛋白对TIGIT的结合。
优选地，所述轻链可变区包含如SEQ ID NO:3所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:4所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:23所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:26所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:24所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:26所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:26所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:23所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:24所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列。
在本发明一较佳实施方案中，所述靶向TIGIT的抗体或抗原结合片段是抗体、Fab、Fab’、F(ab’) 2、Fv、scFv、双特异性抗体、多特异性抗体、单域抗体或单区抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。优选地，所述靶向TIGIT的抗体或抗原结合片段包括人源抗体轻链恒定区和人源抗体重链恒定区。更优选地，所述人源抗体轻链恒定区为κ或者λ型轻链恒定区，和/或，所述人源抗体重链恒定区为hIgG1、hIgG2、hIgG3、 hIgG4的重链恒定区或其突变。
在本发明一较佳实施方案中，所述靶向TIGIT的抗体或抗原结合片段的轻链包含如SEQ ID NO:28、SEQ ID NO:30或SEQ ID NO:32所示的氨基酸序列或其突变，和/或，所述靶向TIGIT的抗体或抗原结合片段的重链包含如SEQ ID NO:29或SEQ ID NO:31所示的氨基酸序列或其突变。
优选地，所述轻链的氨基酸序列如SEQ ID NO:28所示；所述重链的氨基酸序列如SEQ ID NO:29所示；或，所述轻链的氨基酸序列如SEQ ID NO:30所示；所述重链的氨基酸序列如SEQ ID NO:29所示；或，所述轻链的氨基酸序列如SEQ ID NO:30所示；所述重链的氨基酸序列如SEQ ID NO:31所示；或，所述轻链的氨基酸序列如SEQ ID NO:32所示；所述重链的氨基酸序列如SEQ ID NO:31所示。
为解决上述技术问题，本发明提供的技术方案之二为：一种靶向TIGIT的双特异性抗体或多特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中，所述第一蛋白功能区为如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段；所述第二蛋白功能区为非靶向TIGIT的抗体。优选地，所述第一蛋白功能区和第二蛋白功能区分别选自免疫球蛋白、scFv、Fab、Fab’或F(ab’) 2，且所述第一蛋白功能区和第二蛋白功能区中至多只有一个蛋白功能区为免疫球蛋白。更优选地，当所述第二功能区的结构为免疫球蛋白时，所述免疫球蛋白的恒定区包括人源抗体轻链恒定区和人源抗体重链恒定区。进一步更优选地，所述人抗体轻链恒定区为κ链或者λ链，所述人抗体重链恒定区为hIgG1、hIgG2、hIgG3、hIgG4或其突变。
在本发明一较佳实施方案中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为一个或多个scFv，所述scFv包括重链可变区与轻链可变区，所述重链可变区与轻链可变区通过连接子连接。
优选地，所述scFv通过连接子与所述免疫球蛋白连接，所述连接子优选为(G 4S) w或(G 2S) W，所述w优选为0～10之间的整数，更优选为1、2、3或者4。
更优选地，所述scFv为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。
进一步优选地，所述scFv中的连接子为VE(G 2S) WGGVD或(G 4S) w，所述w优选为0～10之间的整数，更优选为1、2、3或者4。
在本发明一较佳实施方案中，所述scFv通过连接子与所述免疫球蛋白连接，所述连接子为(G 4S) 3或(G 2S) 4，和/或，所述scFv的数量为两个，且两个scFv对称地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；所述scFv连接免疫球蛋白的重链。
优选地，所述scFv为轻链可变区-连接子-重链可变区结构，所述scFv的轻链可变区的C末端与连接子连接，所述连接子再与重链可变区的N末端连接，所述scFv的重链可变区的C末端通过所述连接子与所述免疫球蛋白重链的N末端连接；或，所述scFv为重链可变区-连接子-轻链可变区结构，所述scFv的轻链可变区的N末端与所述连接子连接，所述连接子再与重链可变区的C末端连接，所述scFv的重链可变区的N末端与所述免疫球蛋白重链的C末端连接。
更优选地，所述scFv中的连接子为VE(G 2S) 4GGVD或(G 4S) 3。
在本发明一较佳实施方案中，所述第二蛋白功能区靶向PD-1/PD-L1、Claudin18.2、TIM-3或LAG-3。
优选地，所述第二蛋白功能区为靶向PD-1/PD-L1的抗体。
更优选地，所述靶向PD-1/PD-L1的抗体为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab或Avelumab。
进一步更优选地，当所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端由K突变为A；和/或，所述第二蛋白功能区为两个相同的scFv或其突变，其中，所述scFv的轻链可变区为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab或Avelumab的轻链可变区，所述scFv的重链可变区为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab或Avelumab的重链可变区，所述突变优选与原氨基酸序列具有至少99％的序列同一性，并且保持或改善了所述抗体的功能。
在本发明一较佳实施方案中，所述靶向TIGIT的双特异性抗体选自以下组：
(i)所述第一蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:32所示，其重链的氨基酸序列如SEQ ID NO:31所示；所述第二蛋白功能区为两个相同的scFv，所述连接子为(G 4S) 3或VE(G 2S) 4GGVD；
其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端通过(G 4S) 3或(G 2S) 4对称地连接在所述免疫球蛋白的两条重链的N末端，且所述C末端由K突变为A；或，
(ii)所述第一蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:32所示，其重链的氨基酸序列如SEQ ID NO:31所示；所述第二蛋白功能区为两个相同的scFv，所述连接子为(G 4S) 3或VE(G 2S) 4GGVD；
其中，所述scFv的数量为两个；所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3或(G 2S) 4对称地连接在所述免疫球蛋白的两条重链的C末端。
在本发明一具体实施方案中，所述靶向TIGIT的双特异性抗体包括以下氨基酸序列：
氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:33所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:34所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:35所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:36所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:37所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:38所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:39所示的含重链的氨基酸序列。
为解决上述技术问题，本发明提供的技术方案之三为：一种分离的核酸，其编码如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、或如技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体。
为解决上述技术问题，本发明提供的技术方案之四为：一种包含根据技术方案之三所述的分离的核酸的表达载体。
为解决上述技术问题，本发明提供的技术方案之五为：一种宿主细胞，其包含根据技术方案之四所述的表达载体。优选地，所述宿主细胞为原核细胞或真核细胞。
为解决上述技术问题，本发明提供的技术方案之六为：一种靶向TIGIT的抗体或抗原结合片段、或靶向TIGIT的双特异性抗体或多特异性抗体的制备方法，其包含培养如技术方案之五所述的宿主细胞，从培养物中获得所述靶向TIGIT的抗体或抗原结合片段、或靶向TIGIT的双特异性抗体或多特异性抗体。
为解决上述技术问题，本发明提供的技术方案之七为：一种抗体药物偶联物，其包含细胞毒性剂，以及如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、或如技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体。
为解决上述技术问题，本发明提供的技术方案之八为：一种药物组合物，其包含如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、如技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体、和/或如技术方案之七所述的抗体药物偶联物。
优选地，所述药物组合物还包含靶向PD-1/PD-L1的抗体，所述靶向PD-1/PD-L1的抗体为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab和/或Avelumab。
更优选地，所述药物组合物包含靶向TIGIT的抗体或抗原结合片段、和靶向PD-1/PD-L1的抗体，所述靶向TIGIT的抗体或抗原结合片段的轻链氨基酸序列如SEQ ID NO:32所示，重链氨基酸序列如SEQ ID NO:34所示，所述靶向PD-1/PD-L1的抗体为Atezolizumab。
为解决上述技术问题，本发明提供的技术方案之九为：一种套装药盒，其包含药盒A和药盒B，其中：
所述药盒A包含如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、如技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体、和/或如技术方案之七所述的抗体药物偶联物；
药盒B包含靶向PD-1/PD-L1的抗体，所述靶向PD-1/PD-L1的抗体为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab和/或Avelumab。
优选地，所述药盒A包含靶向TIGIT的抗体或抗原结合片段，所述靶向TIGIT的抗体或抗原结合片段的轻链氨基酸序列如SEQ ID NO:32所示，重链氨基酸序列如SEQ ID NO:34所示，所述药盒B包含靶向PD-1/PD-L1的抗体，所述靶向PD-1/PD-L1的抗体为Atezolizumab。
为解决上述技术问题，本发明提供的技术方案之十为：如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体、技术方案之七所述的抗体药物偶联物、技术方案之八中所述的药物组合物和/或如技术方案之九所述的药物组合在诊断、治疗和/或预防癌症的药物中的应用。
优选地，所述癌症为实体肿瘤或液体肿瘤，所述实体肿瘤例如结肠癌、胃癌、肺癌、乳腺癌、头颈癌、肝癌、上皮鳞状细胞癌、食道癌、直肠癌、肾癌、卵巢癌、膀胱癌、胰腺癌、黑色素瘤和神经胶质瘤，所述液体肿瘤例如急性淋巴细胞白血病、B细胞淋巴瘤、T细胞淋巴瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和弥漫大B细胞淋巴瘤。
为解决上述技术问题，本发明提供的技术方案之十一为：如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体、技术方案之七所述的抗体药物偶联物、技术方案之八中所述的药物组合物和/或如技术方案之九所述的药物组合在诊断、治疗和/或预防癌症中的应用。
优选地，所述癌症包括实体肿瘤和/或液体肿瘤，所述实体肿瘤例如结肠癌、胃癌、肺癌、乳腺癌、头颈癌、肝癌、上皮鳞状细胞癌、食道癌、直肠癌、肾癌、卵巢癌、膀胱癌、胰腺癌、黑色素瘤和神经胶质瘤，所述液体肿瘤例如急性淋巴细胞白血病、B细胞淋巴瘤、T细胞淋巴瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和弥漫大B细胞淋巴瘤。
为解决上述技术问题，本发明提供的技术方案之十二为：用于诊断、治疗和/或预防癌症的如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体、技术方案之七所述的抗体药物偶联物、技术方案之八中所述的药物组合物和/或如技术方案之九所述的药物组合。
优选地，所述癌症包括实体肿瘤和/或液体肿瘤，所述实体肿瘤例如结肠癌、胃癌、肺癌、乳腺癌、头颈癌、肝癌、上皮鳞状细胞癌、食道癌、直肠癌、肾癌、卵巢癌、膀胱癌、胰腺癌、黑色素瘤和神经胶质瘤，所述液体肿瘤例如急性淋巴细胞白血病、B细胞淋巴瘤、T细胞淋巴瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和弥漫大B细胞淋巴瘤。
为解决上述技术问题，本发明提供的技术方案之十三为：一种如技术方案之一所述的靶向TIGIT的抗体或抗原结合片段、或如技术方案之二所述的靶向TIGIT的双特异性抗体或多特异性抗体的制剂，
所述制剂包括柠檬酸-柠檬酸钠、吐温80和所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体。优选地，所述制剂还包括精氨酸、谷氨酸和海藻糖中的一个或多个。
在本发明一具体实施方案中，所述制剂的pH为5.5-6.5。
在本发明一优选实施方案中，所述制剂包括20mM的柠檬酸-柠檬酸钠、50mM的精氨酸、50mM的谷氨酸、200mM的海藻糖、0.02％的吐温80和14.2mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0。
在本发明一具体实施方案中，所述制剂包括20mM的His-HCl、50mM的精氨酸或谷氨酸、200mM的海藻糖、0.02％PS80和11.4mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0。
在本发明一具体实施方案中，所述制剂包括20mM的His-HCl、125mM的甘氨酸、125mM的海藻糖、0.02％PS80和11mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0。
在本发明一具体实施方案中，所述制剂包括20mM的His-HCl、50mM的精氨酸盐酸盐、150mM的海藻糖、0.02％吐温80和11.6mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0。
本发明中，“靶向TIGIT”和“抗TIGIT”均指对TIGIT具有结合特异性，可以互相替代使用。
应了解，本发明“第一”、“第二”均无实际意义，仅为区分相同的术语。在提及scFv 或细胞因子或细胞因子受体或Fab’或F(ab’) 2的数量时，“一对”和“两个”、“两对”和“四个”具有相同的含义。在提及轻链或重链或轻链可变区或重链可变区的数量时，“一个”和“一条”、“两个”和“两条”具有相同的含义。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”，通常是指由两对多肽链(每对具有一条轻(L)链和一条重(H)链)组成的免疫球蛋白。从一般意义上，重链可以理解为抗体中分子量较大的多肽链，轻链是指抗体中分子量较小的多肽链。轻链可分类为κ和λ轻链。重链通常可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域[称为互补决定区(CDR)]，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末排列的3个CDR和4个FR组成。各重链/轻链对应的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat EA.Et al.,Sequences of Proteins of Immunological Interest[National Institutes of Health,Bethesda,Md.(1987and 1991)]，或Chothia&Lesk 1987)].Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:877-883的定义。特别地，重链还可以包含3个以上CDR，例如6、9或12个。例如在本发明的双特异性抗体中，重链可以是IgG抗体的重链的N端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特 异性结合，其也被称为“抗原结合部分”。通常参见Fundamental Immunology,Ch.7，Paul,W.,ed.,第2版，Raven Press，N.Y.(1989)，其全文通过引用合并入本文。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab’、F(ab’) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链结合片段(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段；术语“Fab”意指由VL、VH、CL和CH1(或者CH)结构域组成的抗体片段；术语“F(ab’) 2”意指包含通过铰链区上的二硫键连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链结合片段(例如，scFv)，其中VL和VH通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见例如Bird等人，Science242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的G 4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G 4S) 4或(G 4S) 3接头，但也可使用其变体。
本领域技术人员可使用常规技术(例如重组DNA技术或酶促或化学断裂法)从给定的抗体获得其抗原结合片段(例如上述抗体片段)，并且用与筛选完整抗体的方式相同的方式对抗原结合片段进行特异性筛选。
如本文中所使用的，术语“分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌等原核细胞，如酵母细胞等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞和HEK 293细胞等的人或动物细胞。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数(KD)，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于约10 -5M，例如小于约10 -6M、10 -7M、 10 -8M、10 -9M或10 -10M或更小的解离平衡常数结合抗原，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。例如用KINEXA方法在KINEXA 400仪器上检测到的抗体和细胞结合的亲和力。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明所述靶向TIGIT的抗体和双特异性抗体具有相比于现有技术更好的亲和力，和更好的功能。本发明所述靶向TIGIT的抗体人源化程度高，免疫原性低，与非人灵长类抗原结合，临床前安全性评价便利。本发明所述双特异抗体的结构简单，类似常规抗体IgG的结构的双特异抗体，使得纯化简单易行；分子稳定，为后期开发提供极大便利；表达量高，成本低；保留了双靶点的结合及阻断活性；增强T细胞功能活性及小鼠体内药效较佳，优于联合给药。
附图说明
图1为本发明部分优选人源化抗体阻断hTIGIT与PVR结合的活性。
图2为本发明部分优选双特异抗体SDS-PAGE图。
图3为本发明优选双特异抗体40℃处理28天后SDS-PAGE检测结果。
图4为本发明优选双特异性抗体诱导活化T细胞释放IL-2的活性。
图5为本发明优选人源化抗体和双特异抗体抗肿瘤活性。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1：抗原，抗体的克隆，表达和纯化
本发明所用的抗原或购自如下公司：北京义翘神州科技有限公司cyno TIGIT-hFc(Cat#MB12OC2203)，mouse TIGIT-his(Cat#MB09AP0804)，或泰州市百英生物科技有限公司PD-L1-mFc(Cat#1576)，或由本发明表达纯化得到。表达的人TIGIT(-his、-Fc Tag或者-mFc tag)序列为NCBI Reference Sequence：NP_776160.2，全长244个氨基酸,其中1-21位氨基酸为信号肽；胞外(ECD)区域为22-141位氨基酸；胞外(ECD)区域 32-42位氨基酸为同源二聚体形成区，39-127位氨基酸为Ig区，32位和101位为糖基化位点。表达的恒河猴TIGIT(RhTIGIT-mFc tag)序列为NCBI Reference Sequence:XP_014985302.2，全长为245个氨基酸，其中1-21位氨基酸为信号肽；39-128位氨基酸为Ig区。
人CD155(PVR，hFc tag)蛋白序列为GenBank:AAA36462.1，全长392个氨基酸,其中1-20位氨基酸为信号肽；胞外(ECD)区域为21-343位氨基酸。其中43-142位氨基酸为Ig1_PVR_like区，145-240位氨基酸为Ig2_Nectin-2_like区。
人PD-1(hFc/his tag)蛋白序列为NCBI Reference Sequence:NP_005009.2，全长288个氨基酸，其中第1-20位为信号肽；ECD为第21-167位氨基酸。
人PD-L1(hFc/his tag)蛋白序列为NCBI Reference Sequence:NP_054862.1，全长290个氨基酸，其中第1-18位为信号肽；ECD为第19-239位氨基酸。
本发明所用hFc tag抗原均是在C-端连接IgG1Fc区域，his tag抗原为在C-端连接6×his。mFc tag抗原均是在C-端连接mIgG2a Fc区域。
本发明所用抗体，包括阳性对照抗体Ref1(即罗氏RG6058,序列来自WO2015009856A2，#19轻链，#17重链)和阳性对照抗体Ref2(即BMS的22G2,序列来自WO2016/106302A1，#9轻链，#7重链)均由本发明表达纯化。
表达所用pTT5载体(Bio Vector，Cat#102762)。将所表达的重组蛋白，抗体轻、重链序列克隆到pTT5载体上，经瞬时转染HEK293E细胞(Life Technologies，Cat#11625019)表达，后纯化得到。
具体地，293细胞在SMM 293-TⅡ Expression Medium(北京义翘神州科技股份有限公司，Cat#M293TⅡ)培养基中扩培。瞬转开始48h前，调节细胞浓度至1×10 6cells/ml，于摇床中培养48h，培养条件为36.5℃，6％CO 2，120rpm。转染前再次镜检存活率>95％，细胞浓度在4×10 6cells/mL。
准备300mL细胞，15mL Gibco FreeStyle 293 Expression Medium(Gibco，Cat#12338018)溶入重链、轻链质粒分别为90μg和60μg(如果是重组蛋白，单个质粒用量为150μg)，0.22μm过滤除菌。再取15mL FreeStyle 293 Expression Medium溶入1mg/mL PEI(Polysciences Inc，Cat#23966-2)600μL后静置5min。把PEI缓慢加入质粒中，室温孵育10min，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，37℃，8％CO 2摇床培养5天收样，8000g离心10min取上清进行纯化。
抗体或-Fc融合蛋白纯化：将样品高速离心去除杂质，用PBS pH7.4平衡含有Protein A(Mabselect,GE Healthcare Life Science，Cat#71-5020-91AE)的重力柱(生工生物，Cat# F506606-0001)，2-5倍柱体积冲洗。将样品过柱，控制流速，保留时间为5min。用5-10倍柱体积的PBS(生工生物，Cat#B548117-0500)冲洗柱子。再用pH 3.5 0.1M乙酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，酶标仪测定浓度。
His Tag蛋白纯化：将样品高速离心去除杂质；平衡镍柱(Ni smart beads 6FF，常州天地人和生物科技有限公司，Cat#SA036010)，用含有10mM咪唑0.5M NaCl的PBS(pH7.4)溶液平衡镍柱，2-5倍柱体积冲洗。将待纯化上清过柱。控制流速，使其保留时间为5min。漂洗杂蛋白：使用含有10mM咪唑0.5M NaCl的PBS(pH7.4)溶液冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。用含有250mM咪唑0.5M NaCl的PBS(pH7.4)洗脱目的蛋白。
Buffer置换：将洗脱、中和后的hFc/mFc tag或洗脱的his tag蛋白过超滤管，12000g离心10min，(超滤管，Merck Millipore，Cat#UFC500308)，如体积较大，可反复离心直至所有蛋白均浓缩，加入PBS，离心2次，尽量去除残留的buffer，将超滤管倒置在新的收集管中，低速离心1min后，再补加1mL PBS，测定浓度，分装、储存备用。
实施例2：过表达细胞株构建及细胞活性(ELISA)检测
本发明所用的过表达细胞株CHO-K1-T1001系本发明人通过本公司的稳定细胞株构建平台自行构建完成，具体构建过程如下：实验开始第1天，将293T细胞(中国科学院典型培养物保藏委员会细胞库，Cat#GNHu17)接种于两个6cm培养皿，7.5×10 5细胞/皿。第2天将包装质粒pGag-pol和pVSV-G(BioVector质粒载体菌种细胞基因保藏中心)和克隆有人TIGIT基因的质粒pTBE1001各4μg加入OPTI-MEM(Thermofisher Scientific，Cat#31985070)，使最终体积为200μL，另准备200μL OPTI-MEM加入36μL转染试剂fectin(上海源培生物科技股份有限公司，Cat#F210)，二者混匀，室温放置5min，然后将混合物(每皿各200μL)滴加入培养好的293T细胞。第3天将293T细胞培养液换为4mL DMEM高糖培养基(上海源培生物科技股份有限公司，Cat#L130KJ)。第4天将5×10 5个CHO-K1细胞(中国科学院典型培养物保藏委员会细胞库，Cat#SCSP-507)接种于10cm培养皿。第5天收集293T细胞上清(病毒)，用0.45μm滤膜过滤至CHO-K1细胞，同时加入10μg/mL polybrene(上海翊圣生物科技有限公司，Cat#40804ES76)，混匀后放置培养箱，3～4h后将培养基换为含10％FBS的DMEM/F12(上海源培生物科技股份有限公司，Cat#L310KJ)。第7天将细胞传代，从第8天开始，细胞中加入10μg/mL puromycin进行筛选(上海源培生物科技股份有限公司，Cat#S250J0)。2-3天后细胞大量死亡，更换培养基继续培养，直到细胞不再死亡时，细胞大量扩增，筛选单克隆细胞株， 扩培，冻存保种。
本实施例子所用的人TIGIT(pTBE1001)的氨基酸序列NP_776160.2，全长244个氨基酸,其中1-21位为信号肽；第22-244位为本发明构建CHO-K1 TIGIT+细胞系所表达的蛋白序列。
TIGIT+细胞结合活性(ELISA)检测：
将上述实施例得到的人TIGIT过表达的单克隆细胞株扩培后，按10×10 4细胞/孔铺96孔板，37℃培养箱过夜贴壁后去除上清，每孔加入100μL免疫染色固定液(上海碧云天生物技术有限公司，Cat#P0098)，室温固定半小时。100μL 1×PBS(上海源培生物科技股份有限公司，Cat#B320)洗一次，加入230μL 5％牛奶，37℃封闭3小时，230μL 0.05％PBST洗3次。每孔加入50μL，10μg/mL起始，5倍梯度稀释的待测抗体或阳性对照抗体。37℃孵育1小时，230μL 0.05％PBST洗5次。加Anti-human HRP(Jackson Immuno Research，Cat#109-035-003)1:2500，50μL/孔，37℃孵育1小时，PBST洗5遍，每孔加入50μL TMB(Surmodic，Cat#TTMB-1000-01)，室温显色，加入50μL/孔1M H 2SO 4终止反应。酶标仪MultiskanGO(Thermo，型号：51119300)读OD450，Graphpad prism 5进行数据分析。
实施例3：抗TIGIT/PD1/PD-L1抗体和TIGIT/PD1/PD-L1结合实验(ELISA)
用pH7.4的PBS缓冲液将人TIGIT-hFc、TIGIT-his、恒河猴猴TIGIT-mFc(RhTIGIT-mFc)、食蟹猴TIGIT-hFc(cyno TIGIT-hFc)、小鼠TIGIT-his(muTIGIT-his)或PD1和PD-L1等重组蛋白稀释至5μg/mL或2μg/mL，以50μL/孔的体积加入96孔酶标板(Corning，Cat#CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液230μL/孔，37℃孵育箱中孵育3小时或4℃放置过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(1×PBS，pH7.4，含0.05％Tweeen20)洗板5次后，每孔加入50μL上清(含检测抗体)或10μg/mL起始，5倍梯度稀释的待测抗体，37℃孵育1小时，PBST洗板5次，加入1：2500稀释的Anti-mouse Fc-HRP或Anti-human Fc-HRP二抗(Jackson Immuno Research，Cat#115-035-003或109-035-003)，50μL/孔，37℃孵育1小时。用PBST洗板5次后，每孔加入50μL TMB显色底物(KPL，Cat#52-00-03)，室温显色10-15min，每孔加入50μL 1M H 2SO 4终止反应，用MULTISKAN Go酶标仪(ThermoFisher,型号：51119300)读OD450，根据OD值挑选结合活性高的克隆或者计算EC50值(对浓度已知的抗体)。
实施例4：抗TIGIT抗体阻止TIGIT和CD155结合活性实验
用pH7.4的PBS缓冲液将人TIGIT-hFc稀释至5μg/mL，以50μL/孔加入96孔酶标板(Corning，Cat#CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用1×PBS稀释的5％脱脂牛奶封闭液230μL/孔，37℃孵育箱中封闭3小时或4℃放置过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(1×PBS，pH7.4，含0.05％Tweeen20)洗板5次后，稀释Bio-CD155至4μg/mL，取30μL Bio-CD155和30μL上清(含检测抗体)或30μL 100μg/mL起始，3倍梯度稀释的待测抗体混匀后，取50μL至包被的TIGIT-hFc板中，37℃孵育箱孵育1小时，洗板5次后每孔加入50μL 1：1000稀释的Streptavidin-HRP二抗(Genscript，Cat#M00091)，37℃孵育1小时，后PBST洗5遍，每孔加入50μL TMB(Surmodic，Cat#TTMB-1000-01)显色，加入50μL/孔1M H 2SO 4终止反应。酶标仪(MultiskanGO Thermo，型号：51119300)读OD450，Graphpad prism 5进行数据分析。
实施例5：抗PD-1或PD-L1抗体阻断PD1和PD-L1结合活性实验
用pH7.4的PBS缓冲液将人PD1稀释至2μg/mL，以50μL/孔加入96孔酶标板(Corning，Cat#CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用1×PBS稀释的5％脱脂牛奶封闭液230μL/孔，37℃孵育箱中封闭3小时或4℃过夜(16-18小时)进行封闭。弃去封闭液，并用PBST缓冲液(1×PBS，pH7.4，含0.05％Tweeen20)洗板5次后，稀释PD-L1-mFc至0.4μg/mL，取30μL稀释的PD-L1-mFc和30μL适当浓度起始，3倍梯度稀释的待测抗体混匀后，取50μL至包被的PD1板中，37℃孵育箱中孵育1小时，洗板5次后每孔加入50μL 1：2500稀释的Goat anti-mouse IgG(H+L)-HRP二抗(Jackson，Cat#115-035-003)，37℃孵育1小时，后PBST洗5遍，每孔加入50μL TMB(Surmodic，Cat#TTMB-1000-01)显色，加入50μL/孔1M H 2SO 4终止反应。酶标仪MultiskanGO(Thermo，型号：51119300)读OD450，Graphpad prism5进行数据分析。
实施例6：双夹心ELISA方法检测双特异抗体同时结合PD-L1和TIGIT活性
用pH7.4的PBS稀释LT023(TIGIT-his，上海健信生物医药科技有限公司自产)至7μg/mL，加至96孔ELISA板，100μL/孔，4℃过夜孵育。弃去液体后，PBST(1×PBS，pH7.4，含0.05％Tweeen20)洗板1次，250μL/孔。用230μL 5％的脱脂乳，37℃封闭3h。弃去封闭液，PBST洗板3次，250μL/孔。96孔ELISA板封闭的同时，稀释待测抗 体，起始浓度为10μg/mL，5倍梯度稀释，稀释7个点，第8个点以稀释液为空白对照，将稀释好的待测抗体按100μL/孔加至ELISA板中，1个复孔，37℃孵育1.5h。PBST洗板4次后，将PD-L1-mFc用抗体稀释液稀释至0.1μg/mL，加至ELISA板中，100μL/孔，37℃孵育1h。PBST洗板4次，用抗体稀释液将anti-mFc-HRP(Jackson Immuno Research，Cat#115-035-003)按1：2500的比例稀释，加至ELISA板中，100μL/孔，37℃孵育1h。PBST洗板4次，加入TMB(Surmodic，Cat#TTMB-1000-01)显色液100μL/孔，室温显色8min后，加入1M的H 2SO 4 100μL/孔以终止反应。酶标仪MultiskanGO(Thermo，型号：51119300)读OD450，Graphpad prism 5进行数据分析。
实施例7：抗人TIGIT抗体的发现
本发明用人TIGIT-his(北京义翘神州生物技术有限公司，Cat#10917-H08H)作为抗原，弗式佐剂免疫A/J小鼠4次后充免，三天之后电融合并筛选融合杂交瘤，从数千株杂交瘤克隆中筛选，意外发现该次融合中多达467株杂交瘤克隆和TIGIT有较好的结合活性。本发明用实施例4中的方法进一步筛选得到能很好地阻断TIGIT和PVR结合的单克隆细胞株，从中得到鼠源抗体。具体地，实验用A/JGpt小鼠，雌性，4周龄，购自江苏集萃药康生物科技有限公司，动物品系编号：N000018。小鼠购进后，实验室环境饲养1周，白天光/夜晚暗周期调节，温度20-25℃；湿度40-60％。小鼠分成3只/组/笼。用购买的TIGIT-his抗原进行免疫。首免佐剂为弗式完全佐剂(Sigma-Aldrich，Cat#SIGMA F5506-10M)，从二免开始佐剂为弗式不完全佐剂(Sigma-Aldrich，Cat#SIGMA F5881-10M)。抗原与佐剂比例为1：1。100μL/25μg/只首免，100μL/12.5μg/只二免、三免、四免，小腿肌肉注射。融合前3天，100μL/25μg/只加强免疫。免疫时间为第0、14、28、42和56天(加强免疫)。于第36，50天，用上述实施例3的ELISA方法检测小鼠血清抗体滴度，选择血清中抗体滴度高并且滴度处于平台期的小鼠进行脾细胞融合，将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2022142471-appb-000001
CRL-8287 TM)进行融合得到杂交瘤细胞铺96孔板，筛选，优选克隆。
对杂交瘤细胞株进行初次筛选，用实施例3的ELISA方法检测杂交瘤细胞株分泌上清中的抗体和人TIGIT的结合活性，并选择活性好的克隆，取上清用实施例4所述方法检测所分泌的抗体阻断hTIGIT和hPVR的结合活性(Blocking活性)，发现有56株克隆均具有很好的阻断效果，部分结果见表1。本发明综合结合活性及阻断活性，优选14株杂交瘤细胞株进行3轮有限稀释，且每轮有限稀释均利用实施例2、实施例3和实施例4中的方法进行优选，最终得到6株单克隆细胞株，结果如表2所示。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
表1杂交瘤融合筛选单克隆细胞结合活性及阻断活性(OD450)
Figure PCTCN2022142471-appb-000002
表1中列出了部分初次筛选数据。数据显示，很多杂交瘤融合细胞在初始筛选时表现出了很高的结合活性及很好的阻断效果(接近ref甚至更好，ELISA binding值越高表示亲和力越好，blocking数值越低，表明blocking活性越好)，比如3A10、6H4、7H4、11A4、17A10和23H3等克隆。对优选的14株克隆(包括以上6株)进行多次有限稀释，每一轮稀释后7-10天待克隆增殖后，用ELISA方法重新检测各个克隆所分泌抗体(上清)的结合活性和blocking活性，并利用实施例2中的方法检测融合细胞与稳定表达人TIGIT的CHO-K1细胞的结合情况，进一步优选，最终得到6株单克隆细胞株分泌的上清(抗体)对人TIGIT有很好的结合活性，且能很好地阻断人TIGIT与人PVR的结合(结果如表2所示)。
将上述6株单克隆细胞扩增后计数，取1×10 7cells/200μL注射入小鼠腹腔，小鼠饲养6天后收腹水，用实施例1中的方法纯化后分别得到IF071、IF081、IF091、IF101、IF112和IF122 6个鼠源抗体。然后利用实施例2、3和4中的方法，评价6个鼠源抗体与人TIGIT、食蟹猴TIGIT和鼠TIGIT及稳定表达TIGIT的CHO-K1细胞(CHO-K1-T1001)的结合活性及阻断活性，结果如表3所示。结果显示，IF071、IF081、IF091、IF101、IF112和IF122均不与鼠TIGIT结合，IF081不与cynoTIGIT结合，且IF091与hTIGIT、cyno TIGIT和CHO-K1-T1001的结合活性及阻断hTIGIT与hPVR结合的活性均，在6个鼠源 抗体中均为最优，更佳地，IF091与CHO-K1-T1001的结合活性较Ref1好1.73倍，IF091阻断hTIGIT结合hPVR的活性较Ref1好1.83倍。提取IF091的序列，即为本发明优选鼠源mab22的抗体序列。
表2杂交瘤融合筛选单克隆细胞活性(OD450)
克隆号 与hTIGIT结合活性 阻断hTIGIT与hPVR结合的活性
3A10B3D6B3 2.06 0.0586
23H3G9B6D3 1.53 0.0604
17A10D2B3F9 1.28 0.0611
6H4E3C4D5 2.01 0.0578
7H4E11E8F3 1.76 0.0703
11A4B11D2G9 2.00 0.113
表3优选鼠源抗体活性
Figure PCTCN2022142471-appb-000003
ND：未能检测到信号。
实施例8：本发明鼠源抗人TIGIT抗体mab22抗体序列提取、分析鉴定
从杂交瘤优选得到的单克隆细胞株中提取抗体序列过程为本领域技术人员常用的方法。具体地，收集上述单克隆细胞株，扩增培养后，取1×10 6个细胞，用Trizol(Invitrogen，Cat#15596-018)提取RNA(按照试剂盒说明书步骤)，将提取的RNA用反转录试剂盒(生工生物，Cat#B532435)反转录成cDNA，以反转录得到的cDNA为模板，进行PCR扩增。扩增产物测序，分别得到mab22抗体轻、重链可变区碱基/编码序列(如下)。所用引物参阅Novagen发表的手册TB326 Rev.C0308。
本发明优选的杂交瘤细胞株中获得的鼠源单克隆抗体mab22轻链可变区碱基序列(划线部分为编码序列)：
Figure PCTCN2022142471-appb-000004
Figure PCTCN2022142471-appb-000005
本发明优选的杂交瘤细胞株中获得的鼠源单克隆抗体mab22重链可变区碱基序列(划线部分为编码序列)：
Figure PCTCN2022142471-appb-000006
本发明所得到的上述鼠源单克隆抗体mab22轻、重链可变区的碱基序列所编码的氨基酸序列为如下SEQ ID NO:3和SEQ ID NO:4。
本发明优选的杂交瘤单克隆细胞株中获得的鼠源单克隆抗体mab22轻链可变区氨基酸序列：
Figure PCTCN2022142471-appb-000007
本发明优选的杂交瘤细胞单克隆株中获得的鼠源单克隆抗体mab22重链可变区氨基酸序列：
Figure PCTCN2022142471-appb-000008
本发明上述抗体轻、重链可变区序列和IgG不同型的恒定区，例如人hIgG1、hIgG2、hIgG3、hIgG4，人轻链κ、λ型；鼠mIgG1、mIgG2、mIgG3，鼠轻链κ、λ型等重组表达纯化得到完整的人鼠嵌合抗体或鼠抗体。本发明以重链恒定区为hIgG1，轻链κ型为例子，按实施例1表达纯化方法得到嵌合抗体mab22c，用实施例2、实施例3和实施例4之方法检测了mab22c与hTIGIT、RhTIGIT、hTIGIT+细胞的结合活性及阻断hTIGIT与hPVR结合的活性，并和对照抗体进行了比较，结果见表4。
表4本发明抗体mab22c活性分析
Figure PCTCN2022142471-appb-000009
表4结果表明，本发明抗体mab22c较Ref1和Ref2活性均较好。mab22c与hTIGIT的结合活性较Ref1和Ref2均好(0.270nM vs 0.462nM vs 0.396nM)，其结合EC50分别是Ref1和Ref2结合EC50的0.58倍和0.68倍，mab22c与hTIGIT+细胞结合EC50是Ref2EC50的0.47倍，与Ref1的EC50接近(0.101nM vs 0.0.216nM vs 0.138nM)；mab22c阻断hTIGIT结合hPVR的IC50是Ref2 IC50的0.51倍，是Ref1 IC50的0.46倍(0.541nM vs 1.07nM vs 1.19nM)；mab22c与RhTIGIT的结合活性较强，在Ref1和Ref2结合EC50的2倍左右(0.658nM vs 0.243nM vs 0.391nM)。
上述结果表明，本发明意外发现的抗体mab22c是优于Ref1和Ref2的一种新的抗体。mab22c结合hTIGIT活性及阻断hTIGIT结合hPVR的活性较Ref1和Ref2均较好，可用于肿瘤治疗产品的开发，包括单克隆抗体、双特异抗体、多特异抗体、CAR、ADC等。且mab22c能与恒河猴TIGIT结合(恒河猴TIGIT序列Ig-like区域同食蟹猴完全一致)，结合EC50与Ref1和Ref2相差在2倍左右，这一特点为该抗体临床前在非人灵长类动物中进行安全性评价提供便利。
实施例9：本发明鼠源抗体人源化
为了避免用于药物开发过程中的免疫原性等方面的风险，对本发明的鼠源抗体mab22进行了人源化设计和筛选，以及序列优化，具体过程描述如下。
抗体的CDR定义本领域还有多种不同的方法，这些标记CDR方法可总结如下表5。
表5本领域抗体CDR定义不同方法汇总*
Loop CCG定义 Kabat定义 AbM定义 Chothia定义 Contact定义
轻链CDR1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36
轻链CDR2 L50-L56 L50-L56 L50-L56 L50-L56 L45-L55
轻链CDR3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96
重链CDR1 H26-35 H31-35 H26-35 H26-32 H30-35
重链CDR2 H50-65 H50-65 H50-58 H52-56 H47-H58
重链CDR3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101
*更多信息可以参阅网站： http://www.bioinf.org.uk/abs/#cdrdef
上述所述针对TIGIT靶点的鼠源抗体mab22可变区按照表5各种定义方法，其CDR序列标记/注释如下表6-表10所示。
表6本发明抗hTIGIT(anti-hTIGIT)抗体mab22按CCG定义的CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 GFTFSSYTMS(SEQ ID NO:8)
重链CDR2 EISSSGGSTYYPDTVKG(SEQ ID NO:9)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表7本发明抗hTIGIT(anti-hTIGIT)抗体mab22按Kabat定义的CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 SYTMS(SEQ ID NO:11)
重链CDR2 EISSSGGSTYYPDTVKG(SEQ ID NO:9)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表8本发明抗hTIGIT(anti-hTIGIT)抗体mab22按AbM定义的CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 GFTFSSYTMS(SEQ ID NO:8)
重链CDR2 EISSSGGSTY(SEQ ID NO:12)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表9本发明抗hTIGIT(anti-hTIGIT)抗体mab22按Chothia定义的CDR序列
抗体 mab22 CDRs
轻链CDR1 RSSQSIVHNSGNTYLE(SEQ ID NO:5)
轻链CDR2 KVSNRFS(SEQ ID NO:6)
轻链CDR3 FQFSHVPRT(SEQ ID NO:7)
重链CDR1 GFTFSSY(SEQ ID NO:13)
重链CDR2 SSSGGS(SEQ ID NO:14)
重链CDR3 PGLGAWFAY(SEQ ID NO:10)
表10本发明抗hTIGIT(anti-hTIGIT)抗体mab22按Contact定义的CDR序列
抗体 mab22 CDRs
轻链CDR1 VHNSGNTYLEWY(SEQ ID NO:15)
轻链CDR2 LLIYKVSNRF(SEQ ID NO:16)
轻链CDR3 FQFSHVPR(SEQ ID NO:17)
重链CDR1 SSYTMS(SEQ ID NO:18)
重链CDR2 LVAEISSSGGSTY(SEQ ID NO:19)
重链CDR3 ARPGLGAWFA(SEQ ID NO:20)
对本发明鼠源抗体mab22的CDR序列做上述分析、标记、定义后，如本领域许多文献公示的方法进行人源化。将鼠源抗体序列和人抗体种系数据库(v-base)比较，找出同源性高的人抗体轻、重链种系，在此基础上，计算机建模，模拟抗体结构中可能影响和抗原结合的位点，回复突变关键位点和组合，筛选出活性优选的人源化抗体分子。
具体地，通过序列同源性比较分析，发现和mab22轻链同源性比较好的人抗体种系包含IGKV2-40*01、IGKV2D-40*01、IGKV2-28*01、IGKV2-29*02、IGKV2-29*03、IGKV2/OR22-4*01、IGKV2D-28*01、IGKV2D-29*01、IGKV2-29*01、IGKV2D-29*02等。进一步比较、分析，优选人抗体种系轻链IGKV2-28*01。序列比对发现mab22轻链的J 基因区和人抗体种系hJk1、hJk2.1、hJk2.2、hJk2.3、hJk2.4、hJk3、hJk4.1、hJk4.2、hJk5同源性高，进一步比较、分析，优选hJk4.1用于mab22轻链人源化抗体J区，进行人源化设计、筛选和序列优化。
通过序列同源性比较分析，发现和mab22重链同源性比较好的人抗体种系包含有IGHV3-7*01、IGHV3-7*02、IGHV3-7*03、IGHV3-48*01、IGHV3-48*02、IGHV3-48*03、IGHV3-48*04、IGHV3-66*01、IGHV3-66*02、IGHV3-66*04等。进一步比较、分析，优选人种系重链IGHV3-66*01序列用于本发明抗体人源化。序列比对发现和mab22的重链J基因区和人抗体种系重链J基因hJh1、hJh2、hJh3.1、hJh3.2、hJh4.1、hJh4.2、hJh4.3、hJh5.1、hJh5.2、hJh6.1、hJh6.2、hJh6.3、hJh6.4等同源性高，进一步比较、分析，优选hJh4.1用于本发明鼠源抗体mab22重链人源化抗体J区，进行人源化设计、筛选和序列优化。
将本发明抗体mab22的CDR区(见上述CDR之定义)移植到所选择的人源化轻、重链人抗体种系模板上，再与IgG轻、重链恒定区重组。然后，以鼠源抗体的三维结构为基础，对人源化抗体中包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，筛选这些突变以及突变组合，看对抗体活性的影响，并对CDR区化学不稳定氨基酸残基优化，得到结构、活性等优化的抗体分子序列，即完成本发明鼠源抗体的人源化。
以下结合mab22的具体序列，以hIgG1重链，κ型轻链(序列如下)为例子进行说明。
人抗体κ型轻链恒定区：
Figure PCTCN2022142471-appb-000010
人IgG1的重链恒定区：
Figure PCTCN2022142471-appb-000011
本发明人源化轻链可变区优选序列：
>mab22-hL1
Figure PCTCN2022142471-appb-000012
>mab22-hL2
Figure PCTCN2022142471-appb-000013
>mab22-hL3
Figure PCTCN2022142471-appb-000014
本发明人源化重链可变区优选序列：
>mab22-hH1
Figure PCTCN2022142471-appb-000015
>mab22-hH2
Figure PCTCN2022142471-appb-000016
本发明鼠源抗体轻链的人源化序列中回复突变的个数较少，回复突变位点数目只有0个、1个或2个，如上述所列序列，因此本发明抗体轻链可变区序列的人源化程度很高。这些序列与人抗体轻链恒定区κ链或λ链的恒定区序列组合得到本发明抗体的轻链序列，比如本发明轻链用κ型轻链恒定区，如SEQ ID NO:21。同样，人源化所用重链可变区回复突变的位点数也较少，回复突变位点数目只有2个或0个，如上述所列重链可变区序列。因此本发明人源化抗体重链可变区序列人源化程度也很高。这些含不同数量回复突变的重链可变区序列，同任选人IgG1、IgG2、IgG3、IgG4链恒定区序列重组得到本发明的重链序列，比如本发明重链用hIgG1作为恒定区序列为例子加以说明。将抗体人源化后各轻链序列与各重链序列组合得到各人源化抗体。
本发明部分优选人源化抗体序列，以及所选抗体的表达量及活性评估(本发明实施 例子3之ELISA检测方法)结果如下表11所示。
表11本发明人源化抗体表达量及结合活性
Figure PCTCN2022142471-appb-000017
上述结果表明，本发明人鼠嵌合抗体mab22c与hTIGIT的结合活性较对照分子Ref1好，其结合EC50仅是Ref1EC50的0.517倍(0.585nM vs 1.132nM)。由本发明抗体鼠源序列mab22不同人源化程度的轻、重链序列组合得到的上述人源化抗体分子均保留了与嵌合抗体几乎一致的结合活性。
更佳地，不同人源化程度的组合抗体基本保留了嵌合抗体的表达量水平，是Ref1表达量的4.4-5.4倍，除mab22-h4的表达量下降约2倍外，其他抗体表达量水平均在100mg/mL左右。
更加特别地，用实施例4所述的实验方法，检测了本发明人源化抗体阻断hTIGIT与hPVR结合的效果，部分结果如表12和图1，本发明人源化抗体基本保留了嵌合抗体mab22c能很好的阻断hTIGIT与hPVR结合的特性，且人源化抗体阻断hTIGIT与hPVR结合的活性均较Ref1好，以mab22-h6为例，其阻断IC50仅是Ref1IC50的0.44倍(1.443nM vs 3.30nM)。且用实施例2中的方法检测各人源化抗体与hTIGIT+细胞的结合活性发现，本发明人源化抗体与hTIGIT+细胞的结合活性基本一致(mab22-h3稍弱)，均较Ref1结合活性较好，以mab22-h6为例，其结合EC50仅是Ref1结合EC50的0.50倍(0.0133nM vs 0.0268nM)。
这一结果表明，本发明抗体，人源化抗体，人源化优选抗体分子不仅同hTIGIT蛋白结合，与hTIGIT+细胞结合，而且能有效的阻断hTIGIT与hPVR的结合。
表12本发明人源化抗体结合hTIGIT+细胞活性及阻断活性
Figure PCTCN2022142471-appb-000018
表12所述部分优选人源化抗体的轻、重链氨基酸(包括恒定区)序列如下。
人源化mab22-h1抗体氨基酸序列：
轻链：
Figure PCTCN2022142471-appb-000019
重链：
Figure PCTCN2022142471-appb-000020
人源化mab22-h2抗体氨基酸序列：
轻链：
Figure PCTCN2022142471-appb-000021
人源化抗体mab22-h2重链序列同SEQ ID NO:29。
人源化mab22-h5抗体氨基酸序列：
人源化抗体mab22-h5轻链序列同SEQ ID NO:30。
重链：
Figure PCTCN2022142471-appb-000022
人源化mab22-h6抗体氨基酸序列：
轻链：
Figure PCTCN2022142471-appb-000023
人源化抗体mab22-h6重链序列同SEQ ID NO:31。
综合本发明数据表明，发明人通过创新性筛选，意外发现了一株抗人TIGIT抗体，它们结合活性好，能很好地结合人TIGIT蛋白和hTIGIT+细胞，能有效的阻断人TIGIT和人PVR的结合，比目前临床上的抗体(对照抗体Ref1)有更好的活性；能和非人灵长类TIGIT结合，为非临床安全性评价提供便利；人源化程度高，为后期药物开发降低了免疫原性的风险；人源化后抗体表达量高，为下游生产及工艺提供便利，节约成本。本发明抗体独特的特性，使其更适合用于针对人TIGIT靶点抗体药物开发，并且作为候选药物可单独或者联合，特别是为联合PD-1抗体治疗肿瘤提供了新的，甚至是更好的选择。
实施例10：针对TIGIT靶点的双特异抗体设计
基于上述发现的抗TIGIT抗体，本发明进行了多种双特异抗体的设计。所设计的双特异抗体的通式如下。
表13基于本发明抗Sirpα抗体进行的双特异设计(通式1)
方案 含轻链的序列 含重链的序列
1 T2(scFv) n1-T1VL-LC-T2(scFv) n2 T2(scFv) n3-T1VH-HC-T2(scFv) n4
2 T1(scFv) n1-T2VL-LC-T1(scFv) n2 T1(scFv) n3-T2VH-HC-T1(scFv) n4
3 T2(scFv) n1-T1VL-LC-T1(scFv) n2 T2(scFv) n3-T1VH-HC-T1(scFv) n4
4 T1(scFv) n1-T2VL-LC-T2(scFv) n2 T1(scFv) n3-T2VH-HC-T2(scFv) n4
表13中，含轻链的序列指该序列除了包括轻链序列以外，还可以包括与轻链序列连 接的scFv；含重链的序列指该序列除了包括重链序列以外，还可以包括与重链序列连接的scFv。其中，T1代表针对靶点1(比如TIGIT)的第一蛋白功能区，T2代表针对靶点2(非TIGIT)的第二蛋白功能区。T1(scFv)代表针对靶点1抗体的scFv序列；T2(scFv)代表针对靶点2的scFv序列。
(scFv) n1，(scFv) n2，(scFv) n3，(scFv) n4中的n1，n2，n3，n4分别为自然数，可以是0、1、2、3等，在本发明的具体实施例中，n1，n2，n3，n4中其中至少1个数值为1，其余为0。VL，代表针对靶点1或者2的抗体轻链可变区序列；VH，代表针对靶点1或者2的抗体重链可变区序列。LC，代表轻链(κ或者λ)的恒定区序列，优选人轻链恒定区序列；HC代表重链的恒定区序列，包括IgG1、IgG2、IgG3、IgG4等的恒定区序列(缩写为HC-IgG1、HC-IgG2、HC-IgG3、HC-IgG4)，优选人的重链恒定区序列(HC-hIgG)。重链恒定区C末端连接scFv或其它蛋白序列的时候，其C末端末位氨基酸K可以突变，优选突变为A。由此，在方案1中，T1为免疫球蛋白，T2为scFv；在方案2中，T2为免疫球蛋白，T1为scFv；scFv针对的靶点相同；在方案3、4中，两端的scFv针对两个不同的靶点。
表13中，所述scFv为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端。
需说明的是，当上述scFv为轻链可变区-连接子-重链可变区时，其连接方式为轻链可变区的C末端与连接子连接，所述连接子再与重链可变区的N末端连接，从而将scFv轻链可变区的N末端和重链可变区的C末端暴露出来，使其可以通过连接子与免疫球蛋白的轻链和或重链连接。在本发明中，当其连接免疫球蛋白的轻链时，在一些具体的实施例中优选地使用scFv的重链可变区的C末端通过连接子与免疫球蛋白轻链的N末端连接；当其连接免疫球蛋白的重链时，在一些具体的实施例中优选地使用scFv的轻链可变区的N末端与免疫球蛋白重链的C末端连接。
当所述scFv为重链可变区-连接子-轻链可变区时，其连接方式为轻链可变区的N末端与连接子连接，所述连接子再与重链可变区的C末端连接，从而将scFv轻链可变区的C末端和重链可变区的N末端暴露出来，使其可以通过连接子与免疫球蛋白的轻链和/或重链连接。在此情况下，当其连接免疫球蛋白的轻链时，在一些具体的实施例中优选地使用scFv的轻链可变区的C末端与免疫球蛋白轻链的N末端连接；当其连接免疫球蛋 白的重链时，在一些具体的实施例中优选地使用scFv的重链可变区的N末端与免疫球蛋白重链的C末端连接。
所述scFv与所述免疫球蛋白连接的连接子优选为(G 4S) m或(G 2S) m，所述的m优选为0～10之间的整数。进一步优选地，所述连接子为(Gly-Gly-Gly-Gly-Ser) 3或(Gly-Gly-Ser) 4。所述scFv的数量为一对，对称地连接在所述的免疫球蛋白轻链和/或重链的C末端和或N末端。所述scFv中的连接子为VE(G 2S) mGGVD或(G 4S) m，所述的m优选为0～10之间的整数。进一步优选地，所述连接子为VE(G 2S) 4GGVD或(G 4S) 3。
上述双特异设计涉及的各个靶点抗体序列，除了本发明所述anti-TIGIT抗体序列外，其它的靶点抗体序列来自已经公开的抗体序列。包括，抗PD-1抗体Nivolumab/Opidivo(简称Nivo)和Pembrolizumab/Keytruda(简称Pem)；抗PD-L1抗体Atezolizumab/Tecentriq(简称Atezo)、Durvalumab/Imfinzi(简称Durv)和Avelumab/Bavencio(简称Avel)等。Nivolumab、Pembrolizumab、Atezolizumab、Durvalumab和Avelumab等序列都能从www.drugbank.ca等公开资源查到。
实施例11：针对TIGIT和PD-1/PD-L1双靶点的双特异抗体设计和活性评价
本发明针对TIGIT和PD-1/PD-L1两个靶点设计了不同序列结构的双特异抗体，见下表14。
表14针对TIGIT和PD-1或PD-L1双靶点设计的双特异抗体
Figure PCTCN2022142471-appb-000024
Figure PCTCN2022142471-appb-000025
*：κ链表示轻链为人IgG的κ型轻链恒定区。#：IgG的C末端连接连接子的时候，其最末端氨基酸K突变为A。以下重链C末端引入scFv的设计均将最末端K突变为A。
按本发明实施例1中所述的克隆表达纯化方法，分别克隆表达、纯化上述双特异抗体，并用前述实施例3中的方法分别检测各双特异抗体分子和人TIGIT以及PD-1或PD-L1的结合活性。发现不同设计的双特异抗体表达量、质量和活性差异显著。部分优选双特异抗体分子的质量、表达量及活性数据如图2和表15所示。
表15针对TIGIT和PD-1/PD-L1双靶点设计的双特异抗体的结合活性评价
Figure PCTCN2022142471-appb-000026
Figure PCTCN2022142471-appb-000027
注：NA，不适用，表示该抗体无表达。ND，由于质量不好或与另一抗原结合活性差而未检测。括号里面的数值为同实验条件下，同靶点对应的单克隆抗体的结合活性EC 50。*：同样实验条件下，双特异抗体和对应的单克隆抗体的结合活性EC 50的比值。比值越大，说明所设计的双特抗体对单靶点的结合力减弱越多，比如比值为2，则说明所设计的双特异抗体对靶点结合活性和对应的单克隆抗体相比减弱了1倍。比值在2以内(实验误差范围)，说明结合活性没有受到影响。
表15中是本发明抗TIGIT抗体mab22-h6的scFv在PD-1抗体Pem和Nivo、抗PD-L1抗体Atezo、Durv或Avel的重链N末端、C末端；轻链N末端、C末端设计成的双特异分子，或者PD-1抗体Pem或Nivo的scFv、抗PD-L1抗体Atezo、Durv或Avel的scFv与本发明抗TIGIT抗体mab22-h6设计成的双特异分子的表达量、与PD-1或PD-L1和TIGIT的结合活性。
结果表明，相同的抗PD-1抗体或抗PD-L1抗体和抗TIGIT抗体scFv，不同的位置设计成的双特异抗体，表达量和质量不同，如LB601-LB603无表达vs LB604表达量为3.22mg/L、LB609无表达vs LB606表达量为7.68mg/L、LB612 10.54mg/L vs LB613 1.59mg/L，且LB612质量较好，LB613聚体较多；相同的TIGIT抗体和抗PD-L1抗体scFv，不同的位置设计的双特异抗体，表达量不同，质量也不同，如LB622 vs LB623，LB615(12.4mg/L)vs LB621(40mg/L)(图2所示，LB615 SEC纯度远低于LB621)；相同的抗TIGIT抗体和抗PD-L1抗体，IgG形式和scFv形式抗体不同，表达量不同，活性不同，如LB611-LB614 vs LB621，mab22-h6以scFv连接于抗PD-L1抗体上时，抗体表达量低，抗体活性丢失，抗PD-L1抗体以scFv连接在mab22-h6完整抗体分子上时，基本能保留对两个靶点的活性，如LB621，表达量为40mg/L，与两个靶点的结合活性与相应单抗接近；相同的TIGIT抗体，相同的连接位置，不同的PD-1或PD-L1抗体scFv，对活性影响不同，如LB605 vs LB610 vs LB621，说明Pem scFv连接在mab22-h6重链的N端影响了mab22-h6的活性，而scFv形式的Nivo和Atezo连接在mab22-h6重链的N端形成的双特异抗体分子(LB610和LB615)基本保留了两个靶点的活性。
上述数据表明，同样的TIGIT抗体(本发明)以scFv形式连接在非TIGIT抗体上， 所设计的双特异抗体分子针对TIGIT抗原的活性丢失。同样的TIGIT抗体(本发明)，连接不同的PD-1抗体scFv或PD-L1抗体的scFv，或者scFv的位置不同，所设计的双特异抗体分子活性差别也很大。
这些数据表明，基于本发明TIGIT抗体序列所设计的双特异抗体，其序列不同，scFv和抗体位置等不同，活性不同。合适的位置、合适的序列设计得到能针对双靶点活性很好的双特异抗体，这些双特异抗体结构类似常规IgG，有完整的Fc，本发明称之为Sequence-based IgG like bispecific antibody format，即序列特异的IgG结构相似的双特异抗体(SBody)。这些双特异抗体分子具有正常抗体一样完整的Fc，让其纯化工艺可以按照正常的抗体进行，因而工艺简单，具有生产成本低的优势。
将上述保留了双靶点活性的SBody利用实施例4和实施例5中的方法分别针对两个靶点进行功能(阻断抗原和相应配体结合实验)评估，结果见表16a。
表16a针对TIGIT和PD-1/PD-L1双靶点设计的双特异抗体功能活性评价
Figure PCTCN2022142471-appb-000028
注：括号里面的数值为同实验条件下，同靶点对应的单克隆抗体阻断抗原和配体结合活性IC 50。*：IC 50改变倍数，即双特异抗体和对应的单克隆抗体(对照抗体)的IC 50的比值。比值越大，说明所设计的双特抗体对单靶点的功能活性减弱越多，比如比值为2，则说明所设计的双特异抗体对靶点功能活性和对应的单克隆抗体相比减弱了1倍。比值在2以内为实验误差范围，即活性没有受到影响。
用Biacore T200(厂家：Cytiva)仪器对本发明优选双特异抗体的亲和力进行测定。运行缓冲液pH 7.4的HBS-EP+(10mM HEPES、150mM NaCl、3mM EDTA和0.05％的P20)。待测样品1μg/mL。抗原购自北京义翘神州TIGIT-his，货号：10917-H08H，PD-L1-his货号：10084-H08H。捕获ProteinA芯片，Cat#29-1275-56，Cytiva。稀释的分析物(TIGIT-his或PD-L1-his)依次流经芯片表面Fc1，Fc2通道。流速30μL/分钟，结合时间为180秒，解离时间为800秒。实验后，用10mM Glycine-HCl，pH 1.5，30μL/min，30s清洗芯片。实验数据用Biacore T200 evaluation version 3.1(GE)软件以1:1Langmuir模型进行拟合，得出亲和力数值KD。结果如表16b所示。
表16b针对TIGIT和PD-1/PD-L1双靶点设计的双特异抗体的亲和力检测
Figure PCTCN2022142471-appb-000029
Figure PCTCN2022142471-appb-000030
上述功能活性结果表明，本发明设计的双特异抗体(SBody)，保留了对双靶点的结合、亲和力活性。且其阻断抗原与相应配体结合的活性变化与结合活性变化一致，如LB605，其结合人TIGIT的活性稍有减弱，阻断人TIGIT和人PVR的结合活性也稍有减弱(与相对应的单抗相比，变化倍数分别为3.548和3.729)。
综合数据表明，本发明抗TIGIT抗体mab22-h6和PD1、PD-L1抗体设计的双特异抗体SBody不仅活性、功能，而且表达量、质量也是序列特异的。
本发明抗TIGIT抗体mab22-h6和PD1、PD-L1抗体设计的双特异抗体SBody部分优选序列如下：
LB610轻链序列：
LB610轻链序列同SEQ ID NO:32。
LB610重链序列：
Figure PCTCN2022142471-appb-000031
LB621轻链序列：
LB621轻链序列同SEQ ID NO:32。
LB621重链序列：
Figure PCTCN2022142471-appb-000032
Figure PCTCN2022142471-appb-000033
LB605轻链序列：
LB605轻链序列同SEQ ID NO:32。
LB605重链序列：
Figure PCTCN2022142471-appb-000034
LB615轻链序列：
LB615轻链序列同SEQ ID NO:32。
LB615重链序列：
Figure PCTCN2022142471-appb-000035
Figure PCTCN2022142471-appb-000036
LB623轻链序列：
LB623轻链序列同SEQ ID NO:32。
LB623重链序列：
Figure PCTCN2022142471-appb-000037
LB624轻链序列：
LB624轻链序列同SEQ ID NO:32。
LB624重链序列：
Figure PCTCN2022142471-appb-000038
LB625轻链序列：
LB625轻链序列同SEQ ID NO:32。
LB625重链序列：
Figure PCTCN2022142471-appb-000039
实施例12：本发明TIGIT和PD-1/PD-L1双特异性抗体在不同制剂处方中的稳定性 评价
将本发明优选双特异性抗体LB621用实施例中的方法表达纯化的样品用脱盐离心柱(Thermo，Cat#89890)置换至各制剂处方中，制剂处方如表17所示。各制剂buffer置换过程如下，对脱盐离心柱先进行预处理，以1000g离心2min，去除储存液后，向脱盐离心柱中加入1mL各制剂缓冲液，以1000g离心2min，重复进行3次，弃掉收集管中的缓冲液；将脱盐离心柱置于一个新的收集管中，将适量的LB621缓慢地加到柱子中，并加入20μL制剂缓冲液压层，以1000g离心2min，收集离心后的样品，混匀后用0.2μm滤膜过滤；将过滤后的各LB621制剂样品按80μL/管分装，其中4管置于40℃水浴锅中，分别在第7，14，21，28天进行SEC-HPLC和SDS-PAGE检测，即为40℃处理7天、14天、21天和28天的样品；另取1管无菌分装后进行SEC-HPLC检测，即为40℃处理0天的样品。不同制剂处方的SEC-HPLC检测结果如表18所示。
表17本发明优选双特异性抗体LB621制剂处方方案
Figure PCTCN2022142471-appb-000040
表18本发明双特异性抗体LB621各制剂处方样品SEC-HPLC检测结果
Figure PCTCN2022142471-appb-000041
Figure PCTCN2022142471-appb-000042
上述结果表明，本发明双特异抗体LB621在较高浓度下，40℃处理，随时间的延长，在4种制剂处方中，除LB621在处方2中有少量聚体增加，在其他处方中聚体均无进一步增加；在4种处方中，从第21天开始，LB621有部分降解片段产生。40℃处理28天时，片段和聚体均无进一步增加，SEC-HPLC检测LB621的纯度下降至85％左右。LB621在4种制剂处方中的稳定性差别较小，40℃处理28天后的样品SDS-PAGE检测发现(如图3所示)，制剂处方1和制剂处方2中处理28天，LB621产生的降解片段明显少于处方3和处方4，且在处方1中，LB621产生聚体量较少。综合纯度变化及SDS-PAGE发现，LB621在处方1中稳定性较好。
上述结果表明本发明双特异抗体LB621在较高浓度下(14.2mg/mL)，在制剂缓冲液(20mM柠檬酸-柠檬酸钠，50mM精氨酸，50mM谷氨酸，200mM海藻糖，0.02％吐温80，pH6.0)中经40℃处理28天后SEC-HPLC纯度仅下降至86.22％，说明LB621稳定性较好。
实施例13：本发明TIGIT和PD-L1双特异性抗体活化T细胞活性评估
通过SEB活化人PBMC同时与本发明优选双特异抗体LB621、对应的单克隆抗体Atezo(抗PD-L1抗体)和Ref1(抗TIGIT抗体)单独以及联合孵育，对比各抗体诱导IL-2释放的增加量来评价其体外活性。
人PBMC细胞(妙顺生物科技有限公司，货号：PB010C，批号：P121040901C)从液氮中取出，复苏计数并用添加10％热灭活胎牛血清(Gibco，货号：10270-106)的RPMI1640培养基(含1μg/mL SEB)(Hyclone，货号：SH30809.01B)调整密度至2×10 6/mL后按照每孔100μL添加到96孔培养板(Corning，货号：3599)中，活化3天后，同时继续用该培养基依次配制梯度浓度的Atezo，Ref1，Atezo和Ref1的联合组及LB621，阴性对照为human IgG1 isotype，以每孔100μL添加到96孔培养板中。在二氧化碳恒温培养箱中培养3天后，离心收集上清。上清中的IL-2水平通过human IL-2 precoated ELISA kit(达优，货号：1110203)进行测量。
如图4所示，在1μg/mL SEB的作用下，除阴性对照组以及Ref1单抗引起的IL-2释放微弱升高外，其余组均引起了明显的IL-2释放，并存在浓度梯度依赖。同时，同一浓度水平下，本发明双抗LB621组各浓度诱导的IL-2释放水平均高于Atezo和Ref1抗体单独给药组及联合给药组。说明本发明优选双抗LB621同时靶向PD-L1和TIGIT后能很好地协同增强T细胞活性。
实施例14：本发明TIGIT和PD-L1双特异性抗体优化设计分子动物体内药效评估
用BALB/cJGpt-Tigit em1Cin(hTIGIT)/Gpt雌性小鼠(购自浙江维通利华实验动物技术有限公司，生产许可证编号：SCXK(浙)2019-0001)建立动物药效模型，对本发明双特异性抗体LB621进行了体内药效评估。
CT26细胞(购自上海中科院)培养于含10％胎牛血清(Gibco，货号：10270-106)的RPMI1640培养基中(Hyclone，货号：SH30809.01B)，在含5％CO 2的37℃的细胞培养箱中连续培养。BALB/cJGpt-Tigi tem1Cin(hTIGIT)/Gpt雌性小鼠，5只/笼饲养于SPF级环境，温度20～25℃；湿度40％～60％，自由进食进水，定期更换垫料。待CT26细胞长至对数生长期(汇合率在80％-90％)时，用0.25％胰酶消化，收集细胞，用RPMI1640培养基洗涤细胞两次，并用RPMI1640培养基进行重悬计数，调整细胞密度为10×10 6/mL用于接种。接种CT26细胞悬液(1×10 6个)100μL于小鼠左肋部皮下，挑选肿瘤细胞长至体积约120-130mm 3大小后随机分组，每组7只。
待测样品与阳性对照用PBS无菌配制。Blank组为PBS，Atezo(PD-L1抗体)为单抗阳性对照组，Atezo+Ref1为联合用药阳性对照组，LB621为双特异抗体药物待测组。给药方式为腹腔注射。Atezo给药剂量为10mg/kg，注射体积为200μL/只；Atezo+Ref1给药剂量为(10+10)mg/kg，注射体积为(100+100)μL/只；LB621给药剂量为13.3mg/kg(和联合组药物等摩尔)，注射体积为200μL/只。各组给药频率均为2次/周，共3次给药。
各注射样品给药当天为第0天。每次给药前测量体重，肿瘤体积，记录数据。在首次给药后第18天结束试验。药效数据分析统计到第18天。
肿瘤大小计算公式：肿瘤体积TV(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)；肿瘤相对体积(RTV)＝T/T 0或者C/C 0。相对肿瘤增长率(T/C％)＝100％×(T-T 0)/(C-C 0)；抑瘤率(TGI)＝(1-T/C)×100％；其中T 0、T分别为样品组实验开始时及实验结束时的肿瘤体积；C 0、C分别为对照组实验开始时及实验结束时的肿瘤体积。
图5和表19结果表明，BALB/cJGpt-Tigit em1Cin(hTIGIT)/Gpt雌性小鼠CT26结肠癌动物模型中，本发明优选双特异性抗体分子LB621和人源化单克隆抗体Atezo+Ref1联合给药组对肿瘤生长表现出明显的抑制作用，对肿瘤生长的抑制效果(TGI)分别达到76％和62％，显著优于Atezo给药组(TGI为46％)，且LB621给药组对肿瘤生长的抑制作用优于联合给药组。
表19给药18天后肿瘤相对体积分析结果及TGI计算
Figure PCTCN2022142471-appb-000043
Figure PCTCN2022142471-appb-000044
*代表p<0.05；**代表p<0.01；***代表p<0.01。
实施例15：本发明TIGIT和PD-L1双特异性抗体PK评价
用同实施例11中一样的BALB/cJGpt-Tigit em1Cin(hTIGIT)/Gpt雌性小鼠，同样饲养条件进行本发明双特异性抗体的PK评价。随机挑选3只小鼠组成一组。小鼠尾静脉注射mab22-h6和LB621，注射剂量分别为10mg/kg和13.3mg/kg，注射体积为200μL/只。分别于注射前0小时、注射后0.25、0.5、2、8、24、48、72、96、120、144、168、192、216、240、264、288、312、336、360、384小时眼眶取血。所取血样离心，取上清，-20℃保存。待收集完所有时间点血液样本后，用实施例6中双夹心ELISA法检测血清中LB621(双特异性抗体可同时结合PD-L1和TIGIT)的浓度，同时用实施例3中的ELISA方法检测血清中mab22-h6的浓度，来评价LB621和mab22-h6的PK特点。用EXCEL软件分析PK数据，计算LB621和mab22-h6的T 1/2，结果见表20。
表20本发明优选人源化抗体和双特异性抗体的PK评价
抗体 LB621 mab22-h6
抗原 TIGIT&PD-L1 TIGIT
T max(h) 0.25 0.25
C max(μg/mL) 136.57 224.82
T 1/2(h) 35.21 64.83
AUC(μg/mL*h) 5070 5794.5
上述结果表明，本发明优选人源化抗体mab22-h6和双特异性抗体LB621单次尾静脉注射小鼠体内后，血药浓度于0.25h达到峰值，LB621给药组的C max和AUC分别为136.57μg/mL和5070μg/mL*h，T 1/2为35.21小时；mab22-h6给药组的C max和AUC分别为224.82μg/mL和5794.5μg/mL*h，T 1/2为64.83小时。该结果说明本发明优选人源化抗体和优选双特异性抗体在小鼠体内PK参数在正常范围，具可开发性。
综合以上本发明数据表明，发明人通过创新性筛选，意外发现了一种抗人TIGIT抗体，它们和TIGIT结合活性好；和非人灵长类食蟹猴TIGIT蛋白有很好的结合活性。能有效地阻断人TIGIT和人PVR的结合。比目前临床上的抗体(对照抗体Ref1)有更好的活性，体内实验显示，比Ref2具有更优的抗肿瘤活性；且人源化程度高，为后期药物开发降低了免疫原性的风险。人源化后抗体表达量高，为下游生产及工艺提供便利，节约成本。此外，基于本发明TIGIT抗体序列所设计的双特异抗体，能够保留双靶点抗体的 功能活性，对两个靶点的结合活性、亲和力均与其相对应的单抗接近，且阻断抗原与相应的配体结合的活性也和对应的单抗结合活性一致，稳定性好，能协同增强T细胞活性，具有很好的抗肿瘤活性。这些双特异抗体(本发明称之为SBody)结构类似常规IgG，具有正常抗体一样完整的Fc，让其纯化工艺可以按照正常的抗体进行，因而工艺简单，具有生产成本低的优势。本发明抗体独特的特性，使其更适合用于针对人TIGIT靶点抗体药物开发，并且作为候选药物可单独或者联合给药，特别是为联合PD-1、PD-L1抗体治疗肿瘤提供了新的，甚至是更好的选择，且本发明优选出的双特异性抗体为肿瘤的多靶点治疗提供了另一种选择。

Claims (18)

  1. 一种靶向TIGIT的抗体或抗原结合片段，其包括轻链可变区和/或重链可变区；所述抗体或其抗原结合片段结合人TIGIT，并具有阻断PVR和TIGIT结合的功能；所述轻链可变区包含：如SEQ ID NO:5所示的氨基酸序列的CDR1、如SEQ ID NO:6所示的氨基酸序列的CDR2和如SEQ ID NO:7所示的氨基酸序列的CDR3；所述重链可变区包含：如SEQ ID NO:8所示的氨基酸序列的CDR1、如SEQ ID NO:9所示的氨基酸序列的CDR2和如SEQ ID NO:10所示的氨基酸序列的CDR3。
  2. 如权利要求1所述的靶向TIGIT的抗体或抗原结合片段，其特征在于，所述轻链可变区包含如SEQ ID NO:3、SEQ ID NO:23-25或其突变所示的氨基酸序列；所述重链可变区包含如SEQ ID NO:4、SEQ ID NO:26-27或其突变所示的氨基酸序列；
    优选地，所述轻链可变区包含如SEQ ID NO:3所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:4所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:23所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:26所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:24所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:26所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:26所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:23所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:24所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列；或，所述轻链可变区包含如SEQ ID NO:25所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:27所示的氨基酸序列。
  3. 如权利要求1或2所述的靶向TIGIT的抗体或抗原结合片段，其特征在于，其是抗体、Fab、Fab’、F(ab’) 2、Fv、scFv、双特异性抗体、多特异性抗体、单域抗体或单区抗体，或由上述抗体制得的单克隆抗体或多克隆抗体；优选地，其包括人源抗体轻链恒定区和人源抗体重链恒定区；更优选地，所述人源抗体轻链恒定区为κ或者λ型轻链恒定区，和/或，所述人源抗体重链恒定区为hIgG1、hIgG2、hIgG3、hIgG4的重链恒定区或其突变。
  4. 如权利要求3所述的靶向TIGIT的抗体或抗原结合片段，其特征在于，其轻链包含如SEQ ID NO:28、SEQ ID NO:30或SEQ ID NO:32所示的氨基酸序列或其突变，和/或，其重链包含如SEQ ID NO:29或SEQ ID NO:31所示的氨基酸序列或其突变；
    优选地，所述轻链的氨基酸序列如SEQ ID NO:28所示；所述重链的氨基酸序列如 SEQ ID NO:29所示；或，所述轻链的氨基酸序列如SEQ ID NO:30所示；所述重链的氨基酸序列如SEQ ID NO:29所示；或，所述轻链的氨基酸序列如SEQ ID NO:30所示；所述重链的氨基酸序列如SEQ ID NO:31所示；或，所述轻链的氨基酸序列如SEQ ID NO:32所示；所述重链的氨基酸序列如SEQ ID NO:31所示。
  5. 一种靶向TIGIT的双特异性抗体或多特异性抗体，其包括第一蛋白功能区和第二蛋白功能区，其中，所述第一蛋白功能区为如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段；所述第二蛋白功能区为非靶向TIGIT的抗体；优选地，所述第一蛋白功能区和第二蛋白功能区分别选自免疫球蛋白、scFv、Fab、Fab’或F(ab’) 2，且所述第一蛋白功能区和第二蛋白功能区中至多只有一个蛋白功能区为免疫球蛋白；更优选地，当所述第二功能区的结构为免疫球蛋白时，所述免疫球蛋白的恒定区包括人源抗体轻链恒定区和人源抗体重链恒定区；进一步更优选地，所述人抗体轻链恒定区为κ链或者λ链，所述人抗体重链恒定区为hIgG1、hIgG2、hIgG3、hIgG4或其突变。
  6. 如权利要求5所述的靶向TIGIT的双特异性抗体或多特异性抗体，其特征在于，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为一个或多个scFv，所述scFv包括重链可变区与轻链可变区，所述重链可变区与轻链可变区通过连接子连接；
    优选地，所述scFv通过连接子与所述免疫球蛋白连接，所述连接子优选为(G 4S) w或(G 2S) W，所述w优选为0～10之间的整数，更优选为1、2、3或者4；
    更优选地，所述scFv为轻链可变区-连接子-重链可变区，其轻链可变区N末端或重链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；或所述scFv为重链可变区-连接子-轻链可变区，其重链可变区N末端或者轻链可变区C末端通过连接子相应地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；
    进一步优选地，所述scFv中的连接子为VE(G 2S) WGGVD或(G 4S) w，所述w优选为0～10之间的整数，更优选为1、2、3或者4。
  7. 如权利要求6所述的靶向TIGIT的双特异性抗体或多特异性抗体，其特征在于，所述scFv通过连接子与所述免疫球蛋白连接，所述连接子为(G 4S) 3或(G 2S) 4，和/或，所述scFv的数量为两个，且两个scFv对称地连接在所述的免疫球蛋白轻链和/或重链的C末端或N末端；所述scFv连接免疫球蛋白的重链；
    优选地，所述scFv为轻链可变区-连接子-重链可变区结构，所述scFv的轻链可变区的C末端与连接子连接，所述连接子再与重链可变区的N末端连接，所述scFv的重链可变区的C末端通过所述连接子与所述免疫球蛋白重链的N末端连接；或，所述scFv为 重链可变区-连接子-轻链可变区结构，所述scFv的轻链可变区的N末端与所述连接子连接，所述连接子再与重链可变区的C末端连接，所述scFv的重链可变区的N末端与所述免疫球蛋白重链的C末端连接；
    更优选地，所述scFv中的连接子为VE(G 2S) 4GGVD或(G 4S) 3。
  8. 如权利要求5-7中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体，其特征在于，所述第二蛋白功能区靶向PD-1/PD-L1、Claudin18.2、TIM-3或LAG-3；
    优选地，所述第二蛋白功能区为靶向PD-1/PD-L1的抗体；
    更优选地，所述靶向PD-1/PD-L1的抗体为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab或Avelumab；
    进一步更优选地，当所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端由K突变为A；和/或，所述第二蛋白功能区为两个相同的scFv或其突变，其中，所述scFv的轻链可变区为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab或Avelumab的轻链可变区，所述scFv的重链可变区为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab或Avelumab的重链可变区，所述突变优选与原氨基酸序列具有至少99％的序列同一性，并且保持或改善了所述抗体的功能。
  9. 如权利要求5-8中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体，其特征在于，其选自以下组：
    (i)所述第一蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:32所示，其重链的氨基酸序列如SEQ ID NO:31所示；所述第二蛋白功能区为两个相同的scFv，所述连接子为(G 4S) 3或VE(G 2S) 4GGVD；
    其中，所述scFv的数量为两个；所述scFv为轻链可变区-连接子-重链可变区结构，两个scFv的重链可变区的C末端通过(G 4S) 3或(G 2S) 4对称地连接在所述免疫球蛋白的两条重链的N末端，且所述C末端由K突变为A；或，
    (ii)所述第一蛋白功能区为免疫球蛋白，其轻链的氨基酸序列如SEQ ID NO:32所示，其重链的氨基酸序列如SEQ ID NO:31所示；所述第二蛋白功能区为两个相同的scFv，所述连接子为(G 4S) 3或VE(G 2S) 4GGVD；
    其中，所述scFv的数量为两个；所述scFv为重链可变区-连接子-轻链可变区结构，两个scFv的重链可变区的N末端分别通过(G 4S) 3或(G 2S) 4对称地连接在所述免疫球蛋白的两条重链的C末端；
    优选地，所述靶向TIGIT的双特异性抗体包括以下轻链和含重链的氨基酸序列：
    氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:33所示的含重链的氨基 酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:34所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:35所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:36所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:37所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:38所示的含重链的氨基酸序列；或，氨基酸序列如SEQ ID NO:32所示的轻链，如SEQ ID NO:39所示的含重链的氨基酸序列。
  10. 一种分离的核酸，其编码如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段、或如权利要求5-9中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体。
  11. 一种包含根据权利要求10所述的分离的核酸的表达载体。
  12. 一种宿主细胞，其包含根据权利要求11所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞。
  13. 一种靶向TIGIT的抗体或抗原结合片段、或靶向TIGIT的双特异性抗体或多特异性抗体的制备方法，其包含培养如权利要求12所述的宿主细胞，从培养物中获得所述靶向TIGIT的抗体或抗原结合片段、或靶向TIGIT的双特异性抗体或多特异性抗体。
  14. 一种抗体药物偶联物，其包含细胞毒性剂，以及如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段、或如权利要求5-9任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体。
  15. 一种药物组合物，其包含如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段、如权利要求5-9中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体、和/或如权利要求14所述的抗体药物偶联物。
  16. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A包含如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段、如权利要求5-9中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体、和/或如权利要求14所述的抗体药物偶联物；所述药盒B包含靶向PD-1/PD-L1的抗体，所述靶向PD-1/PD-L1的抗体为Nivolumab、Atezolizumab、Pembrolizumab、Durvalumab和/或Avelumab；
    优选地，所述药盒A包含靶向TIGIT的抗体或抗原结合片段，所述靶向TIGIT的抗体或抗原结合片段的轻链氨基酸序列如SEQ ID NO:32所示，重链氨基酸序列如SEQ ID NO:34所示，所述药盒B包含靶向PD-1/PD-L1的抗体，所述靶向PD-1/PD-L1的抗体为 Atezolizumab。
  17. 如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段、权利要求5-9中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体、权利要求14所述的抗体药物偶联物、权利要求15所述的药物组合物和/或如权利要求16所述的药物组合在制备诊断、治疗和/或预防癌症的药物中的应用；
    优选地，所述癌症为实体肿瘤或液体肿瘤，所述实体肿瘤例如结肠癌、胃癌、肺癌、乳腺癌、头颈癌、肝癌、上皮鳞状细胞癌、食道癌、直肠癌、肾癌、卵巢癌、膀胱癌、胰腺癌、黑色素瘤和神经胶质瘤，所述液体肿瘤例如急性淋巴细胞白血病、B细胞淋巴瘤、T细胞淋巴瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和弥漫大B细胞淋巴瘤。
  18. 一种如权利要求1-4中任一项所述的靶向TIGIT的抗体或抗原结合片段、或如权利要求5-9中任一项所述的靶向TIGIT的双特异性抗体或多特异性抗体的制剂，其特征在于，
    所述制剂包括柠檬酸-柠檬酸钠、吐温80和所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体；优选地，所述制剂还包括精氨酸、谷氨酸和海藻糖中的一个或多个；更优选地，所述制剂包括20mM的柠檬酸-柠檬酸钠、50mM的精氨酸、50mM的谷氨酸、200mM的海藻糖、0.02％的吐温80和14.2mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0；
    或，所述制剂包括20mM的His-HCl、50mM的精氨酸或谷氨酸、200mM的海藻糖、0.02％PS80和11.4mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0；
    或，所述制剂包括20mM的His-HCl、125mM的甘氨酸、125mM的海藻糖、0.02％PS80和11mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0；
    或，所述制剂包括20mM的His-HCl、50mM的精氨酸盐酸盐、150mM的海藻糖、0.02％吐温80和11.6mg/mL的所述靶向TIGIT的抗体或抗原结合片段、或所述靶向TIGIT的双特异性抗体或多特异性抗体，所述制剂的pH为6.0。
PCT/CN2022/142471 2021-12-27 2022-12-27 靶向tigit的抗体和双特异性抗体及其应用 WO2023125561A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111617350.9A CN116355095A (zh) 2021-12-27 2021-12-27 靶向tigit的抗体和双特异性抗体及其应用
CN202111617350.9 2021-12-27

Publications (1)

Publication Number Publication Date
WO2023125561A1 true WO2023125561A1 (zh) 2023-07-06

Family

ID=86937506

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/142471 WO2023125561A1 (zh) 2021-12-27 2022-12-27 靶向tigit的抗体和双特异性抗体及其应用

Country Status (2)

Country Link
CN (1) CN116355095A (zh)
WO (1) WO2023125561A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025140497A1 (zh) * 2023-12-29 2025-07-03 百奥泰生物制药股份有限公司 Pvrig结合蛋白、双特异性抗体及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116789836B (zh) * 2023-08-14 2024-01-05 浙江时迈药业有限公司 针对dll3的抗体及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180169238A1 (en) * 2016-08-17 2018-06-21 Mark White Anti-tigit antibodies, anti-pvrig antibodies and combinations thereof
CN112661854A (zh) * 2020-12-03 2021-04-16 安徽安科生物工程（集团）股份有限公司 抗pd-l1与tigit双特异性抗体及其制备与应用
CN113563470A (zh) * 2020-04-29 2021-10-29 广州昂科免疫生物技术有限公司 结合tigit抗原的抗体及其制备方法与应用
WO2021238932A1 (zh) * 2020-05-26 2021-12-02 百奥泰生物制药股份有限公司 多特异性抗体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180169238A1 (en) * 2016-08-17 2018-06-21 Mark White Anti-tigit antibodies, anti-pvrig antibodies and combinations thereof
CN113563470A (zh) * 2020-04-29 2021-10-29 广州昂科免疫生物技术有限公司 结合tigit抗原的抗体及其制备方法与应用
WO2021238932A1 (zh) * 2020-05-26 2021-12-02 百奥泰生物制药股份有限公司 多特异性抗体及其应用
CN112661854A (zh) * 2020-12-03 2021-04-16 安徽安科生物工程（集团）股份有限公司 抗pd-l1与tigit双特异性抗体及其制备与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIAO YANG, CHEN PEIRAN, LUO CHENG, XU ZIYANG, LI XUE, LIU LIQIONG, ZHAO LIWEN: "Discovery of a novel anti PD-L1 X TIGIT bispecific antibody for the treatment of solid tumors.", CANCER TREATMENT AND RESEARCH COMMUNICATIONS, vol. 29, 1 January 2021 (2021-01-01), pages 100467, XP093067109, ISSN: 2468-2942, DOI: 10.1016/j.ctarc.2021.100467 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025140497A1 (zh) * 2023-12-29 2025-07-03 百奥泰生物制药股份有限公司 Pvrig结合蛋白、双特异性抗体及其应用

Also Published As

Publication number Publication date
CN116355095A (zh) 2023-06-30

Similar Documents

Publication Publication Date Title
CN111867630B (zh) 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用
JP7345578B2 (ja) 新規抗ｐｄ－ｌ１抗体
CN113166246B (zh) 一种抗体及其用途
WO2021185273A1 (zh) 靶向Sirpα的抗体或其抗原结合片段及其制备和应用
CN110431231B (zh) 抗-cd3抗体和包含所述抗体的分子
WO2018137576A1 (zh) 抗pd-1单克隆抗体及其制备方法和应用
CN107922492B (zh) 结合cd79的抗体分子
WO2021170082A1 (zh) 抗cd47/抗pd-l1抗体及其应用
WO2019091436A1 (zh) 4-1bb抗体及其制备方法和应用
CN110606891A (zh) 一种针对人cldn18.2的新型抗体分子, 抗原结合片段及其医药用途
BR112019016204A2 (pt) anticorpo ou fragmento de ligação a antígeno do anticorpo, polinucleotídeo, vetor, célula, imunócito artificial, métodos para produzir um anticorpo ou um fragmento de ligação a antígeno do anticorpo, para produzir uma molécula que se liga ao cd3 humano e cd3 de macaco cinomolgo e ao gprc5d humano, composição medicinal para tratamento e/ou prevenção, moléculas tendo atividade de ligação a antígeno e que se ligam ao cd3 humano e cd3 de macaco cinomolgo e ao gprc5d humano, e, usos para preparar um medicamento para tratar e/ou prevenir um câncer, para induzir citotoxicidade para as células expressando gprc5d e para redirecionamento de células t para as células expressando gprc5d
CN114502591B (zh) 靶向bcma的抗体、双特异性抗体及其用途
WO2020007240A1 (zh) 一种双特异性抗体及其用途
TWI821699B (zh) 抗b7h4抗體及其雙抗和應用
CN110172099B (zh) 抗lag-3人源化单克隆抗体分子，抗原结合片段及其医药用途
WO2022002036A1 (zh) 一种双特异性抗体及其用途
WO2020249041A1 (zh) 靶向lag-3的抗体和双特异性抗体及其用途
WO2023125561A1 (zh) 靶向tigit的抗体和双特异性抗体及其应用
CN116848135A (zh) 新颖的抗gremlin1抗体
CN118240082A (zh) Pd-1结合分子及其应用
CN116209680A (zh) 与人CD3ε结合的新型人抗体
WO2022247826A1 (zh) 靶向pd-l1和cd73的特异性结合蛋白
WO2019174637A1 (zh) 一种针对tim-3的全人源化抗体分子、抗原结合片段及其医药用途
WO2024165031A1 (zh) 三特异性抗原结合分子及其应用
HK40048164A (zh) 一種抗體及其用途

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: 22914813

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22914813

Country of ref document: EP

Kind code of ref document: A1