WO2023125941A1 - Anticorps à domaine unique anti-tigit et anticorps bispécifique basé sur celui-ci - Google Patents

Anticorps à domaine unique anti-tigit et anticorps bispécifique basé sur celui-ci Download PDF

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WO2023125941A1
WO2023125941A1 PCT/CN2022/143967 CN2022143967W WO2023125941A1 WO 2023125941 A1 WO2023125941 A1 WO 2023125941A1 CN 2022143967 W CN2022143967 W CN 2022143967W WO 2023125941 A1 WO2023125941 A1 WO 2023125941A1
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antibody
amino acid
heavy chain
seq
acid sequence
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秦玉蓉
黄潇
孙建明
李静
丁宓
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南京维立志博生物科技有限公司
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    • A—HUMAN NECESSITIES
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    • 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
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
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    • 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 provides a monoclonal antibody specifically binding to TIGIT and a bispecific antibody constructed based on the TIGIT monoclonal antibody. Also provided are nucleic acid molecules encoding the antibodies, expression vectors for expressing the antibodies, host cells and production methods. The invention also provides methods of treatment using the antibodies of the invention.
  • TIGIT T-cell immunoreceptor with Ig and ITIM domains
  • IgV domain immunoglobulin V IgV-like domain
  • ITIM tyrosine-based immunoreceptor inhibitory motif
  • ITT immunoglobulin tyrosine tail
  • TIGIT is mainly expressed in effector CD4+ T cells, follicular helper CD4+ T cells, regulatory T cells (Treg), effector CD8+ T and NK cells, and has become a popular target for cancer immunotherapy.
  • TIGIT has multiple ligands, including: PVR (Necl-5 or CD155), Nectin2 (CD112), Nectin3 (CD113) and Nectin4 (PVRL4), but the interaction between TIGIT and CD155 is the strongest, and the affinity has been reported About 1nM, the affinity with Nectin2 and 3 is very low, and the affinity with Nectin4 is close to that of CD155 (Reches A., Ophir Y., et al.(2020). "Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity "J. Immunother. Cancer. 8.”).
  • CD155 plays an immunoregulatory role by interacting with TIGIT, CD226, and CD96. Since the affinity of CD155 to TIGIT is much higher than that of CD226 and CD96, TIGIT and CD155 will preferentially bind to activate the two motifs present in the cytoplasmic tail of TIGIT. Inhibitory signals mediated: tyrosine-based immunoreceptor inhibitory motif (ITIM) and immunoglobulin tyrosine tail (ITT)-like motif.
  • ITIM tyrosine-based immunoreceptor inhibitory motif
  • ITT immunoglobulin tyrosine tail
  • Tumor cell surface ligands bind to TIGIT on the surface of NK cells and T cells to inhibit NK cytotoxicity and T cell activity, thereby mediating the immune escape mechanism of tumor cells.
  • Karsten Mahnke et al showed that since this inhibitory signaling pathway exists outside of the canonical PD1/PD-L1 co-suppressive pathway, blockade of both signaling pathways by bispecific antibodies results in melanoma-specific cytotoxic T cells The effect function is greatly enhanced.
  • the PD1/PD-L1 signaling pathway axis has been identified in melanoma, and the second inhibitory pathway characterized by TIGIT/CD155 interaction also exists in melanin (Karsten Mahnke and Alexander H. Enk (2015). "TIGIT -CD155 Interactions in Melanoma: A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention "Journal of Investigative Dermatology 136(1): 9-11.).
  • TIGIT TIGIT's inhibitory signaling pathway exerts powerful inhibitory effects in different immune cell subsets and its ligand CD155 is widely expressed in a variety of solid tumors
  • targeting TIGIT is a very promising therapeutic strategy.
  • TIGIT is highly expressed on the surface of T cells and NK cells, while other immune checkpoints such as PD1 are only expressed on the surface of T cells, this determines that TIGIT has greater advantages as a therapeutic target. There is a need to develop bispecific antibody drugs based on TIGIT antibodies.
  • the invention provides an anti-TIGIT single domain antibody (sdAb, single domain antibody) and a bispecific antibody constructed using the same.
  • the anti-TIGIT single domain antibody of the present invention has high affinity to human TIGIT and can recognize human and cynomolgus TIGIT.
  • the anti-TIGIT single domain antibody can effectively block the binding of TIGIT and PVR protein, and the blocking activity is significantly better than that of the control antibody Tiragolumab.
  • the TIGIT single domain antibody of the present invention can also effectively activate T cells to release cytokines.
  • the anti-PD1/TIGIT bispecific antibody of the present invention has blocking activity on both TIGIT/CD155 and PD1/PD-L1, and has a brighter therapeutic prospect in indications where both PD1/PD-L1 and TIGIT/CD155 exist .
  • the anti-TIGIT/anti-CTLA4 bispecific antibody of the present invention has one or more of the following activities/functions: first, it can bind well to TIGIT and CTLA4, and has a higher affinity to TIGIT than to CTLA4 (for example, a higher affinity to CTLA4) order of magnitude), which in turn leads to the localization of anti-TIGIT and anti-CTLA4 bifunctional antibodies to the tumor site, reducing the stay of the bifunctional antibodies in the peripheral system, thereby reducing the peripheral toxicity of the CTLA4 antibody end and increasing the dosage of CTLA4 antibodies; second, TIGIT and CTLA4 in It is highly expressed on Treg cells in the tumor, and the bifunctional antibody of the present invention can eliminate immunosuppressive Treg cells in the tumor through the Fc effect; thirdly, the bifunctional antibody of the present invention can specifically relieve TIGIT and CTLA4 on effector T cells. Immunosuppressive, activates T cells, thereby exerting the effect of suppressing tumors, and has a good application prospect.
  • the invention relates to the following specific embodiments:
  • a VHH antibody specifically binding to TIGIT comprising
  • CDRs complementarity determining regions
  • said CDR sequences are defined according to IMGT.
  • VHH antibody of embodiment 1 comprising the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein
  • VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3
  • VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
  • VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8
  • VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
  • VHH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 11 or consists of it
  • VHH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or consists of it
  • VHH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence or consisting of it.
  • VHH antibody of embodiment 1 comprising or consisting of a heavy chain variable region, said heavy chain variable region
  • amino acid changes comprising one or more (preferably no more than 10, more preferably The amino acid sequence of no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region.
  • a heavy chain antibody specifically binding to TIGIT comprising the VHH antibody of any one of embodiments 1-3.
  • the heavy chain antibody of embodiment 4 which comprises the VHH antibody of any one of embodiments 1-3 linked to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1 , human IgG2, human IgG3 or human IgG4, optionally, the VHH antibody is connected to the Fc region through a hinge region or part thereof, preferably, the amino acid sequence of the hinge region part is EPKSS (SEQ ID NO: 43 ).
  • the heavy chain antibody of embodiment 4 which comprises the VHH antibody of any one of embodiments 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1 or IgG4, preferably , the Fc region
  • amino acid changes compared to the amino acid sequence shown in SEQ ID NO: 40 or 42 (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence.
  • the Fc region comprises a mutation that improves the effector function of the Fc region, such as a mutation that increases ADCC
  • the mutation is a combination of the following mutations: S239D, A330L and I332E( EU numbering), preferably, it comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 41
  • amino acid changes comprising one or more (preferably no more than 10, More preferably no more than 5, 4, 3, 2, 1) of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region.
  • a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds TIGIT, and comprises the VHH antibody of any one of embodiments 1-3 and 9 , or the heavy chain antibody of any one of embodiments 4-9.
  • the second antigen binding region specifically binds PD-1, PD-L1 or PD-L2 or CTLA-4, preferably, the second antigen binding region specifically binds PD -1 comprising a PD1 antibody from WO2019219064A or an antigen-binding fragment thereof, such as a single-chain Fv, Fab, Fab', (Fab)2, single domain antibody, VHH or heavy chain antibody; preferably, the second antigen-binding region specifically binds to CTLA-4, which comprises an antibody from Ipilimumab or an antigen-binding fragment thereof, such as a single-chain Fv of the anti-CTLA-4 antibody, Fab, Fab', (Fab)2, single domain antibody, VHH or heavy chain antibody.
  • CTLA-4 which comprises an antibody from Ipilimumab or an antigen-binding fragment thereof, such as a single-chain Fv of the anti-CTLA-4 antibody, Fab, Fab', (Fab)2, single domain antibody, VHH or heavy chain antibody.
  • bispecific antibody of embodiment 12, wherein said bispecific antibody has the following structure:
  • Heavy chain from N-terminal to C-terminal, heavy chain variable region VH of the second antigen antibody-heavy chain constant region CH1-heavy chain constant region Fc-anti-TIGIT VHH; or
  • Light chain from N-terminal to C-terminal, the light chain variable region of the second antigen antibody-light chain constant region CL;
  • the second antigen is selected from PD-1 or CTLA-4.
  • bispecific antibody of embodiment 10 or 11, wherein said bispecific antibody has the following structure:
  • Heavy chain 1 from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc of the second antigen antibody;
  • Heavy chain 2 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
  • Light chain from N-terminal to C-terminal, the light chain variable region of the second antigen antibody-light chain constant region CL;
  • the second antigen is selected from PD-1 or CTLA-4, such as CTLA-4.
  • linker peptide is from the hinge region of human IgG1, 2, 3 or 4 or a portion thereof, including a native or mutated hinge region or portion thereof, for example from a human IgG1 hinge region, for example the connecting peptide is EPKSS (SEQ ID NO: 43).
  • the heavy chain constant region CH1 of the second antigen antibody is from IgG, such as IgG1, IgG2, IgG3 or IgG4; preferably, the heavy chain constant region CH1 From IgG1 or IgG4, more preferably, the heavy chain constant region CH1 comprises or consists of the amino acid sequence described in SEQ ID NO: 28 or 31.
  • the first Fc region contains a junction mutation, which
  • the second Fc region contains a button mutation, which
  • Heavy chain from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc-anti-TIGIT VHH of the second antigen antibody;
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • the second antigen is PD-1
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • the second antigen is PD-1
  • Heavy chain from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc of anti-TIGIT VHH-second antigen antibody;
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • the second antigen is CTLA-4
  • Heavy chain 1 from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc of the second antigen antibody;
  • Heavy chain 2 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • a nucleic acid molecule encoding the VHH antibody of any one of embodiments 1-3 and 9, or the heavy chain antibody of any one of embodiments 4-9, or the bispecific antibody of any one of embodiments 10-25 The heavy chain and/or light chain in the antibody, or consists of said nucleic acid sequence.
  • An expression vector comprising the nucleic acid molecule according to embodiment 26, preferably, the expression vector is pCDNA, such as pCDNA3.1.
  • a host cell comprising the nucleic acid molecule of embodiment 26 or the expression vector of embodiment 27, preferably, said host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293FT cells or CHO-S cells.
  • a method for preparing the VHH antibody of any one of embodiments 1-3 and 9, or the heavy chain antibody of any one of embodiments 4-9, or the bispecific antibody of any one of embodiments 10-25 comprising, under conditions suitable for expression of said VHH antibody or heavy chain antibody or bispecific antibody chain, culturing an embodiment comprising a nucleic acid encoding a VHH antibody or a heavy chain antibody, or each encoding a bispecific antibody
  • An immunoconjugate comprising the VHH antibody of any one of embodiments 1-3 and 9, or the heavy chain antibody of any one of embodiments 4-9, or the antibody of any one of embodiments 10-25 bispecific antibody.
  • a pharmaceutical composition or medicament or preparation comprising the VHH antibody of any one of embodiments 1-3 and 9, or the heavy chain antibody of any one of embodiments 4-9, or any of embodiments 10-25
  • the bispecific antibody of one aspect, or the immunoconjugate of embodiment 30 and optionally a pharmaceutical excipient is optionally a pharmaceutical excipient.
  • a pharmaceutical combination product comprising the VHH antibody of any one of embodiments 1-3 and 9, or the heavy chain antibody of any one of embodiments 4-9, or the bismuth antibody of any one of embodiments 10-25 Specific antibodies, or immunoconjugates of embodiment 30, and other therapeutic agents.
  • a method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of the VHH antibody of any one of embodiments 1-3 and 9, or the heavy antibody of any one of embodiments 4-9. Chain antibody, or the bispecific antibody of any one of embodiments 10-25, or the immunoconjugate of embodiment 30, or the pharmaceutical composition or preparation of embodiment 31; or the pharmaceutical combination product of embodiment 32.
  • Figure 1 shows that anti-human TIGIT heavy chain antibody blocks the binding of human TIGIT protein to CD155 (EC50, nM).
  • Fig. 2 shows the binding activity (EC50, nM) of heavy chain antibody against human TIGIT to HEK293-human TIGIT cells.
  • Fig. 3 shows the binding activity (EC50, nM) of heavy chain antibody against human TIGIT to HEK293-cynomolgus monkey TIGIT cells.
  • FIG. 4 shows that anti-human TIGIT heavy chain antibody activates CD8+ T cells to release IFN ⁇ factor.
  • Figure 5 shows that the anti-human TIGIT heavy chain antibody after sequence optimization and Fc engineering blocks the binding of human TIGIT protein to CD155 (IC50, nM).
  • Fig. 6 shows a schematic diagram of the structure of the anti-PD1/TIGIT bispecific antibody of the present invention
  • Fig. 6A is a schematic diagram of E4
  • Fig. 6B is a schematic diagram of D1 and D4.
  • Figure 7 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to human TIGIT protein.
  • Figure 8 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to human PD1 protein.
  • Figure 9 shows the activity of anti-PD1/TIGIT bispecific antibody blocking the binding of human TIGIT protein to CD155 (IC50, nM).
  • Figure 10 shows the activity of anti-PD1/TIGIT bispecific antibody blocking the binding of human PD1 protein to PD-L1 (IC50, nM).
  • Figure 11 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to HEK293-human TIGIT cells.
  • Figure 12 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to Jurkat-NFAT-human PD1 cells.
  • Figure 13 shows the ADCC killing activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody on activated CD4+T or CD8+T cells.
  • Figure 14 shows the schematic structure of the anti-TIGIT/CTLA-4 bispecific antibody of the present invention
  • Figure 14A is a schematic diagram of THC4
  • Figure 14B is a schematic diagram of CT1KH
  • Figure 14C is a schematic diagram of CT2KH.
  • Figure 15 shows the activity of anti-TIGIT/CTLA-4 bispecific antibody blocking the binding of human TIGIT protein to CD155.
  • Figure 16 shows the activity of anti-TIGIT/CTLA-4 bispecific antibody blocking the binding of human CTLA-4 protein to CD80.
  • Figure 17 shows the binding activity of anti-TIGIT/CTLA-4 bispecific antibody to HEK293-human TIGIT cells
  • VHH antibody TIGIT-binding single domain antibody
  • heavy chain antibody TIGIT-binding single domain antibody
  • the invention relates to an antibody that binds TIGIT.
  • an antibody or antigen-binding fragment thereof of the invention binds mammalian TIGIT, eg, human TIGIT or cynomolgus TIGIT.
  • the anti-TIGIT antibodies of the invention are single domain antibodies, particularly VHH antibodies.
  • Single domain antibodies or VHH antibodies, have a molecular weight approximately one-tenth that of a human IgG molecule, and a physical diameter of only a few nanometers. Due to the small molecular size, single-domain mAbs have the following advantages over conventional four-chain antibodies: high stability and solubility, and the ability to recognize hidden antigenic sites. In addition, single-domain antibodies are also cheaper to produce than conventional four-chain antibodies. In addition to their application as individual molecules, single domain antibodies are also suitable building blocks for the construction of multispecific molecules.
  • the single domain antibody of the invention is a VHH antibody comprising or consisting of a heavy chain variable region, which typically has the following structure: FR1-VHH CDR1- FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1-3.
  • the CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, preferably the boundaries of the three CDRs in the VHH sequence can be defined by IMGT.
  • the anti-TIGIT VHH antibodies of the invention comprise
  • CDRs three complementarity determining regions (CDRs) contained in the VH set forth in any one of SEQ ID NOS: 1, 6, 9, 14, 16, 18 and 21, or
  • said CDR sequences are defined according to IMGT.
  • an anti-TIGIT VHH antibody of the invention comprises or consists of a heavy chain variable region comprising
  • CDRs three complementarity determining regions (CDRs) contained in the VH set forth in any one of SEQ ID NOS: 1, 6, 9, 14, 16, 18 and 21, or
  • said CDR sequences are defined according to IMGT.
  • the anti-TIGIT VHH antibodies of the invention comprise the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.
  • the anti-TIGIT VHH of the invention comprises or consists of a heavy chain variable region comprising the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3.
  • the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 3, 8 or 11, or the VHH CDR1 comprises an amino acid sequence selected from SEQ ID NO: 3, 8 or 11
  • An amino acid sequence with one, two or three changes preferably amino acid substitutions, preferably conservative substitutions) compared to the sequence.
  • the VHH CDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 4, 12 or 23, or the VHH CDR2 comprises, compared to the amino acid sequence of SEQ ID NO: 4, 12 or 23, One, two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences.
  • the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 5 or 13, or the VHH CDR3 comprises an amino acid sequence selected from SEQ ID NO: 5 or 13 compared to a , two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences.
  • an anti-TIGIT VHH antibody of the invention comprises or consists of a heavy chain variable region comprising
  • Complementarity Determining Regions CDRs
  • HCDR1, HCDR2 and HCDR3 wherein HCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 5 or 13, or HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 5 or 13
  • An amino acid sequence with one, two or three changes preferably amino acid substitutions, preferably conservative substitutions
  • the VHH CDR2 of the anti-TIGIT VHH antibody of the present invention comprises or consists of the following amino acid sequence:
  • ITTSXSA preferably, X is selected from D or S (SEQ ID NO: 57).
  • the anti-TIGIT VHH antibody of the present invention comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein
  • VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3
  • VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
  • VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8
  • VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
  • VHH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 11 or consists of it
  • VHH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or consists of it
  • VHH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence or consisting of it.
  • an anti-TIGIT VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises the complementarity determining regions (CDRs) VHHCDR1, VHHCDR2 and VHHCDR3, wherein
  • VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3
  • VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
  • VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8
  • VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
  • VHH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 11 or consists of it
  • VHH CDR2 comprises the amino acid sequence shown in SEQ ID NO: 12 or consists of it
  • VHH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence or consisting of it.
  • the anti-TIGIT VHH antibody of the present invention comprises or consists of a heavy chain variable region, said heavy chain variable region
  • amino acid changes comprising one or more (preferably no more than 10, more preferably The amino acid sequence of no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region.
  • the anti-TIGIT VHH antibody of the present invention comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21.
  • a VHH antibody of the invention comprises CDR amino acid sequences and/or framework (FR) amino acid sequences derived from a camelid heavy chain antibody produced by immunizing a camelid (eg, an alpaca).
  • VHH mAbs of the invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework regions derived from human amino acid sequences (i.e., human antibodies) or other non-camelidae mammalian species sequence.
  • the VHH antibodies of the invention are chimeric antibodies.
  • the VHH antibodies of the invention are humanized antibodies. Humanization can be achieved by replacing one or more amino acid residues, especially the framework region sequences, in a non-human native VHH sequence (such as a VHH sequence from camelids or alpaca immunization) with those from Residues at the corresponding positions of the heavy chain VH of a conventional human antibody.
  • Methods for humanizing VHHs are well known in the art, such as those described in Example 3.
  • humanizing substitutions are made in a manner that preserves the favorable binding properties of single domain antibodies. Tests for determining the biological properties of humanized single domain antibodies, such as binding affinity, etc., are well known in the art to determine and select appropriate humanized residue mutations or combinations of mutations.
  • the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:
  • VHH antibody back-mutate the key amino acids in the FR region to the corresponding amino acids of the nanobody (VHH antibody) to ensure the original affinity, that is, to obtain a humanized anti-TIGIT VHH antibody, and optionally sequence the VHH antibody.
  • the backmutation site is selected from one or more of T28, L78, W103, R83, V37, G44, L45, and W47. In some embodiments, the backmutation is selected from one or more of T28P; L78V; W103K; R83K; V37F; G44E; L45R; W47F. In some embodiments, the combination of backmutated sites in the humanized VHH antibody is selected from:
  • the combination of back mutations in the humanized VHH antibody is selected from:
  • T28P, L78V and W103K (eg for heavy chain template IGHJ4*01);
  • V37F, G44E, L45R and W47F (eg for heavy chain template IGHV3-23*01).
  • the heavy chain variable region germline genes suitable for humanization of the VHH antibodies of the invention are selected from IGHJ4*01, IGHV3-48*01 or IGHV3-23*01.
  • the invention also provides functional variants of the single domain antibodies (particularly VHH antibodies) of the invention.
  • the functional variant can be introduced into the coding nucleic acid sequence of the exemplary single domain antibody of the present invention, such as into the CDR sequence and/or FR sequence, by using a method well known in the present invention, for example, by random or site-directed mutagenesis, And subsequent screening (eg, by phage display library screening) of variants retaining the desired properties, to obtain functional variants.
  • functional variants retain significant sequence identity to the parental single domain antibody (or VHH).
  • the functional variant retains the desired biological properties of the parental single domain antibody (or VHH), e.g., the variant has comparable (e.g., at least 50%, 60%, 70%, 80%) biological activity relative to the parent. , preferably above 90%) biological activity, or improved biological activity (eg 110-150% or higher).
  • the desired biological properties include, for example, but not limited to, the binding affinity of the target antigen (such as CD155) (as measured by KD value), the activity of blocking the binding of the target antigen to the receptor (such as measured by IC50 value), in vitro Or activation of T cell activity (as measured by released cytokines, for example) in vivo, inhibition of tumor growth/survival in vitro or in vivo.
  • the invention provides affinity variants of the VHH polypeptides of the invention.
  • the affinity variant exhibits one or more amino acid changes in amino acid sequence relative to the parent single domain antibody from which it is derived, wherein the affinity variant has an altered binding affinity for the antigen of interest compared to the parent antibody.
  • antibody stability can also be engineered, for example, by mutating the asparagine in the antibody to eliminate deamidation.
  • the VHH antibody of the invention comprises a D63S mutation in CDR2 to eliminate deamidation.
  • the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.
  • a single domain antibody or VHH of the invention can be linked to a constant region of a human antibody, or a portion thereof, such as an Fc region, to generate a antibody comprising a VHH-constant region.
  • heavy chain antibody of VHH-CH1-Fc or VHH-Fc comprises a VHH antibody of the present invention and an Fc region at its C-terminus.
  • the VHH is linked to the Fc via a hinge region or a portion thereof, eg, from an IgG hinge region (eg, IgGl, 2, 3 or 4 hinge region) or a portion thereof.
  • an anti-TIGIT heavy chain antibody of the invention comprises a VHH as defined herein or a heavy chain variable region therein, and a heavy chain constant region or the Fc region of a heavy chain constant region.
  • a linker peptide is included between the VHH or its heavy chain variable region and the heavy chain constant or Fc region, such as an antibody hinge region or portion thereof, such as from an IgG hinge region or portion thereof (including native or Mutated IgG hinge region or part thereof).
  • the connecting peptide is from a human IgG1, 2, 3 or 4 hinge region or portion thereof, including a native or mutated hinge region or portion thereof, such as from a human IgG1 hinge region, such as the connecting peptide is EPKSS (SEQ ID NO: 43).
  • the heavy chain antibody comprises an Fc portion from a camelid (eg, alpaca).
  • said heavy chain antibody is produced and isolated by immunizing said camelid, eg, alpaca.
  • Various means are known in the art for immunizing camelids and isolating the VHH antibodies or heavy chain antibodies produced against the antigen of interest.
  • the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, eg, a constant region from a human IgGl, human IgG2, human IgG3, or human IgG4.
  • a human or non-human primate (eg, cynomolgus monkey) antibody eg, a constant region from a human IgGl, human IgG2, human IgG3, or human IgG4.
  • the heavy chain antibody comprises an Fc portion from a human or non-human primate (eg, cynomolgus monkey).
  • the heavy chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3 or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region, such as a human IgG1 Fc region.
  • a heavy chain antibody according to the present invention can dimerize with another polypeptide chain comprising an Fc region (eg another heavy chain antibody that is the same or different) via the Fc region.
  • the invention also provides a homo- or heteromultimeric protein comprising a heavy chain antibody of the invention.
  • the protein comprises a heavy chain antibody formed by the pairing of two identical heavy chain antibody chains.
  • the Fc region of the invention can be mutated to obtain desired properties. Mutations to the Fc region are known in the art.
  • the Fc region is modified in a manner characteristic of its effector function. In one embodiment, said effector function has been increased relative to the wild isotype Fc region.
  • the effector function of the Fc region eg ADCC
  • ADCC is improved by making mutations in the Fc region, for example by making mutations S239, A330 and I332 (according to EU numbering) at one or more of the following positions.
  • ADCC is improved by mutation of the following combination of sites: S239, A330 and I332 (according to EU numbering).
  • the mutation is selected from 1, 2 or 3 of S239D, A330L and I332E.
  • the mutation that changes the effector function is the following mutation combination: S239D, A330L and I332E (reference "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14; 103 (11 ): 4005-10.”).
  • the Fc region is an Fc region from IgGl containing mutations S239D, A330L, and I332E (according to EU numbering).
  • the Fc region :
  • (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or
  • the Fc region is from IgG1 comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 40 or 41 , 96%, 97%, 98% or 99% identical amino acid sequence or consists of it.
  • the Fc region is from IgG1 comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 41 %, 97%, 98% or 99% identical amino acid sequence or consisting thereof, and comprising the following combination of mutations: S239D, A330L and I332E (EU numbering).
  • the Fc region is from IgG4 comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence shown in SEQ ID NO: 42 %, 97%, 98% or 99% identical amino acid sequences or consist thereof.
  • an antibody TIGIT antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain comprising a heavy chain variable region, an Fc region, and a linker peptide linking the heavy chain variable region and the Fc region.
  • the connecting peptide comprises the amino acid sequence shown in SEQ ID NO: 43, or consists of said amino acid sequence.
  • the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention comprises or consists of a heavy chain comprising or consisting of a heavy chain variable region, a connecting peptide and an Fc region of a VHH of the present invention, where the heavy chain
  • amino acid changes comprising one or more (preferably no more than 10, More preferably no more than 5, 4, 3, 2, 1) of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region.
  • the invention also encompasses multispecific molecules, such as bispecific antibodies, comprising a VHH or heavy chain antibody of the invention or fragments thereof.
  • a multispecific antibody molecule may, for example, be a trispecific antibody molecule comprising a first binding specificity for TIGIT and a second and third binding specificity for one or more of the molecules.
  • Another aspect of the invention relates to a bispecific antibody comprising
  • a first antigen-binding region and a second antigen-binding region wherein the first antigen-binding region specifically binds TIGIT, and the second antigen-binding region specifically binds a tumor-associated antigen or an immune checkpoint molecule, such as PD-1, PD-L1 or PD -L2 or CTLA4.
  • the first antigen binding region comprises an anti-TIGTI VHH antibody or heavy chain antibody, particularly a VHH antibody, described herein.
  • the first antigen binding region comprises or consists of an anti-TIGIT VHH of the invention, more preferably said VHH is a humanized VHH.
  • the second antigen binding region comprises a PD1 antibody from WO2019219064A, or an antigen-binding fragment or domain thereof, eg, a fragment or domain comprising a PD1 antibody of WO2019219064A.
  • the second antigen-binding region of the bispecific antibody applicable to the present invention may comprise or consist of a full-length anti-PD-1 antibody or an antigen-binding fragment thereof, as long as it can specifically bind to PD-1, including but not Limited to, for example, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy-chain antibodies that specifically bind to PD-1.
  • the second antigen binding region comprises an antibody from Ipilimumab or an antigen binding fragment or domain thereof, eg, a fragment or domain comprising an antibody to Ipilimumab.
  • the second antigen-binding region of the bispecific antibody suitable for the present invention may comprise or consist of a full-length anti-CTLA-4 antibody or an antigen-binding fragment thereof, as long as it is capable of specifically binding to CTLA-4, including but not Limited to, for example, full length antibodies, single chain Fv, Fab, Fab', (Fab)2, single domain antibodies, VHH or heavy chain antibodies, etc. that specifically bind CTLA-4.
  • the anti-TIGIT VHH of the present invention is used as the first antigen-binding region, which can be connected to the N-terminal or C-terminal of the second antigen-binding region, for example, connected to the Fc fragment of the second antigen-binding region.
  • the first and second antigen binding regions are linked by a linker (eg, when the anti-TIGIT VHH is inserted between the Fab fragment and the Fc fragment of the second antigen antibody).
  • the linker is a peptide of about 3 to about 20 amino acids in length.
  • bispecific antibodies of the invention have the following structure:
  • Heavy chain from N-terminal to C-terminal, heavy chain variable region VH of the second antigen antibody-heavy chain constant region CH1-heavy chain constant region Fc-anti-TIGIT VHH; or
  • Light chain from N-terminal to C-terminal, light chain variable region-light chain constant region CL of the second antigen antibody.
  • the second antigen is PD-1, such as human PD-1.
  • the second antigen is CTLA-4, such as human CTLA-4.
  • the bispecific antibodies of the invention have two heavy chains and two light chains, preferably the same two heavy chains and two light chains.
  • the structure of the bispecific antibody is as shown in Figure 6A or Figure 6B or Figure 14A.
  • the anti-TIGIT single domain antibody VHH is connected to the Fc part of the heavy chain constant region at the N-terminal or C-terminal through a linker.
  • the linker is a peptide of about 3 to about 20 amino acids in length.
  • the anti-TIGIT VHH of the present invention serves as the first antigen-binding region, which can be combined with Fc to form a heavy chain antibody, and heterodimerized with the second antigen-binding region to form a bispecific antibody.
  • bispecific antibodies of the invention have the following structure:
  • Heavy chain 1 from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc of the second antigen antibody;
  • Heavy chain 2 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
  • Light chain from N-terminal to C-terminal, light chain variable region-light chain constant region CL of the second antigen antibody.
  • the bispecific antibody of the invention has 1 heavy chain 1 , 1 heavy chain 2 and 1 light chain.
  • the structure of the bispecific antibody is shown in Figure 14B or 14C.
  • the anti-TIGIT single domain antibody VHH is linked to the N-terminal of the heavy chain constant region Fc via a linker peptide.
  • the connecting peptide is from a human IgG1, 2, 3 or 4 hinge region or portion thereof, including a native or mutated hinge region or portion thereof, such as from a human IgG1 hinge region, such as the connecting peptide is EPKSS (SEQ ID NO: 43).
  • each tandem VHH can be connected by a linker.
  • the linker is a peptide of about 3 to about 20 amino acids in length.
  • the second antigen is CTLA-4, eg, human CTLA-4.
  • the anti-TIGIT VHH of the invention is as defined above.
  • the heavy chain variable region and/or the light chain variable region of the anti-PD1 antibody in the bispecific antibody of the present invention is from the PD1 antibody of WO2019219064A.
  • the heavy chain variable region VH of the anti-PD1 antibody of the present invention comprises three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3. In some embodiments, the light chain variable region of the anti-PD1 antibody of the invention comprises three complementarity determining regions VLCDR1, VLCDR2 and VLCDR3.
  • the complementarity determining regions VHCDR1, VHCDR2 and VHCDR3 of the three heavy chain variable regions of the present invention are from a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 24.
  • Region VH, preferably said CDRs are defined by Kabat.
  • the complementarity determining regions VLCDR1, VLCDH2 and VLCDR3 of the three light chain variable regions of the present invention are derived from a light chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 34.
  • Region VL, preferably said CDRs are defined by Kabat.
  • the complementarity determining region VHCDR1 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 25.
  • the complementarity determining region VHCDR2 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 26.
  • the complementarity determining region VHCDR3 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 27.
  • the complementarity determining region VLCDR1 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 35.
  • the complementarity determining region VLCDR2 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 36.
  • the complementarity determining region VLCDR3 of the anti-PD1 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 37.
  • the heavy chain variable region VH of the anti-PD1 antibody comprises VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 comprises or consists of the sequence shown in SEQ ID NO: 25; VHCDR2 comprises the sequence shown in SEQ ID NO: 26 and/or VHCDR3 comprises or consists of the sequence shown in SEQ ID NO: 27.
  • the light chain variable region VL of the anti-PD1 antibody comprises VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 comprises or consists of the sequence shown in SEQ ID NO: 35; VLCDR2 comprises the sequence shown in SEQ ID NO: 36 sequence or consists of it; and/or VLCDR3 comprises or consists of the sequence shown in SEQ ID NO: 37.
  • the heavy chain variable region VH of the anti-PD1 antibody of the present invention comprises the amino acid sequence described in SEQ ID NO: 24, or comprises at least 90% of the amino acid sequence described in SEQ ID NO: 24 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:24.
  • the heavy chain variable region VH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1) mutated amino acid sequences, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, eg, VHCDR1, VHCDR2 or VHCDR3.
  • the light chain variable region VL of the anti-PD1 antibody of the present invention comprises the amino acid sequence described in SEQ ID NO: 34, or comprises at least 90% of the amino acid sequence described in SEQ ID NO: 34 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:34.
  • the light chain variable region VL comprises one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1) mutated amino acid sequences, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, eg, VLCDR1, VLCDR2 or VLCDR3.
  • the heavy chain variable region and/or the light chain variable region of the anti-CTLA-4 antibody in the bispecific antibody of the present invention is from Ipilimumab.
  • the heavy chain variable region VH of the anti-CTLA-4 antibody of the present invention comprises three complementarity determining regions VHCDR1, VHCDR2 and VHCDR3.
  • the light chain variable region of an anti-CTLA-4 antibody of the invention comprises three complementarity determining regions, VLCDR1, VLCDR2 and VLCDR3.
  • the complementarity determining regions VHCDR1, VHCDR2 and VHCDR3 of the three heavy chain variable regions of the present invention are from a heavy chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 62.
  • Region VH, preferably said CDRs are defined by Kabat.
  • the complementarity determining regions VLCDR1, VLCDR2 and VLCDR3 of the three light chain variable regions of the present invention are derived from a light chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 67.
  • Region VL, preferably said CDRs are defined by Kabat.
  • the complementarity determining region VHCDR1 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO:59.
  • the complementarity determining region VHCDR2 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 60.
  • the complementarity determining region VHCDR3 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 61.
  • the complementarity determining region VLCDR1 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 64.
  • the complementarity determining region VLCDR2 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 65.
  • the complementarity determining region VLCDR3 of the anti-CTLA-4 antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 66.
  • the heavy chain variable region VH of the anti-CTLA-4 antibody comprises VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 comprises the sequence shown in SEQ ID NO: 59 or consists of it; VHCDR2 comprises the sequence shown in SEQ ID NO: 60 and/or VHCDR3 comprises or consists of the sequence shown in SEQ ID NO: 61.
  • the light chain variable region VL of the anti-CTLA-4 antibody comprises VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 comprises the sequence shown in SEQ ID NO: 64 or consists of it; VLCDR2 comprises the sequence shown in SEQ ID NO: 65 and/or VLCDR3 comprises or consists of the sequence shown in SEQ ID NO: 66.
  • the heavy chain variable region VH of the anti-CTLA-4 antibody of the present invention comprises the amino acid sequence described in SEQ ID NO: 62, or comprises the amino acid sequence described in SEQ ID NO: 62 at least An amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of the amino acids set forth in SEQ ID NO:62.
  • the heavy chain variable region VH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1) mutated amino acid sequences, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, eg, VHCDR1, VHCDR2 or VHCDR3.
  • the light chain variable region VL of the anti-CTLA-4 antibody of the present invention comprises the amino acid sequence described in SEQ ID NO: 67, or comprises and the amino acid sequence described in SEQ ID NO: 67 has at least An amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of the amino acids set forth in SEQ ID NO:67.
  • the light chain variable region VL comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1) mutated amino acid sequences, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, eg, VLCDR1, VLCDR2 or VLCDR3.
  • the heavy chain constant region CH1 of the second antigen antibody in the bispecific antibody of the invention is from IgG, such as IgG1, IgG2, IgG3 or IgG4.
  • the heavy chain constant region CH1 is from IgG1 or IgG4.
  • the heavy chain constant region CH1 is the heavy chain constant region CH1
  • the heavy chain constant region CH1 and Fc region in the bispecific antibody of the present invention are from the same IgG, such as IgG1, IgG2, IgG3 or IgG4, preferably, both are from IgG1 or IgG4.
  • the light chain constant region CL of the bispecific antibody of the present invention is a Lambda or Kappa light chain constant region, preferably a Kappa light chain constant region. In some embodiments, the light chain constant region CL
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38;
  • amino acid sequence comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 38
  • the amino acid sequence that is altered is or consists of said amino acid sequence.
  • the Fc region applicable to the heavy chain antibody of the invention is also applicable to the bispecific antibody of the invention.
  • the heavy chain constant region Fc in the bispecific antibody of the invention is from IgG, such as IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region is from IgGl or from IgG4.
  • the two Fc regions in the bispecific antibody of the invention dimerize to form a dimeric Fc.
  • the first and second Fc regions are identical. In other embodiments, eg where the bispecific antibody comprises different heavy chains, the first and second Fc regions are different, pair and heterodimerize.
  • An Fc region fragment suitable for use in an antibody molecule of the invention may be any antibody Fc region.
  • Fc regions can include native sequence Fc regions and variant Fc regions.
  • Native sequence Fc domains encompass the naturally occurring Fc sequences of various immunoglobulins, such as the Fc regions of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014 , doi: 10.3389/fimmu.2014.00520.).
  • the Fc region of an antibody of the invention may comprise two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the antibody Fc region may also have an IgG hinge region or a part of an IgG hinge region at the N-terminus, eg, an IgG1 hinge region or a part of an IgG1 hinge region. Mutations may be contained in the hinge region.
  • the hinge region can be EPKSS or EPKSC.
  • the Fc region of the antibody of the present invention includes from N-terminus to C-terminus: CH2-CH3, or from N-terminus to C-terminus: hinge region-CH2-CH3.
  • the Fc region suitable for use in an antibody or bispecific antibody of the invention is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 or human IgG4 Fc.
  • amino acid changes compared to the amino acid sequence shown in SEQ ID NO: 40 or 42 (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence.
  • the Fc region of the invention can be mutated to obtain desired properties. Mutations to the Fc region are known in the art.
  • the Fc region is modified in a manner characteristic of its effector function. In one embodiment, said effector function has been increased relative to the wild isotype Fc region.
  • the effector function of the Fc region eg ADCC
  • ADCC is improved by making mutations in the Fc region, for example by making mutations S239, A330 and I332 (according to EU numbering) at one or more of the following positions.
  • ADCC is improved by mutation of the following combination of sites: S239, A330 and I332 (according to EU numbering).
  • the mutation is selected from 1, 2 or 3 of S239D, A330L and I332E. In one embodiment, the mutation that alters effector function is a combination of the following mutations: S239D, A330L and I332E.
  • the Fc region comprised by the bispecific antibody of the present invention may contain mutations that favor heterodimerization.
  • mutations are introduced in the CH3 region of both Fc regions.
  • the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to associate with
  • the complementary engineered other CH3 domains heterodimerize such that the first and second CH3 domains heterodimerize without forming homodimers between the two first CH3 domains or the two second CH3 domains).
  • knob mutations and hole mutations are respectively introduced into the Fc region of the first monomer and the Fc region of the second monomer.
  • This technique is described, for example, in Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG.” Nat Biotechnol 16(7):677-681.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knot mutation); while in the CH3 region of another Fc region
  • the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (deduction mutation)
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the Leucine residues were replaced with alanine residues (L368A) (numbering according to EU index).
  • the knot mutation comprises or consists of the following: the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine at position 354 residue is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with cysteine residue replacement); and in the CH3 region of another Fc region, the buckle mutation comprises or consists of the following: the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally at position 366 The threonine residue at position 368 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index), optionally a tyrosine residue at position 349 Amin
  • one Fc region comprises amino acid substitutions S354C and T366W (knot mutation) and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A and Y407V (deduction mutation) (numbering according to the EU index).
  • the bispecific antibody of the invention comprises two Fc regions that are heterodimerized, wherein
  • one Fc-region polypeptide comprises mutation T366W and the other Fc-region polypeptide comprises mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises mutations T366W and Y349C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or
  • Fc-region polypeptide comprises mutations T366W and S354C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and Y349C.
  • the Fc region further comprises other mutations that facilitate heterodimer purification.
  • the H435R mutation Eric J. Smith, Scientific Reports
  • the Fc region with a Hole mutation can be introduced into one of the Fc regions of the heterodimer (e.g., the Fc region with a Hole mutation) to facilitate Protein A was used to purify heterodimers.
  • mutations such as C220S can also be introduced in the hinge region to facilitate the formation of heterodimers.
  • the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein
  • the first Fc region comprises a junction mutation comprising or consisting of the amino acid sequence of SEQ ID NO: 54 or an amino acid sequence at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical
  • the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 54 and comprises a knot mutations (eg, S354C and T366W); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC;
  • the second Fc region comprises a button mutation comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to the amino acid sequence SEQ ID NO: 75 .
  • the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 75 and comprises a buckle mutation;
  • the Fc region may or may not comprise a hinge region EPKSS or EPKSC.
  • the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein
  • the first Fc region comprises a junction mutation comprising or consisting of the amino acid sequence SEQ ID NO: 78 or an amino acid sequence at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical Composition;
  • the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 78 and comprises S239D , A330L and I332E mutations and junction mutations (eg S354C and T366W); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC;
  • the second Fc region comprises a button mutation comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to the amino acid sequence SEQ ID NO: 77 .
  • the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 77 and comprises S239D, A330L and I332E mutations and button mutations; in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC.
  • the anti-TIGIT single domain antibody VHH in the bispecific antibody of the present invention comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are from the VHH shown in SEQ ID NO: 18 or 21.
  • the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3
  • HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 23 or 57
  • HCDR3 comprises SEQ ID NO: The amino acid sequence shown in 5 or consists of it.
  • the anti-TIGIT single domain antibody VHH in the bispecific antibody of the present invention comprises or consists of a heavy chain variable region VH, said heavy chain variable region VH comprising SEQ ID NO : The amino acid sequence shown in 18 or 21 or consists of said amino acid sequence.
  • the bispecific antibodies of the invention comprise
  • Heavy chain from N-terminal to C-terminal, heavy chain variable region VH of the second antigen antibody-heavy chain constant region CH1-heavy chain constant region Fc-anti-TIGIT VHH; or
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • the second antigen is PD-1
  • (iii) consists of the amino acid set forth in SEQ ID NO: 30, 32 or 33, or
  • a mutated amino acid sequence such as a substitution, deletion or addition, preferably a substitution, such as a conservative substitution, preferably, the mutation is absent from the CDRs of the anti-TIGIT VHH, or absent from the anti-TIGIT VHH, or not present in the heavy chain variable region CDR of an anti-PD1 antibody, or preferably not present in the heavy chain variable region;
  • the bispecific antibodies of the invention comprise
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 30, 32 or 33, or comprising at least 90%, 91%, or 92% of the amino acid sequence described in said SEQ ID NO: 30, 32 or 33 , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said sequences;
  • a light chain comprising the amino acid sequence of SEQ ID NO: 39, or comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence described in said SEQ ID NO: 39 Amino acid sequences that are %, 96%, 97%, 98% or 99% identical.
  • the bispecific antibodies of the invention comprise
  • Heavy chain from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc of anti-TIGIT VHH-second antigen antibody;
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • the second antigen is CTLA-4
  • (iv) comprising one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutated amino acids compared to the amino acid sequence shown in SEQ ID NO: 69 Sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution, preferably, said mutation does not exist in the CDR of anti-TIGIT VHH, or does not exist in the VHH of anti-TIGIT, or does not exist in in, or preferably absent from, the heavy chain variable region CDRs of an anti-CTLA-4 antibody;
  • (iii) consists of the amino acid set forth in SEQ ID NO: 68, or
  • the bispecific antibodies of the invention comprise
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 69, or comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence described in said SEQ ID NO: 69 %, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said sequences;
  • a light chain comprising the amino acid sequence of SEQ ID NO: 68, or comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence described in said SEQ ID NO: 68 Amino acid sequences that are %, 96%, 97%, 98% or 99% identical.
  • the bispecific antibodies of the invention comprise
  • Heavy chain 1 from N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-heavy chain constant region Fc of the second antigen antibody;
  • Heavy chain 2 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
  • Light chain from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
  • the second antigen is CTLA-4
  • (ii) comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 72 or 74 the amino acid sequence of
  • (iv) comprising one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 72 or 74
  • the amino acid sequence of the amino acid sequence, the mutation such as substitution, deletion or addition, preferably a replacement, such as a conservative substitution; preferably, the mutation does not exist in the CDR of the anti-TIGIT VHH, or does not exist in the VHH of the anti-TIGIT;
  • the bispecific antibodies of the invention comprise
  • Heavy chain 1 which comprises the amino acid sequence of SEQ ID NO: 71, or comprises at least 90%, 91%, 92%, 93%, 94%, Amino acid sequences that are 95%, 96%, 97%, 98% or 99% identical, or consist of said sequences;
  • Heavy chain 2 which comprises the amino acid sequence of SEQ ID NO: 72 or 74, or comprises at least 90%, 91%, 92%, 93% of the amino acid sequence described in said SEQ ID NO: 72 or 74 , 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • a light chain comprising the amino acid sequence of SEQ ID NO: 68, or comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence described in said SEQ ID NO: 68 Amino acid sequences that are %, 96%, 97%, 98% or 99% identical.
  • the VHH or heavy chain antibody or bispecific antibody described herein comprises one or more amino acid mutations.
  • amino acid mutations include amino acid substitutions, insertions or deletions.
  • amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid mutations described in the present invention occur in regions outside the CDRs (eg in FRs). In some embodiments, the amino acid mutations described in the present invention occur in the heavy chain constant region of the antibody, such as the Fc region. In a preferred embodiment, the amino acid mutations in the Fc region enhance the ADCC effect of the antibody.
  • one or more amino acid mutations can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to alter one or more functional properties of the antibody, such as serum half-life, Complement fixation, complement dependent cytotoxicity, Fc receptor binding and/or antibody dependent cytotoxicity.
  • Fc region variants may include human Fc region sequences (eg, human IgGl, IgG2, IgG3 or IgG4 Fc regions) comprising amino acid mutations (eg, substitutions) at one or more amino acid positions.
  • variable region of an antibody may be desirable to mutate the variable region of an antibody to prevent deamidation, for example, by mutating 1 or 2 amino acids (e.g., aspartic acid) in the variable region, e.g., a CDR, that are susceptible to deamidation mutation.
  • 1 or 2 amino acids e.g., aspartic acid
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art.
  • Moieties suitable for such derivatization include, but are not limited to, water soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerin), polyvinyl alcohol, and mixture
  • the VHH antibody or heavy chain antibody or bispecific antibody of the present invention has one or more of the following properties.
  • anti-TIGIT VHH or heavy chain antibodies of the invention have one or more of the following properties
  • TIGIT capable of specifically binding to TIGIT, such as human TIGIT or cynomolgus TIGIT;
  • (iii) capable of specifically binding cynomolgus TIGIT, e.g. with high affinity, e.g., as determined by the Fortebio detection system, e.g., as determined by the method described in Example 4, with a KD of less than 6, 5, 4, 3 , 2 or 1nM, also for example less than 0.9nM or 0.8nM, also for example less than 5 ⁇ 10 -12 M, 4 ⁇ 10 - 12 M, 3 ⁇ 10 -12 M, 2 ⁇ 10 -12 M, or 1 ⁇ 10 - 12M ;
  • TIGIT such as human TIGIT
  • CD155 such as human CD155
  • the blocking activity is higher than that of known TIGIT antibodies, such as Tiragolumab analog of WO2017053748A2;
  • TIGIT such as human or cynomolgus TIGIT
  • TIGIT antibodies such as Tiragolumab analog of WO2017053748A2
  • T cells such as primary T cells, such as CD8+ T cells
  • cytokines such as IFN ⁇
  • the bispecific antibody of the invention is capable of specifically binding TIGIT and PD-1, eg human TIGIT and human PD-1, eg with high affinity. In some embodiments, the bispecific antibodies of the invention are capable of specifically binding TIGIT and CTLA-4, eg human CTLA-4 and human TIGIT, eg with high affinity.
  • the bispecific antibody that specifically binds TIGIT and PD-1 of the present invention has one or more of the following properties:
  • TIGIT capable of specifically binding to TIGIT, such as human and/or cynomolgus TIGIT, for example with high affinity
  • TIGIT such as human TIGIT
  • CD155 such as human CD155
  • TIGIT antibodies such as Tiragolumab analog of WO2017053748A2
  • TIGIT e.g. human or cynomolgus TIGIT
  • TIGIT e.g., human or cynomolgus TIGIT
  • PD1 e.g., human PD1
  • (vii) have better structural safety, such as no obvious killing of killer T cells (such as CD4+T and CD8+T cells), and preferably cannot significantly induce NK cells to kill activated CD4+T and CD8+T cells , ADCC killing activity is equivalent to known TIGIT antibody, such as Tiragolumab analog of WO2017053748A2;
  • PD1 antibody such as known PD1 antibody (such as the PD1 antibody disclosed in WO2019219064A); have the activity of TIGIT VHH or heavy chain antibody of the present invention;
  • the bispecific antibodies of the invention that specifically bind TIGIT and CTLA-4 have one or more of the following properties:
  • Bispecific antibodies that specifically bind TIGIT and CTLA-4 have one or more of the following properties:
  • TIGIT capable of specifically binding to TIGIT, such as human and/or cynomolgus TIGIT, for example with high affinity
  • CTLA-4 capable of specifically binding to CTLA-4, such as human CTLA-4, for example with high affinity
  • TIGIT such as human TIGIT
  • CD155 such as human CD155
  • the blocking activity is higher than that of known TIGIT antibodies, such as Tiragolumab analog of WO2017053748A2;
  • TIGIT e.g. human or cynomolgus TIGIT
  • (vii) have better structural safety, such as no obvious killing of killer T cells (such as CD4+T and CD8+T cells), and preferably cannot significantly induce NK cells to kill activated CD4+T and CD8+T cells , ADCC killing activity is equivalent to known TIGIT antibody, such as Tiragolumab analog of WO2017053748A2;
  • CTLA-4 antibody such as known CTLA-4 antibody (such as Ipilimumab);
  • (x) have the activity of TIGIT VHH or heavy chain antibody of the present invention
  • the invention provides a nucleic acid encoding any of the above VHH or heavy chain antibodies or bispecific antibodies or any chain thereof.
  • the nucleic acid of the present invention comprises a coding sequence selected from any one of SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 14-22, 30, 32, 33, 69, 71, 72 or 74.
  • Nucleic acid showing an amino acid sequence, or encoding any one selected from SEQ ID NO: 1, 2, 6, 7, 8, 9, 10, 14-22, 30, 32, 33, 69, 71, 72 or 74 Nucleic acids having amino acid sequences at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences shown.
  • each antibody or polypeptide amino acid sequence may be encoded by multiple nucleic acid sequences due to codon degeneracy.
  • Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, eg, by de novo solid-phase DNA synthesis, or by PCR amplification.
  • the invention provides a nucleic acid encoding any of the above single domain antibodies or VHHs.
  • the polypeptide encoded by the nucleic acid is capable of displaying human TIGTI antigen (and/or cynomolgus) binding ability when expressed from a suitable expression vector.
  • the nucleic acid is operably linked in-frame to a nucleic acid encoding another peptide/polypeptide such that when expressed from a suitable expression vector, a single domain antibody or VHH comprising the single domain antibody or VHH and another peptide/polypeptide is produced. fusion protein or chimeric polypeptide.
  • the nucleic acid is operably linked in-frame to a nucleic acid encoding an Fc region (e.g., a human Fc region) such that when expressed from a suitable expression vector, a single domain antibody or VHH comprising the single domain antibody or VHH is produced and Heavy chain antibodies in the Fc region.
  • an Fc region e.g., a human Fc region
  • single domain antibodies or VHH can be fused with a secretory signal peptide at the N-terminus, and/or a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin tag or an hFc tag.
  • the invention provides a nucleic acid encoding any of the above bispecific antibodies.
  • the polypeptide encoded by the nucleic acid When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of displaying human TIGIT and a second antigen (eg, human PD-1 or human CTLA-4) binding ability.
  • the nucleic acids encoding the heavy and light chains of the bispecific antibody may be in the same vector or in different vectors.
  • the nucleic acids encoding the heavy and light chains of the bispecific antibody can be introduced into the same or different host cells for expression.
  • the method for producing a bispecific antibody of the invention comprises the step of culturing a nucleic acid comprising a nucleic acid encoding said heavy and light chain under conditions suitable for expressing said heavy and light chain of said molecule. host cells to produce the bispecific antibody of the present invention.
  • a vector comprising said nucleic acid is provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • YACs yeast artificial chromosomes
  • the vector is eg a pcDNA vector, eg pcDNA3.1.
  • a host cell comprising said nucleic acid or said vector is provided, eg for cloning or expressing a vector encoding a VHH or a heavy chain antibody or a bispecific antibody.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells such as CHO cells (eg CHO-S) or 293 cells (eg 293F or HEK293 cells) or other cells suitable for the production of antibodies or fragments thereof.
  • the host cell is prokaryotic, such as a bacterium, such as E. coli.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which glycosylation pathways have been "humanized” result in the production of antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted for growth in suspension can be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7); the human embryonic kidney line (HEK293, 293F or 293T cells), and the like.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as YO, NSO and Sp2/0. Suitable mammalian host cell lines for antibody production are known in the art.
  • a method for preparing a VHH antibody or a heavy chain antibody or a bispecific antibody of the present invention comprises, under conditions suitable for expression of a VHH antibody or a heavy chain antibody or a bispecific antibody or a chain thereof , culturing host cells comprising a nucleic acid encoding said VHH or heavy chain antibody or bispecific antibody (e.g., any polypeptide chain and/or multiple polypeptide chains) or an expression vector comprising said nucleic acid, as provided above , and optionally recovering said VHH or heavy chain antibody or bispecific antibody from said host cell (or host cell culture medium).
  • a nucleic acid encoding said VHH or heavy chain antibody or bispecific antibody e.g., any polypeptide chain and/or multiple polypeptide chains
  • an expression vector comprising said nucleic acid
  • the polynucleotide encoding the polypeptide chain of the VHH antibody or heavy chain antibody or bispecific antibody of the present invention can be inserted into one or more vectors for further cloning and/or expression in host cells.
  • Expression vectors can be constructed using methods well known to those skilled in the art. Once an expression vector comprising one or more nucleic acid molecules of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
  • VHH antibodies or heavy chain antibodies or bispecific antibodies prepared as described herein can be obtained by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography Wait for purification.
  • the actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art.
  • the purity of antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • VHH or heavy chain antibodies or bispecific antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the VHH or heavy chain antibody or bispecific antibody of the present invention is tested for its target (eg antigen) binding activity, for example by known methods such as biofilm thin layer interferometry, ELISA, and the like. Binding to TIGIT and/or PD1 can be assayed using methods known in the art, exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA) or biofilm thin layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) is used to measure.
  • RIA radioimmunoassay
  • BBI biofilm thin layer interferometry
  • ECL electrochemiluminescence
  • SPR surface plasmon resonance
  • FACS flow cytometry
  • the invention also provides assays for identifying the biological activity of a VHH or heavy chain antibody or bispecific antibody.
  • the biological activity is selected from the properties of the VHH or heavy chain antibody or bispecific antibody of the invention.
  • the binding activity of the antibody molecule of the present invention to cells expressing TIGIT and/or a second antigen can be determined by methods known in the art, such as fluorescent reporter molecules and flow cytometry technique, or the exemplary method disclosed in the examples herein, for example, the binding of the antibody molecule of the present invention to TIGIT and/or PD-1 expressed on cells is determined by the method shown in Example 6.
  • the receptor fluorescent reporter can be activated by a method known in the art, such as an ELISA blocking assay. assay, cell proliferation assay, or the exemplary methods disclosed in the Examples herein.
  • an ELISA blocking assay assay, cell proliferation assay, or the exemplary methods disclosed in the Examples herein.
  • the blocking activity of molecules that block the binding of human TIGIT and/or human PD-1 to their associated receptors can be determined using an ELISA blocking assay, such as the method described in Example 5 or 14.
  • the activation activity of the antibody molecules of the present invention on T cells can be determined by methods known in the art, such as T cell activation test systems, such as primary T cell activation test systems, for example, by implementing the method shown in 7, By detecting the amount of cytokines (such as IFN ⁇ ) released by T cells (such as CD8+ T cells).
  • T cell activation test systems such as primary T cell activation test systems
  • T cells such as CD8+ T cells
  • cytotoxicity test systems such as NK cell-dependent cytotoxicity test systems, such as those shown in Example 17.
  • Method to measure for the killing activity or structural safety of the antibody molecules of the present invention, methods known in the art can be used, such as cytotoxicity test systems, such as NK cell-dependent cytotoxicity test systems, such as those shown in Example 17.
  • Cells for use in any of the above in vitro assays are primary cells or cell lines, including those that naturally express or overexpress TIGIT (e.g. human or cynomolgus monkey) or CD155 or secondary antigens (PD1 or PDL1 (e.g. human or cynomolgus PD1 or PDL1) or CTLA-4 (such as human CTLA-4), such as cells overexpressing TIGIT and/or CD155 and/or PD1 and/or PDL1, such as 293 cells or CHO cells or Jurkat cells, such as HEK293 or CHO - K1 or Jurkat/NFAT-Luc cells.
  • TIGIT e.g. human or cynomolgus monkey
  • PD1 or PDL1 e.g. human or cynomolgus PD1 or PDL1
  • CTLA-4 such as human CTLA-4
  • the present invention also provides a method for detecting the druggability of the antibody molecule of the present invention, for example, by detecting the pharmacokinetic characteristics of the antibody molecule.
  • Animals can be obtained by methods known in the art, such as the method shown in Example 18.
  • Pharmacokinetic parameters of the antibody molecule in a model eg, rat model.
  • the invention also provides a fusion protein comprising any of the VHH or heavy chain antibodies or bispecific antibodies described herein, for example comprising a VHH or heavy chain antibody or bispecific antibody of the invention, in combination with Linked other molecules (such as other proteins, such as proteins used in therapy).
  • the invention provides immunoconjugates comprising any of the VHH or heavy chain antibodies or bispecific antibodies described herein.
  • the immunoconjugate comprises one or more additional therapeutic agents (eg, cytotoxins or small molecule compounds) or markers.
  • the invention provides a composition or a medicament or a formulation comprising any of the VHH or heavy chain antibodies or bispecific antibodies described herein, preferably the composition is a pharmaceutical composition.
  • composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises a VHH or heavy chain antibody or bispecific antibody of the invention in combination with one or more other therapeutic agents.
  • composition or drug or preparation of the present invention may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions or medicaments or formulations of the invention can be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injections or eye drops), powders or suspensions, liposomes, and suppositories.
  • liquid solutions eg, injections or eye drops
  • powders or suspensions e.g., liposomes
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • a VHH or heavy chain antibody or bispecific antibody comprising a VHH or heavy chain antibody or bispecific antibody described herein can be prepared by admixing a VHH or heavy chain antibody or bispecific antibody of the invention having the desired purity with one or more optional pharmaceutical excipients.
  • Drugs or formulations of antibodies for example in the form of lyophilized formulations or aqueous solutions.
  • compositions or medicaments or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.
  • the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the VHH antibody or heavy chain antibody or bispecific antibody of the present invention, and one or more other therapeutic agents.
  • the present invention also provides a complete kit containing the drug combination, for example, the complete kit contains in the same package:
  • a first container containing a pharmaceutical composition comprising a VHH antibody or heavy chain antibody or bispecific antibody of the invention
  • VHH or heavy chain antibodies or bispecific antibodies Uses of VHH or heavy chain antibodies or bispecific antibodies and methods of using them.
  • One aspect of the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject the anti-TIGIT VHH or heavy chain antibody or bispecific antibody of the present invention, or an immunoconjugate or combination thereof substances or drugs or preparations.
  • the disease is eg a tumor, eg cancer.
  • Cancer can be early, middle or advanced or metastatic.
  • the cancer can be a solid tumor or a hematological tumor.
  • the tumor is a tumor or cancer that is resistant to a known drug, such as a known anti-PD-1 antibody or anti-CTLA-4 antibody, such as a refractory tumor or cancer.
  • the patient has (e.g., elevated levels, e.g., at the nucleic acid or protein level) of PD1 or PDL1 or PDL2 or CTLA-4, and/or TIGIT (e.g., compared to the same tissue in a healthy individual, or compared to adjacent healthy tissue in the patient).
  • elevated levels e.g., at the nucleic acid or protein level
  • TIGIT e.g., compared to the same tissue in a healthy individual, or compared to adjacent healthy tissue in the patient.
  • the disease treatment would benefit from inhibition of PD1 or PDL1 or PDL2 or CTLA-4 at the nucleic acid or protein level, and/or TIGIT.
  • the cancer is characterized as having elevated protein levels and/or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, or PD-L2, or CTLA-4 and/or having elevated Cancers with high protein levels and/or nucleic acid levels (e.g., elevated expression) of TIGIT, e.g., tumor cells of the cancers with elevated PD-1, PD-L1, and/or PD-L2 or CTLA- 4 protein levels and/or nucleic acid levels (e.g., increased expression), and/or elevated TIGIT protein levels and/or nucleic acid levels (e.g., increased expression) (e.g., compared to the same tissue of a healthy individual, or compared to compared to adjacent healthy tissue in the patient).
  • Anti-TIGIT VHH or heavy chain antibodies or bispecific antibodies of the invention can be administered by any suitable method, including parenteral Administration is administered intrapulmonarily, intranasally, and, if required for local treatment, intralesionally.
  • Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Administration may be by any suitable route, for example by injection, eg intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dose of the anti-TIGIT VHH or heavy chain antibody or bispecific antibody (and immunoconjugates, compositions, pharmaceutical compositions, preparations, combination products, etc.) comprising the present invention (when alone or in combination with one or more other therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, prophylactic or therapeutic administration, previous therapy, patient The clinical history and response to the antibodies, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in one treatment or over a series of treatments.
  • the invention provides the use of an anti-TIGIT VHH or heavy chain antibody or bispecific antibody of the invention, or an immunoconjugate or composition comprising the same, in the manufacture or preparation of a medicament for use as described herein. Uses, such as for the prevention or treatment of related diseases or conditions mentioned herein.
  • an anti-TIGIT VHH or heavy chain antibody or bispecific antibody can also be combined with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents for the uses described herein, for example for the prevention and/or treatment of the related diseases or conditions mentioned herein.
  • the present invention also relates to methods for diagnosis and detection of VHH or heavy chain antibodies or bispecific antibodies and compositions for diagnosis and detection comprising same.
  • the anti-TIGIT VHH or heavy chain antibody antibodies provided herein can be used to detect the presence of TIGIT in a biological sample.
  • the anti-bispecific antibodies provided herein can be used to detect the presence of TIGIT and/or PD1 in a biological sample.
  • the anti-bispecific antibodies provided herein can be used to detect the presence of TIGIT and/or CTLA-4 in a biological sample.
  • the term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR).
  • the biological sample is a bodily fluid, such as blood, serum or plasma.
  • the method comprises contacting a biological sample with a VHH or heavy chain antibody as described herein under conditions that allow it to bind to TIGIT, and detecting whether there is an interaction between the VHH or heavy chain antibody and TIGIT. form a complex. Complex formation indicates the presence of TIGIT.
  • the method can be an in vitro or in vivo method.
  • an antibody of the invention is used to select a subject suitable for treatment with a VHH or heavy chain antibody of the invention, for example wherein TIGIT is the biomarker used to select said subject.
  • the method comprises contacting a biological sample with a bispecific antibody as described herein under conditions that allow it to bind to TIGIT and/or PD-1, and detecting the binding of the antibody to TIGIT and/or PD-1 Or whether a complex is formed between PD-1. Complex formation indicates the presence of TIGIT and/or PD-1.
  • the method can be an in vitro or in vivo method.
  • the antibody of the invention is used to select a subject suitable for treatment with the bispecific antibody of the invention, for example wherein TIGIT and/or PD-1 are biomarkers used to select said subject .
  • the method comprises contacting a biological sample with a bispecific antibody as described herein under conditions that permit its binding to TIGIT and/or CTLA-4, and detecting the binding of the antibody to TIGIT and/or CTLA-4. Or whether a complex is formed between CTLA-4. Complex formation indicates the presence of TIGIT and/or CTLA-4.
  • the method can be an in vitro or in vivo method.
  • an antibody of the invention is used to select a subject suitable for treatment with a bispecific antibody of the invention, for example wherein TIGIT and/or CTLA-4 are biomarkers used to select said subject .
  • labeled VHH or heavy chain antibodies or bispecific antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are detected indirectly, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • the label is, for example, a label such as biotin or hFc.
  • the sample is obtained prior to treatment with a VHH or heavy chain antibody or bispecific antibody of the invention. In some embodiments, the sample is obtained prior to other therapy. In some embodiments, the sample is obtained during or after treatment with the other therapy.
  • TIGIT and/or PD1 are measured prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.
  • TIGIT and/or CTLA-4 are measured prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.
  • a method of treating a disease of the present invention comprising: testing a subject (e.g., a sample) (e.g., a sample from a subject) for the presence of TIGIT, thereby determining a TIGIT value, The TIGIT value is compared to a control value (eg, a value in a normal individual), and if the TIGIT value is greater than the control value, administering to the subject a therapeutically effective amount of a drug according to the invention, optionally in combination with one or more other therapies.
  • VHH antibody or heavy chain antibody thus treating the disease.
  • a method of treating a disease of the invention comprising: testing a subject (e.g., a sample) (e.g., a sample from a subject) for the presence of TIGIT and/or PD1, thereby determining TIGIT and/or PD1 values, comparing the TIGIT and/or PD1 values with control values (eg, values in normal individuals), and if the TIGIT and/or PD1 values are greater than the control values, administering to the subject a therapeutically effective amount of any A bispecific antibody according to the invention is selected in combination with one or more other therapies, thus treating said disease.
  • a subject e.g., a sample
  • control values e.g, values in normal individuals
  • a method of treating a disease of the invention comprising: testing a subject (e.g., a sample) (e.g., a sample from a subject) for the presence of TIGIT and/or CTLA-4, The TIGIT and/or PD1 value is thus determined, the TIGIT and/or CTLA-4 value is compared to a control value (e.g., a value in a normal individual), and if the TIGIT and/or CTLA-4 value is greater than the control value, the subject is given Administration of a therapeutically effective amount of a bispecific antibody according to the invention, optionally in combination with one or more other therapies, thus treats the disease.
  • a control value e.g., a value in a normal individual
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • TIGIT T cell immune receptor with Ig and ITIM domains
  • TIGIT refers to a T-cell immune receptor from any vertebrate source, including mammals such as primates (e.g., humans or cynomolgus monkeys) and rodents ( For example, any native TIGIT of mouse and rat), unless otherwise stated.
  • TIGIT is also known in the art as V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM.
  • the term encompasses "full length", unprocessed TIGIT as well as any form of TIGIT that results from processing in the cell.
  • human TIGIT comprises the amino acid sequence shown in SEQ ID NO: 46, or consists of said sequence.
  • cynomolgus monkey TIGIT comprises the amino acid sequence shown in SEQ ID NO: 48, or consists of said sequence.
  • PD-1 refers to programmed cell death protein 1.
  • the term “PD-1” includes variants, isoforms, homologs, orthologs and paralogs.
  • an antibody specific for human PD-1 protein may cross-react with PD-1 protein from a species other than human (eg, cynomolgus monkey).
  • the antibody specific for human PD-1 protein may be completely specific for human PD-1 protein and not exhibit cross-reactivity with other species or other types, or may be with antibodies from certain other species but not PD-1 cross-reactivity in all other species.
  • human PD-1 refers to a human PD-1 sequence, such as the complete amino acid sequence of human PD-1 having Genbank accession number. NP_005009.2.
  • cytotoxic T lymphocyte associated protein 4" or "CTLA-4" is an inhibitory receptor upregulated on T cells (A1egre et al., 2001, Nature Immunology Reviews (Nat Rev Immunol) 1:220-8).
  • CTLA-4 suppresses the immune response in several ways: it competes with the T-cell co-stimulatory receptor CD28 for its ligands CD80 and CD86, thereby blocking co-stimulation; it sends a negative signal to inhibit T-cell activation; and it can also Trapping of CD80 and CD86 from opposing cells by trans-endocytosis leads to attenuated T cell co-stimulation via CD28. It is constitutively expressed on Treg cells and only expressed on activated traditional T cells.
  • CTLA4 is a key negative regulator of T cells and has the same B7 ligands (CD80, CD86) as CD28, but CTLA4 has molecular affinity with B7 Higher, so CTLA4 and CD28 on T cells compete with B7 molecules on APC cells, thereby inhibiting the activation of T cells (Shunsuke Chikuma.(2017). "CTLA-4, an Essential Immune-Checkpoint for T- Cell Activation "Curr Top Microbiol Immunol 410:99-126.). CTLA4 can also mediate the trans-endocytosis of B7 molecules on the surface of APC cells by Treg cells, thereby reducing the expression of B7 molecules on the surface of APC cells and reducing the activation of CD28 on T cells.
  • CTLA4 can restore the antitumor immune response through two independent but complementary mechanisms, the first is to promote the proliferation and activation of tumor-infiltrating T cells, and the second is to attenuate the function of immunosuppressive Treg cells.
  • Ipilimumab blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), but clinically there are many treatment-related adverse events, especially dose-limiting toxicities at higher doses that prevent its maximal antitumor activity potential.
  • CTLA4 cytotoxic T-lymphocyte-associated antigen 4
  • ipilimumab-induced irAE include: blocking CTLA4 will activate T cell clones that respond to self-antigens, leading to autoimmune disease-like symptoms; the Fc effect of ipilimumab will lead to Depletion of tissue-resident Treg cells hinders peripheral tolerance and predisposes patients to irAEs after CTLA4 antibody treatment; CTLA4 antibody treatment leads to endocytic degradation of CTLA4 receptors, and ipilimumab significantly downregulates CTLA4 on the cell surface receptor.
  • Single domain antibody (single domain antibody, sdAb) is used herein to refer to an antibody polypeptide that recognizes and binds an antigen of interest through a single variable antibody domain, such as a single VH or a single VL.
  • the single variable antibody domain of a single domain antibody does not need to be paired with another antibody variable domain to be able to recognize and bind an antigen of interest.
  • a single domain antibody comprising a heavy chain variable domain is also referred to as VHH.
  • VHH or "VHH antibody” are used interchangeably herein and generally refer to an antibody comprising or consisting of only one heavy chain variable region that has antigen-binding activity.
  • VHH usually contains three CDRs and four highly conserved framework regions, and usually has the structure of the following formula: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4; CDR1 To CDR3 refers to complementarity determining regions 1-3.
  • the CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, preferably the boundaries of the three CDRs in the variable region sequence can be defined by IMGT.
  • VHHs generally comprise only heavy chain variable domains derived from heavy chain antibodies lacking light chains, also known as Nanobodies.
  • the VHH used in the present invention is preferably from a camelid, such as an alpaca, or a humanized or sequence-optimized form thereof (eg, affinity matured to increase binding affinity).
  • a VHH of the invention is a monovalent monospecific polypeptide molecule consisting or consisting essentially of a single heavy chain variable region (eg, the heavy chain variable region of a heavy chain antibody).
  • the single domain antibodies or VHHs of the invention may also be contained within larger polypeptides/proteins.
  • polypeptides/proteins comprising a VHH of the present invention include, but are not limited to, heavy chain antibodies (HcAbs) or bispecific antibodies or fusion proteins.
  • HcAbs heavy chain antibodies
  • the "heavy chain antibody” in the present invention refers to an antibody that does not have a light chain, for example, it can contain VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from N segment to C segment, or can VH-CH1-CH2-CH3 is included; homodimers, such as heavy chain dimer antibodies without light chains, can be encompassed.
  • the heavy chain antibody of the present invention may contain the VH derived from a standard antibody or the VH derived from a single domain antibody.
  • the VH in the heavy chain antibody of the invention can be VHH.
  • the heavy chain antibody of the present invention may be a heavy chain antibody having a framework region and/or a heavy chain constant region derived from a camelid (llama, camel, especially alpaca), which is humanized form or its sequence-optimized form (affinity matured form), or a fragment thereof (eg, a fragment comprising at least part of a constant region).
  • the heavy chain antibodies of the present invention also encompass antibodies formed by fusing the heavy chain variable region or VHH to an Fc region (e.g., a human IgG Fc region, such as a human IgG1 or IgG4 Fc region).
  • an Fc region e.g., a human IgG Fc region, such as a human IgG1 or IgG4 Fc region.
  • the term "monospecific” refers to a polypeptide/protein molecule having one or more antigen binding sites, each of which binds to the same epitope of the same antigen.
  • multispecific antibody refers to an antibody that has at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope. Antigen binding to different epitopes. Multispecific antibodies are antibodies that have binding specificities for at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen.
  • multispecific binding molecule refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule, i.e. the molecule comprises at least a first target binding region and a second target binding region, wherein the The first target binding region binds one target or antigen and the second target binding region binds another antigen or target.
  • the molecules according to the invention comprise specificities for at least two different antigens or targets.
  • Molecules according to the invention also encompass multispecific molecules comprising multiple target binding regions/binding sites, such as trispecific binding molecules.
  • bispecific binding molecules of the invention are bispecific antibodies.
  • linker refers to any molecule that enables the direct linking of different parts of a bispecific binding molecule.
  • linkers that establish covalent linkages between different molecular moieties include peptide linkers and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol copolymer.
  • the linker is a peptide linker (also known as a "linker peptide”), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and/or serine (S) alone or in combination.
  • a peptide linker can link a first target-binding region of a binding molecule to a second target-binding region.
  • a peptide linker can also join one part of an antibody to another part of an antibody, such as joining a light chain variable region to a heavy chain variable region.
  • the peptide linker is of a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the connecting peptide is 5-50 amino acids in length, eg, 10, 15, 20, 25, 30 amino acids in length.
  • the connecting peptide comprises the amino acid sequence (GS)n, (GGS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, wherein n is an integer equal to or greater than 1 , for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • the connecting peptide is a hinge region or portion of a hinge region from an immunoglobulin, including a native hinge region or portion thereof, or a mutated hinge region or portion thereof.
  • the connecting peptide is, for example, the hinge region or part thereof (eg EPKSC) or a mutated hinge region or part thereof of an immunoglobulin (eg IgG, eg IgGl, IgG2, IgG3 or IgG4), eg EPKSS.
  • suitable flexible linker peptides can be rationally designed using computer programs to model the three-dimensional structures of proteins and peptides, or by phage display methods.
  • target binding region refers to any portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds a particular target or antigen.
  • the target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the remainder of the BsAB, and may or may not bind its target as a separate entity.
  • the target binding region can also be a receptor or a ligand, or a domain of a receptor capable of binding a ligand.
  • the "target-binding region” is also referred to as "antigen-binding region”.
  • full-length antibody refers to an antibody molecule having the molecular structure of a native immunoglobulin.
  • a full-length antibody comprises two heavy chains (H) and two light chains (L) inter-connected by disulfide bonds.
  • the full length antibody comprises two heavy chains (H) interconnected by disulfide bonds.
  • the full-length antibody heavy chain usually consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2 and CH3.
  • a full-length antibody light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain, CL.
  • VL light chain variable region
  • CL constant region
  • Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the term "antibody fragment” includes a portion of an intact antibody. In preferred embodiments, antibody fragments are antigen-binding fragments.
  • antigen-binding fragment of an antibody is a molecule that, unlike a full-length antibody, comprises a portion of a full-length antibody, but which is capable of binding to the antigen of the full-length antibody or with the full-length antibody (i.e., with the full-length antibody from which the antigen-binding fragment is derived).
  • Antibodies compete for antigen binding.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, diabody, single domain antibody (sdAb), Nanobody.
  • Fab fragments can be obtained by papain digestion of full-length antibodies. Furthermore, digestion of whole antibodies by pepsin below the disulfide bonds in the hinge region yields F(ab')2, a dimer of Fab', a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting F(ab')2 dimers to Fab' monomers.
  • a Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment consists of the VL and VH domains of a single arm of an antibody.
  • the two domains VL and VH of the Fv fragment can be encoded by separate genes, but recombinant methods can also be used to link the two domains using a synthetic linker peptide so that they are produced as a single protein chain, and in the single protein The VL and VH regions in the chain pair to form a single chain Fv (scFv).
  • scFv single chain Fv
  • Fab fragment or “Fab” are used interchangeably herein to refer to an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, and a light chain variable domain consisting of two polypeptide chains.
  • Immunoglobulin fragments of variable domain VL and light chain constant domain CL wherein one polypeptide chain comprises VH and a constant region selected from CH1 and CL from N-terminus to C-terminus, and the other polypeptide chain runs from N-terminus to C-terminus
  • the VL end comprises a VL and another constant region selected from CL and CH1, wherein the VH and VL domains pair to form an antigen binding site.
  • the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as "Fab heavy chain”; correspondingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as "Fab light chain”.
  • Target refers to a bound substance to which a binding molecule is directed.
  • Targets can be antigens, ligands or receptors.
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • any macromolecule including essentially any protein or peptide, can be used as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen that specifically interacts with an antibody molecule.
  • a "complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point").
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment schemes, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, A1-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S.
  • CDR CDR sequence
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • residue positions in antibody variable regions including heavy chain variable region residues and light chain variable region residues
  • Kabat numbering system Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDRs in the VHH or heavy chain antibodies of the invention are determined according to IMGT.
  • VHCDR and VLCDR in the anti-PD1 antibody of the present invention were determined according to Kabat.
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment schemes are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment scheme rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Antibodies with different specificities have different binding sites for different antigens (under the same assignment scheme).
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.
  • Fc domain or “Fc region” are used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” or “Fc region” comprises two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) of the heavy chain derived from antibodies of the IgG, IgA and IgD classes; and the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the two heavy chains of antibodies of the IgE class.
  • the numbering of amino acid residues in the Fc region or in the heavy chain constant region is according to eg Edelman, G.M. et al., Proc. Natl. Acad.
  • Fc region excludes the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region CL of immunoglobulins , but in some cases may include a hinge region N-terminal to the heavy chain constant region.
  • chimeric antibody is an antibody molecule in which (a) the constant region or portion thereof has been altered, replaced or exchanged such that the antigen binding site is of a different or altered class, effector function and/or species source of constant regions, or with completely different molecules (eg, enzymes, toxins, hormones, growth factors, drugs) that confer new properties on chimeric antibodies; Or altered antigen-specific variable region alterations, substitutions or exchanges.
  • camelid heavy chain antibodies can be modified by exchanging their constant regions with those from human immunoglobulins. Due to the exchange of human constant regions, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans compared to the original camel antibody.
  • a "humanized antibody” is an antibody that retains the antigen-specific reactivity of a non-human antibody (eg, an alpaca monoclonal antibody) while being less immunogenic when administered to a human as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibodies with their human counterparts (i.e., replacing parts of the constant and variable regions that do not participate in binding with the corresponding parts of human antibodies) .
  • a non-human antibody eg, an alpaca monoclonal antibody
  • the term "anti”, “bind” or “specifically binds” means that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of a binding site to bind a particular target or antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or surface plasmon resonance (SPR) assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD assay biofilm thin layer interferometry
  • SPR surface plasmon resonance
  • an effective amount refers to such an amount or dose of the antibody or fragment or composition or combination of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, relative to an untreated subject %, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom.
  • a host cell is any type of cellular system that can be used to produce an antibody molecule of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused.
  • Labels can themselves be detectable (eg, radioisotopic or fluorescent labels) or, in the case of enzymatic labels, can catalyze the chemical alteration of a detectable substrate compound or composition.
  • the term is intended to encompass both direct labeling of a probe or antibody by conjugating (ie, physically linking) a detectable substance to the probe or antibody as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.
  • the label is hFc or biotin.
  • “Individual” or “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.
  • an “isolated” antibody or molecule is one that has been separated from a component of its natural environment.
  • the antibody or molecule is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or determined by reverse phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or determined by reverse phase HPLC.
  • Percent identity (%) refers to after aligning a candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account any When conservative substitutions are taken as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in this specification.
  • the invention contemplates variants of the antibody molecules of the invention having a substantial degree of identity, for example at least 80% identity, with respect to the antibody molecules and their sequences specifically disclosed herein , 85%, 90%, 95%, 97%, 98%, or 99% or higher. Such variants may contain conservative changes.
  • “conservative changes” include substitutions, deletions or additions to the polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the 8 groups of amino acids containing mutually conservative substitutions are listed below: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M ), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), Methionine (M).
  • the term "conservative sequence change” is used to refer to an amino acid modification that does not significantly affect or alter the antigen-binding characteristics of interest of an antibody molecule or binding protein molecule of the invention comprising an amino acid sequence.
  • conservatively modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or higher, eg 100-110% or higher binding affinity for the antigen of interest relative to the parent antibody or binding protein.
  • pharmaceutical excipient refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which are administered together with the active substance.
  • adjuvant such as Freund's adjuvant (complete and incomplete)
  • excipient carrier or stabilizer, etc.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • non-fixed combination means that the active ingredients (e.g., (i) the immunoconjugate of the invention, and (ii) the other therapeutic agent) are combined as separate entities simultaneously, with no particular time limit, or at the same or different times. Interval, sequential administration to the patient, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient.
  • fixed combination means that two or more active agents are administered to a patient simultaneously as a single entity.
  • Dosages and/or intervals of two or more active agents are preferably selected such that the combined use of the parts produces a greater effect in treating a disease or condition than can be achieved by any one component alone.
  • the components may each be in the form of separate formulations, which may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities, such as radiation therapy or surgery, to treat a disease described herein.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule with fixed ratios of the active ingredients.
  • administration includes co-administration for each active ingredient in multiple or in separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration.
  • administration also includes using each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.
  • anti-tumor effect refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancerous and cancerous cells and tissues.
  • tumor-associated antigen refers to an antigenic determinant presented on the surface of a target cell, wherein the target cell is a cell in a tumor, such as a cancer cell, a cell of the tumor stroma.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indications of a disease, alleviating symptoms, or arresting or inhibiting a disease, condition, or disorder. Further development.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses) that incorporate recombinant polynucleotides. virus and adeno-associated virus).
  • tissue sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of tissue or cell samples can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as tears, vitreous humor, cerebrospinal fluid , amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • the tissue sample is tumor tissue.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • antigen hTIGIT-Fc 0.25 mg of antigen hTIGIT-Fc (ACRO, product number TIT-H5254) was mixed with incomplete Freund's adjuvant, emulsified and injected subcutaneously at multiple points. A total of 7 immunizations were carried out with an interval of 3 weeks between each immunization, wherein the fifth and seventh times were mixed with purified cynomolgus TIGIT-11maFc protein (SEQ ID NO: 47) and incomplete Freund's adjuvant.
  • the library was screened using phage display technology.
  • 20 ⁇ g of cynoTIGIT protein ACRO, Product No. TIT-C5223
  • washed 15 times with 0.1% PBST washed 15 times with 0.1% PBST, and finally eluted with 1mL 100mM triethylamine, and neutralized with 500 ⁇ L 1M Tris-HCl pH7.4.
  • the overnight culture was centrifuged at 10,000 rpm for 20 min at 4°C, the supernatant was collected, and the precipitate was discarded.
  • the third round of screening uses 10 ⁇ g of biotin-labeled cynoTIGIT protein adsorption, 0.1% PBST washes 15 times, and finally uses 1 mL of 100 mM Triethylamine was used for elution, and 500 ⁇ L of 1M Tris-HCl at pH 7.4 was used for neutralization.
  • the eluted phages were diluted and infected TG1 in the logarithmic phase, spread on 2YT (50 ⁇ g/mL carbenicillin + 2% glucose) plates, cultured overnight at 37°C, and picked the next day
  • Single clones on the plate were cultured in deep-well plates containing 400 ⁇ L of 2YT (50 ⁇ g/mL carbenicillin) liquid medium at 37 °C and 220 rpm until logarithmic phase, adding 1 mM IPTG, and induced at 30 °C overnight at low temperature.
  • centrifuge the deep well plate at 500 g for 5 minutes, and take the supernatant for ELISA detection.
  • the 1G3-VHH, 6F6-VHH, 5H2-VHH and EPKSS connecting peptides were respectively inserted into the expression vector pcDNA3.1(+) containing the coding gene of the human IgG1 heavy chain constant region Fc sequence (SEQ ID NO: 40), to Plasmids expressing anti-TIGIT heavy chain antibodies (1G3, 6F6, 5H2) were obtained.
  • amino acid sequence of the anti-TIGIT heavy chain antibody 1G3 is as follows (SEQ ID NO: 7), wherein the connecting peptide is shown in bold and underlined, and the constant region Fc is shown in italics.
  • amino acid sequence of the anti-TIGIT heavy chain antibody 6F6 is as follows (SEQ ID NO: 2), wherein the connecting peptide is shown in bold and underlined, and the constant region Fc is shown in italics.
  • amino acid sequence of the anti-TIGIT heavy chain antibody 5H2 is as follows (SEQ ID NO: 10), wherein the connecting peptide is shown in bold and underlined, and the constant region Fc is shown in italics.
  • the single domain antibody 1G3 screened in Example 2 was selected respectively , 6F6 and 5H2 highly homologous heavy chain variable region germline genes were used as templates, and the CDRs of single domain antibodies were transplanted into corresponding human templates respectively, and the formation sequence was FRI-CDR1-FR2-CDR2-FR3-CR3 - the variable region sequence of FR4.
  • the key amino acids in the FR region were back-mutated to the corresponding amino acids of the nanobody (VHH antibody) to ensure the original affinity, that is, a humanized anti-TIGIT VHH antibody was obtained.
  • the determination of the amino acid residues in the CDR region is determined and annotated by the IMGT numbering system.
  • the template for the humanized heavy chain of antibody 1G3 is IGHJ4*01, and humanized antibody 1G3-H1 is obtained after humanization.
  • the sequence of the humanized variable region is as follows:
  • Table 1 lists the sequences and lists the corresponding back mutation sites.
  • the humanized heavy chain template of antibody 6F6 is IGHV3-48*01, and humanized antibody 6F6-H1 is obtained after humanization.
  • the humanized variable region sequence is as follows:
  • Table 2 lists the sequences and lists the corresponding back mutation sites.
  • the humanized heavy chain template of the antibody 5H2 is IGHV3-23*01, and the humanized antibody 5H2-H1V2 is obtained after humanization, and the sequence of the humanized variable region is as follows:
  • Table 3 lists the sequences and lists the corresponding back mutation sites.
  • the nucleic acids encoding 1G3-H1-VH, 6F6-H1-VH, 5H2-H1V2-VH plus EPKSS linking peptide were respectively inserted into the expression vector pcDNA3.1(+) containing the coding gene of the human IgG1 heavy chain constant region Fc sequence, To obtain plasmids expressing anti-TIGIT full-length humanized heavy chain antibodies (1G3-H1, 6F6-H1, 5H2-H1V2). According to the manufacturer's product manual, use the ExpiCHO TM Expression System (ThermoFisher, Cat. No. A29133)
  • the plasmids encoding 1G3; 6F6; 5H2; 1G3-H1; 6F6-H1; 5H2-H1V2 obtained above in Examples 2 and 3 were transfected into ExpiCHO-S cells to express the TIGIT antibody.
  • Cells were cultured for 10-12 days after transfection. When the cell survival rate dropped to 60% to 70%, the supernatant was collected, and the protein expressed in the supernatant was purified using the MabSelect Sure protein A affinity chromatography system (GE healthcare).
  • Antibodies, homodimeric heavy chain antibodies against TIGIT heavy chain antibodies were obtained. The purified antibody was concentrated, sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and molecular exclusion to be greater than 95%. The results showed that the purity of the antibody met the requirements and could be used in the next experiment.
  • control antibody Tiragolumab comes from the patent WO2017053748A2, codon optimization and gene synthesis were performed by General Biosystems (Anhui) Co., Ltd. and cloned into the expression vector pcDNA3.1(+). Then apply the above expression and purification techniques to obtain the control antibody, hereinafter referred to as Tiragolumab analog herein.
  • Example 4 Affinity experiment of anti-human TIGIT heavy chain antibody with human TIGIT protein and cynomolgus monkey TIGIT protein
  • the kinetic experiment method is carried out according to the following steps: a) equilibrate the baseline with Running Buffer for 100s, b) add anti-human TIGIT heavy chain antibody diluted with Running Buffer, the final concentration is 5 ⁇ g/mL, and solidify for 200s, c ) Equilibrate the baseline with Running buffer for 300s, d) Add 100nM human TIGIT protein and cynomolgus monkey TIGIT protein diluted with Running Buffer to each well, combine for 200s, and dissociate for 600s. The experimental data were fitted and calculated using Fortebio Data Ahalysis software 1:1 binding model.
  • Table 4 summarizes the binding affinity of the anti-human TIGIT heavy chain antibody of the present invention to human TIGIT protein and cynomolgus TIGIT protein
  • Table 4 shows that the anti-human TIGIT heavy chain antibody constructed in this application can specifically bind human TIGIT protein and cynomolgus monkey TIGIT protein and has high binding activity.
  • Example 5 ELISA method detects that the heavy chain antibody against human TIGIT inhibits the binding of human TIGIT protein to CD155
  • human TIGIT(M22-P141)-Fc protein (SEQ ID NO: 45) was coated on a 96-well plate at 50 ⁇ L/well, and incubated overnight at 4°C. The plate was incubated at 37° C. with blocking buffer (PBS solution containing 1% BSA) and blocked for 1 h. After blocking, the plate was washed three times with PBST solution (PBS solution containing 0.05% Tween 20).
  • blocking buffer PBS solution containing 1% BSA
  • the anti-human TIGIT heavy chain antibody can block the binding of human TIGIT protein to CD155, and the blocking activity of the humanized TIGIT antibody is higher than that of the positive control antibody Tiragolumab analog (from patent WO2017053748A2, SEQ ID NO: 50, SEQ ID NO: 51) Stronger.
  • HEK293/human TIGIT cells were collected by digestion and centrifugation, resuspended in PBS, and inoculated into a 96-well plate (Corning, Cat.
  • the test antibody (1G3, 6F6, 5H2, 1G3-H1, 6F6-H1 heavy chain antibody prepared in Example 3, the highest concentration is 150nM, diluted 4 times, a total of 8 concentration points) was added to HEK293/human TIGIT cells, 4 After incubation at °C for 30 minutes, centrifuge (1000 rpm, 5 minutes) and discard the supernatant.
  • the tested TIGIT heavy chain antibodies can bind to HEK293/human TIGIT cells, with EC50 in the range of 0.6386nM-0.7873nM, and the binding activity is stronger than that of the positive control Tiragolumab (EC50 is 1.605nM).
  • the nucleotides encoding the TIGIT protein (amino acid sequence SEQ ID NO: 48) of cynomolgus monkeys were constructed on the eukaryotic expression vector, and then transfected After transfection into HEK293 cells, 48 hours after transfection, they were screened with 0.3 ⁇ g/mL puromycin (Puromycin Dihydrochloride, Gibco, Cat. No. A1113802) for 3 to 5 days, and cells highly expressing cynomolgus TIGIT protein were obtained.
  • the antibody to be tested (1G3, 6F6, 5H2, 1G3-H1, 6F6-H1 heavy chain antibody prepared in Example 3, the initial concentration is 150nM, diluted 4 times, 8 points ) was added to the cells and incubated at 4°C for 30 minutes. After washing the cells twice with PBS, add fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific, and incubate at 4°C for 30 minutes. The cells were washed twice with PBS, and the cells were resuspended, and finally the fluorescent signal was detected with a Cytoflex (Beckman) flow cytometer. Tiragolumab analog was used as a positive control.
  • the tested TIGIT heavy chain antibody can bind to the cynomolgus monkey TIGIT protein expressed on HEK293 cells, and the binding activity is stronger than that of the positive control Tiragolumab.
  • PBMCs human peripheral blood mononuclear cells isolated from Technology Co., Ltd.
  • CHO-K1/OKT3/CD155 target cells Using Lipofectamine TM 2000 (Invitrogen, Cat. No. 11668019) transfection reagent, according to the manufacturer's instructions, the nucleotides encoding human CD155 protein (amino acid sequence SEQ ID NO: 49) were constructed into true 48 hours after transfection, use 4 ⁇ g/mL puromycin (Puromycin Dihydrochloride, Gibco, product number A1113802) to select for 3 to 5 days, A CHO-K1/CD155 stable cell line stably expressing CD155 protein was obtained.
  • puromycin Puromycin Dihydrochloride, Gibco, product number A1113802
  • nucleotide amino acid sequence SEQ ID NO: 53
  • a CHO-K1/CD155 stable cell line CHO-K1/OKT3/CD155 cells
  • PBMC cells Resuscitate PBMC cells, isolate CD8+ T cells (Miltenyi Biotec, product number 130-096-495) according to the instructions of the Miltenyi cell sorting kit, and use 1640 complete medium containing 10% FBS (Gibco, product number 10099-141C) ( Gibco, Cat. No. 22400-071) was resuspended, adjusted the cell density to 1E6/mL, and plated in 96-well plates (Corning, Cat. No. 3599) at 100 ⁇ L per well (that is, 100,000 cells per well).
  • FBS Gibco, product number 10099-141C
  • Gibco Gibco, Cat. No. 22400-071
  • the final detection concentrations of the test antibody are 50nM and 5nM, add 50 ⁇ L per well to a 96-well plate, and then put the cells The incubator continued to incubate for 72h. After the incubation, take out the experimental plate and centrifuge at 2000rpm for 3 minutes to make all the cells sink to the bottom of the plate. Carefully pipette 100 ⁇ L of the supernatant into a new 96-well plate, and detect the level of human IFN ⁇ (Cisbio, Cat. No. 62HIFNGPEH) factor in the supernatant. The results are shown in Figure 4, indicating that the anti-TIGIT antibody can activate CD8+ T cells to release cytokines.
  • the VHH sequence was amplified by PCR and connected to the N-terminal of the IgG1-Fc constant region, and the Fc region was mutated at the following sites: S239D, A330L, I332E (according to EU numbering), and the final vector was constructed
  • the antibody 6F6-DLE was expressed by eukaryotic cells (the preparation method is as in Example 3), and its amino acid sequence is shown in SEC ID NO:20.
  • variable region sequence of 6F6 (D63S) was constructed to the N-terminal of the IgG1-Fc constant region according to the aforementioned method, and the Fc region also contained mutation sites S239D, A330L, and I332E (according to Kabat coding), forming
  • the vector was expressed in eukaryotic cells to obtain the antibody 6F6-DS-DLE, the amino acid sequence of which was SEC ID NO: 22.
  • Example 3 The method in Example 3 was used to express the above-mentioned optimized antibody.
  • Example 9 TIGIT heavy chain antibody after sequence optimization and Fc modification, human TIGIT protein, cynomolgus monkey TIGIT protein affinity test
  • the AHC sensor (ForteBio, Cat. No. 18-5060) was put into Running Buffer (1 X PBS Hyclone, Cat. No. SH30256.01, containing 0.02% Tween20, pH7.0), and pre-equilibrated at room temperature for 10 min.
  • the kinetic experiment method was carried out according to the following steps: a) equilibrate the baseline with Running Buffer for 180s, b) add anti-human TIGIT heavy chain antibodies (6F6-DS-DLE, 6F6DLE and 6F6- H1), final concentration 5 ⁇ g/mL, solidify for 200s, c) equilibrate the baseline with Running buffer for 300s, d) add 100nM human TIGIT protein and cynomolgus TIGIT protein diluted with Running Buffer to each well, combine for 200s, and dissociate for 600s.
  • the experimental data were fitted and calculated using Fortebio Data Analysis software 1:1 binding model.
  • Table 5 summarizes the binding affinity of the anti-human TIGIT heavy chain antibody to human TIGIT protein and cynomolgus TIGIT protein after sequence optimization and Fc modification
  • Table 5 shows that the PTM-modified and Fc-modified anti-human TIGIT VHH antibodies constructed in this application can specifically bind to human TIGIT protein and cynomolgus TIGIT protein, and the activity is not significantly different from that before modification.
  • Example 10 TIGIT heavy chain antibody after sequence optimization and Fc modification inhibits the binding of human TIGIT protein to CD155 ELISA method
  • 0.5 ⁇ g/mL human TIGIT(M22-P141)-Fc (SEQ ID NO: 45) was coated on a 96-well plate at 50 ⁇ L/well, and incubated overnight at 4°C. The plate was incubated at 37° C. with blocking buffer (PBS solution containing 1% BSA) and blocked for 1 h. After blocking, the plate was washed three times with PBST solution (PBS solution containing 0.05% Tween 20). Dilute anti-TIGIT antibody (initial concentration is 14nM, 3-fold serial dilution) with diluent, and mix with 1.1 ⁇ g/mL (concentration before mixing) CD155-mFc (ACRO, Cat. No.
  • CD5-H5254 at a volume ratio of 1:1 Mix, add to plate and incubate at 37°C for 1h. After incubation, the plate was washed with PBST solution. Dilute the secondary antibody (horseradish peroxidase HRP-labeled affinity-purified goat anti-mouse IgG, Fc ⁇ , Jackson Immuno Research, Cat. No. 115-035-164) with diluent, add to the plate and incubate at 37°C for 1 h, after the incubation Wash again, develop color with TMB for 15 minutes, then stop with 1M H 2 SO 4 , then read the absorbance value of OD450nm-OD620nm in a microplate reader, the results are shown in Figure 5.
  • the present invention constructs bispecific antibodies with two structures as shown in Figure 6, wherein the anti-TIGIT antibody part of each bispecific antibody is derived from the above-mentioned humanized 6F6-H1 antibody; the anti-PD1 part is derived from the patent WO2019219064A
  • the PD1 antibody 1B12 a bispecific antibody, is constantly differentiated into two forms, IgG1 and IgG4.
  • This type of bispecific antibody is also called "anti-PD1/TIGIT bispecific antibody” herein, and is sometimes referred to as "bispecific antibody, double antibody” in the examples.
  • the order of the heavy chain (D1-H) of bispecific antibody D1 from N-terminal to C-terminal is anti-PD1 antibody heavy chain variable region VH, CH1 constant region, anti-TIGIT antibody VHH and IgG1 Fc constant region, and its amino acid sequence is SEQ ID NO: 30;
  • the light chain (D1-L) of the bispecific antibody D1 is sequenced from the N-terminal to the C-terminal of the anti-PD1 light chain antibody variable region VL and light chain constant region CL, and its amino acid sequence is SEC ID NO: 39;
  • the order of the heavy chain (D4-H) of the bispecific antibody D4 from the N-terminal to the C-terminal is the variable region VH of the anti-PD1 heavy chain antibody, the constant region of CH1, the VHH of the anti-TIGIT antibody, and the Fc constant region of IgG4, and its amino acid sequence is SEQ ID NO: 32;
  • the sequence of the light chain (D4-L) of the bispecific antibody D4 from the N-terminal to the C-terminal is the anti-PD1 antibody variable region VH and the light chain constant region CL, and its amino acid sequence is SEC ID NO: 39;
  • the order of the heavy chain of the bispecific antibody E4 from N-terminal to C-terminal is anti-PD1 antibody variable region, CH1 constant region, IgG4 Fc constant region and anti-TIGIT antibody VHH, and its amino acid sequence is SEC ID NO: 33; bispecific The sequence from the N-terminal to the C-terminal of the antibody E4 light chain is the anti-PD1 antibody variable region and the light chain constant region CL, and its amino acid sequence is SEC ID NO: 39.
  • Each bispecific antibody expression vector was constructed as follows:
  • D1-H Using the PD1 antibody heavy chain coding sequence (SEQ ID NO: 56 corresponding to WO2019219064A in WO2019219064A) as a template to amplify the PD1 heavy chain variable region and constant region CH1
  • the nucleotide sequence of the segment use 6F6-H1 as a template to amplify the nucleotide sequence of the variable region VHH of the TIGIT antibody, and use overlapping PCR technology to splice the above two segments through the linker GGS, and directly connect them to the human IgG1 constant
  • D1-H molecule can be obtained by expression.
  • D1-L Using the sequence encoded by the light chain of the PD1 antibody in WO2019219064A (SEQ ID NO: 39, corresponding to SEQ ID NO: 39 in WO2019219064A) as a template, amplify the nucleotides of the variable region of the light chain of PD1 and the CL segment of the constant region Acid sequence, and connected to the expression vector, expressed to obtain D1-L molecules.
  • D4-H uses the PD1 antibody heavy chain coding sequence in WO2019219064A (SEQ ID NO: 55, corresponding to SEQ ID NO: 24 in WO2019219064A) as a template to amplify the nucleotides of the CH1 segment of the PD1 heavy chain variable region and constant region Sequence; use 6F6-H1 as a template to amplify the nucleotide sequence of TIGIT antibody variable region VHH, and use overlapping PCR technology to splice the above two sequences through the connecting peptide GGS; use human IgG4 heavy chain as a template to amplify CH2-CH3 For the constant region sequence, the above two sequences are connected to the expression vector by overlapping PCR technology (there is no connecting peptide between the two sequences), and the D4-H molecule is obtained by expression.
  • D4-L Using the sequence encoded by the light chain of the PD1 antibody in WO2019219064A (SEQ ID NO: 39, corresponding to SEQ ID NO: 39 in WO2019219064A) as a template, amplify the nucleotides of the variable region of the light chain of PD1 and the CL segment of the constant region Acid sequence, and connected to the expression vector, expressed to obtain D4-L molecules.
  • E4-H using the PD1 antibody heavy chain coding sequence (SEQ ID NO: 56, corresponding to WO2019219064A in WO2019219064A) as a template to amplify the PD1 heavy chain variable region and constant region
  • Nucleotide sequence Amplify the nucleotide sequence of the variable region VHH of the TIGIT antibody using 6F6-H1 as a template, and use overlapping PCR technology to splice the above two sequences, and directly connect them to the expression vector, and express to obtain E4-H molecular.
  • E4-L Using the sequence encoded by the light chain of the PD1 antibody in WO2019219064A (SEQ ID NO: 39, corresponding to SEQ ID NO: 39 in WO2019219064A) as a template, amplify the nucleotides of the variable region of the light chain of PD1 and the CL segment of the constant region Acid sequence, and connected to the expression vector, expressed to obtain E4-L molecules.
  • the above groups of expression vectors encoding the heavy chain and light chain were transfected into ExpiCHO-S cells to express the double antibody.
  • Cells were cultured for 10-12 days after transfection.
  • the cell survival rate dropped to 60% to 70%, the supernatant was collected, and the protein expressed in the supernatant was purified using the MabSelect Sure protein A affinity chromatography system (GE healthcare).
  • Antibodies obtain D1, D4 and E4 double antibodies.
  • the purified double antibody was concentrated, sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and molecular exclusion to be greater than 95%. The results showed that the purity of the antibody met the requirements and could be used in the next experiment.
  • Example 12 Experiment of binding activity of anti-PD1/TIGIT bispecific antibody to human TIGIT protein
  • the relative binding activity of the anti-PD1/TIGIT bispecific antibody to human TIGIT protein was determined by ELISA.
  • Human TIGIT-his (ACRO, Cat. No. TIT-H52H3) was diluted with PBS (HyClone, Cat. No. SH30256.01) to 0.5 ⁇ g/ml, coated on a 96-well plate, 50 ⁇ L/well, and incubated overnight at 4°C. Non-specific binding sites were blocked by incubation with PBS containing 1% BSA for 1 hour at 37°C. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween20).
  • PBST PBS containing 0.05% Tween20
  • Example 11 Dilute the anti-PD1/TIGIT bispecific antibody and Tiragolumab analog (control) prepared in Example 11 with binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA) (initial concentration is 1.5nM, 3-fold serial dilution, 7 concentration points), and incubated with the coated protein at 37°C for 1 hour. After incubation, wash the plate three times with PBST, dilute peroxidase-labeled goat anti-human Fc secondary antibody (Jackson Immuno Research, 109-035-098) to 1:25000 with binding buffer, incubate at 37°C for 1 hour, and wash again , TMB was developed for 15 minutes and then terminated with 1M H2SO4.
  • binding buffer PBS containing 0.05% Tween20 and 0.5% BSA
  • Example 13 Experiment on binding activity of anti-PD1/TIGIT bispecific antibody to human PD1 protein
  • the relative binding activity of the anti-PD1/TIGIT bispecific antibody to human PD1 protein was determined by ELISA.
  • Human PD1-his protein (Sino, Cat. No. 10377-H08H-100) was diluted to 0.2 ⁇ g/ml with PBS (HyClone, SH30256.01), coated on a 96-well plate, 50 ⁇ l/well, and incubated overnight at 4°C. Non-specific binding sites were blocked by incubation with PBS containing 1% BSA for 1 hour at 37°C. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween20).
  • PBS HyClone, SH30256.01
  • the anti-PD1/TIGIT bispecific antibody prepared in Example 11 and 1B12PD1 (IgG1mut) (control) (the IgG1 mutant of 1B12PD1 of WO2019219064A, the sequence is SEQ ID NO: 39, SEQ ID NO: 58) (initial concentration is 1.5nM, 3-fold serial dilution, 7 concentration points), and incubated with the coated protein at 37°C for 1 hour.
  • Example 14 Anti-PD1/TIGIT bispecific antibody inhibits the binding activity of human TIGIT protein to CD155 protein
  • 0.5 ⁇ g/mL of human TIGIT-human IgG1 Fc protein (SEQ ID NO.45) was coated on a 96-well plate at 50 ⁇ L/well, and incubated overnight at 4°C. The plate was incubated at 37° C. with blocking buffer (PBS solution containing 1% BSA) and blocked for 1 h. After blocking, the plate was washed three times with PBST solution (PBS solution containing 0.05% Tween 20).
  • blocking buffer PBS solution containing 1% BSA
  • Example 15 Anti-PD1/TIGIT bispecific antibody blocks the interaction between human PD1 and PD-L1
  • Human PD1 (M1-V170)-human IgG1 Fc protein (SEQ ID NO: 52) was diluted with PBS (HyClone, SH30256.01) to 0.5 ⁇ g/ml, coated on a 96-well plate, 50 ⁇ l/well, and incubated overnight at 4°C. Non-specific binding sites were blocked by incubation with PBS containing 1% BSA for 1 hour at 37°C. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween20).
  • PBS HyClone, SH30256.01
  • Dilute anti-PD1/TIGIT bispecific antibody (concentration is 22.5nM, 3-fold serial dilution, 7 concentration points) with binding buffer (PBS containing 0.05% Tween20 and 0.5% BSA), and dilute to 0.8 ⁇ g/ml respectively
  • binding buffer PBS containing 0.05% Tween20 and 0.5% BSA
  • the PD-L1 protein (Sino, Cat. No. 10084-H05H) was mixed at 1:1 and incubated with the coated protein at 37°C for 1 hour.
  • PD1 monoclonal antibody 1B12PD1 (IgG1mut) was used as a positive control, and IgG (SinoBiological product number HG1K) was used as a negative control.
  • the plate was washed three times with PBST, and the peroxidase-labeled goat anti-mouse Fc secondary antibody (Jackson Immuno Research, 115-035-164) was diluted to 1:10000 with binding buffer, incubated at 37°C for 1 hour, and again After washing, TMB was developed for 15 minutes and then terminated with 1M H2SO4.
  • the absorbance at 450nm-620nm was measured, and the inhibition curve of the anti-human PD1/TIGIT bispecific antibody PD1 is shown in FIG. 10 .
  • Example 16 Binding activity of anti-PD1/TIGIT bispecific antibody to human TIGIT and human PD1 cells
  • Example 6 Cell binding experiments were used to determine whether the anti-PD1/TIGIT bispecific antibody could bind to the human TIGIT protein stably expressed on HEK293 cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat. No. GNHu 43). The experimental process and method are as in Example 6 (1), using Tiragolumab ahalog as a positive control, and PD1 monoclonal antibody 1B12PD1 (IgG1 mut) as a negative control.
  • Example 6 (2) Determine whether the anti-PD1/TIGIT bispecific antibody can be stably expressed on the surface of Jurkat/NFAT-Luc cells (Jurkat cells stably expressing NFAT-Luc reporter gene elements, see WO2019219064A for construction methods) by cell binding experiments PD1 protein binding.
  • the experimental process and method are as in Example 6 (1), using PD1 monoclonal antibody 1B12PD1 (IgG1 mut) as a positive control, and Tiragolumab analog as a negative control.
  • Example 17 ADCC killing of activated CD4+T and CD8+T cells by anti-PD1/TIGIT bispecific antibody Traumatic activity
  • NK cells In order to detect whether anti-PD1/TIGIT IgG1 subtype bispecific antibody mediates the activity of NK cells to kill activated CD4+T and CD8+T cells, we established a primary NK cell-dependent cytotoxicity test system, respectively Activated CD4+T and CD8+T are target cells, and human peripheral blood mononuclear cells (PBMCs) are effector cells.
  • PBMCs peripheral blood mononuclear cells
  • Target cell preparation prepare a plate suspension containing 1 ⁇ g/mL OKT3 (Invitrogen, catalog number 16-0037-85) and 1 ⁇ g/mL anti-CD28 (Biolegend, catalog number 302934) with pre-cooled PBS, take 5 mL and add to 10 cm cells Incubate overnight at 4°C. The next day, discard the coating solution and wash once with pre-cooled PBS for later use. Resuscitate PBMC cells, and separate CD4+T cells (Miltenyi Biotec, product number 130-096-533) or CD8+T cells (Miltenyi Biotec, product number 130-096-495) respectively according to the instructions of the Miltenyi cell separation kit. 1640 Complete Medium (Gibco, Cat. No.
  • %FBS Gibco, Cat. No. 10099-141C
  • PBMC cells Resuscitate PBMC cells, adjust the cell density to 1-2E6/mL with 1640 complete medium containing 10% FBS, add IL2 (Jiangsu Jinsili Pharmaceutical Co., Ltd.) to activate overnight (the final concentration in the system is 100IU/mL), as effector cell suspension.
  • IL2 Jiangsu Jinsili Pharmaceutical Co., Ltd.
  • the activated CD4+T and CD8+T target cells were collected by centrifugation respectively, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the MEM- ⁇ test buffer (Gibco , Cat. No. 41061-029) to adjust the target cell density to 2 ⁇ 10 5 /ml, spread 50 ⁇ L per well (that is, 10,000 cells per well) into a 96-well plate (Corning, Cat. No. 3599).
  • the maximum detection concentration of the antibody is 50nM. Perform 5-fold serial dilutions to obtain a total of 8 concentration points, and add 50 ⁇ L to each well.
  • the PBMC effector cell suspension contained 200IU/mL IL2 (Jiangsu Kingsley Pharmaceutical Co., Ltd.).
  • D1, D4 and E4 were intravenously injected into rats (Pengli Biomedical Technology) at a dose of 10 mg/kg respectively, at different time points from 0 to 336 hours (0 to 14 days) (before administration, given Blood samples were collected at 10min, 30min, 1h, 4h, 8h, 24h, 48h, 7day, 10day, 14day) after the drug. All samples were processed into plasma and stored frozen at -70 to -86°C until analysis.
  • Human TIGIT-his (ACRO, Cat. No. TIT-H52H3) protein was diluted to 0.3 ⁇ g/mL with PBS (Biosharp, Cat. No. BL302A), 50 ⁇ L/well was added to a microtiter plate (Costar, Cat. No. 42592) and incubated overnight at 4°C. Then incubate at 37° C. for 1 hour in PBS solution containing 1% bovine serum albumin (Shanghai Sangong, product number: A500023-0025g). After the blocking, the cells were washed 3 times with PBST (PBS containing 0.05% Tween-20).
  • D1, D4, or E4 were diluted in serum-containing dilution buffer (containing 0.05% Tween-20, 0.5% bovine serum albumin, 2% v/v rat serum) at an initial concentration of 50 nL, 2-fold Doubling dilution of 6 concentration points, a total of 7 concentration points of the antibody solution is the standard curve.
  • serum-containing dilution buffer containing 0.05% Tween-20, 0.5% bovine serum albumin, 2% v/v rat serum
  • dilute D1, D4 or E4 with serum-containing dilution buffer to concentrations of 30ng/mL, 6ng/mL and 1.5ng/mL, respectively, as high, medium and low quality controls.
  • All rat sera were diluted with blank mixed rat serum and dilution buffer (PBS containing 0.05% Tween-20 and 0.5% bovine serum albumin), so that the final antibody concentration was maintained at 30-1.5 ng/mL.
  • Human PD1-mFc protein (Sino, Cat. No. 10377-H05H) was diluted with PBS (Biosharp, Cat. No.: BL302A) to 0.2 ⁇ g/mL, 50 ⁇ L/well was added to a microtiter plate (Costar, Cat. No. 42592) and incubated overnight at 4°C. Then incubate at 37° C. for 1 hour in PBS solution containing 1% bovine serum albumin (Shanghai Sangong, product number: A500023-0025g). After the blocking, the cells were washed 3 times with PBST (PBS containing 0.05% Tween-20).
  • PBS Biosharp, Cat. No.: BL302A
  • 50 ⁇ L/well 50 ⁇ L/well was added to a microtiter plate (Costar, Cat. No. 42592) and incubated overnight at 4°C. Then incubate at 37° C. for 1 hour in PBS solution
  • D1, D4 and E4 were diluted in serum-containing dilution buffer (containing 0.05% Tween-20, 0.5% bovine serum albumin, 2% v/v rat serum), the initial concentration was 50ng/mL, 2 times The antibody solution of 6 concentration points was diluted, and the antibody solution of 7 concentration points in total was used as the standard curve.
  • serum-containing dilution buffer containing 0.05% Tween-20, 0.5% bovine serum albumin, 2% v/v rat serum
  • dilute D1, D4 or E4 with serum-containing dilution buffer to concentrations of 30ng/mL, 6ng/mL and 1.5ng/mL, respectively, as high, medium and low quality controls.
  • All rat sera were diluted with blank mixed rat serum and dilution buffer (PBS containing 0.05% Tween-20 and 0.5% bovine serum albumin) to keep the final concentration at 30-1.5 ng/mL.
  • the above pharmacokinetic experiments show that the antibody of the present invention has the pharmacokinetic characteristics of general antibodies in the body of rats, has good stability and druggability, and is suitable for making medicines.
  • the present invention constructs TIGIT/CTLA4 bispecific antibodies with three structures as shown in Figure 14, wherein the anti-TIGIT antibody variable region part of each bispecific antibody is derived from 6F6-D63S, and the anti-CTLA4 antibody variable region part is derived from In Ipilimumab (Ipilimumab).
  • the bispecific antibody constant region is in the IgG1 (DLE) format.
  • the heavy chain of the bispecific antibody THC4 (named HTC) from the N-terminal to the C-terminal sequence is the heavy chain of the anti-TIGIT antibody Variable region VHH, anti-CTLA4 antibody heavy chain variable region VH, IgG1 constant region CH1, and IgG1 Fc constant region, wherein the Fc constant region is mutated (S239D, A330L, I332E) referred to as "DLE” (reference "Engineered antibody Fc variants with enhanced effector function.Proc Natl Acad Sci USA.2006 Mar 14;103(11):4005-10.”), the amino acid sequence of heavy chain HTC is SEC ID NO:69.
  • the light chain of the bispecific antibody THC4 (named ipilimumab LC) from the N-terminal to the C-terminal sequence is the anti-CTLA4 antibody light chain antibody variable region VL and light chain constant region CL, and its amino acid sequence is SEC ID NO: 68.
  • the order of the heavy chain 1 (named CK) of the bispecific antibody CT1KH from the N-terminal to the C-terminal is the heavy chain variable region VH of the anti-CTLA4 antibody, the IgG1 constant region CH1, and the IgG1 Fc constant region, wherein the Fc constant region is subjected to a point mutation ( S239D, A330L, I332E, T366W, S354C) form ADCC-enhanced IgG1 "knob” chain (reference "Engineered antibody Fc variants with enhanced effector function.Proc Nat1Acad Sci USA.2006 Mar 14; 103(11): 4005 -10 " and Merchant, A.M., et al. (1998).
  • 2TH amino acid sequence is SEQ ID NO: 74.
  • the amino acid sequence of the light chain of the bispecific antibody CT2KH (named ipilimumab LC) is SEQ ID NO: 68.
  • Each bispecific antibody expression vector was constructed as follows:
  • HTC Using the 6F6-DS-DLE plasmid as a template to amplify the 6F6-D63S VHH fragment, using ipilimumab-HC as a template to amplify ipilimumab VH-CH1, using overlapping PCR technology to splicing the above two sequences through the adapter GGS and recombining them in On the expression vector containing Fc (Fc contains S239D, A330L, and I332E point mutations), an expression vector for the complete heavy chain of HTC is formed.
  • Fc contains S239D, A330L, and I332E point mutations
  • CK Use ipilimumab-HC as a template to amplify ipilimumab VHCH1, use ipilimumab-H IgG1wt Rknob (the constant region contains T366W, S354C point mutations to form a knob structure) as a template to amplify IgG1 Fc constant region, and carry out "Fc constant region” DLE" point mutations (S239D, A330L, I332E), and the above fragments were recombined into the expression vector to form a heavy chain CK expression vector.
  • TH and 2TH expression vectors were synthesized by General Biology (Anhui) Co., Ltd.
  • AHC (IgG Fc capture) sensor tips (ForteBio, Cat. No. 18-5060) were pre-equilibrated in PBST (PBS, 0.02% Tween20, pH 7.0) for 10 minutes at room temperature.
  • Kinetic experiments were carried out in 96-well plates as follows: a) equilibrate baseline in PBST for 180 seconds, b) load 5ug/mL anti-TIGIT/CTLA-4 bispecific antibodies (THC4, CT2KH and CT1KH) and The control monoclonal antibody (6F6-H1 and Ipilimumab) was stopped for 200s, c) the baseline was balanced for 300 seconds, d) with His-tagged human TIGIT (SEC ID NO:) or human CTLA-4 (Sino Biological, Cat. No. 11159-H08H) Association at a concentration of 100 nM for 200 s, and e) dissociation in PBST for 600 s, respectively.
  • a 1:1 local fitting model was fitted to
  • Table 12 summarizes the binding affinities of anti-TIGIT/CTLA-4 bispecific antibodies (THC4, CT2KH and CT1KH) and control monoclonal antibodies (6F6-H1 and Ipilimumab analog) to human TIGIT protein and human CTLA-4 protein.
  • THC4, CT2KH, and CT1KH could specifically bind to human TIGIT and CTLA-4, and there was no significant change in the binding activity of TIGIT and CTLA4 compared with monoclonal antibodies.
  • Example 21 Anti-TIGIT/CTLA-4 bispecific antibody inhibits the binding activity of human TIGIT protein to human CD155 protein
  • High-binding clear polystyrene 96-well plates were coated with 0.5 ⁇ g/mL human TIGIT(M22-P141 )-Fc (SEQ ID NO: 45) in phosphate buffered saline PBS at 50 ⁇ L/well incubated overnight at 4°C. Plates were then washed once on an automatic plate washer using PBST solution (0.05% Tween 20 in PBS). Add 200 ⁇ L of blocking buffer (PBS solution containing 1% BSA) to each well and incubate at 37° C. for 1 hour.
  • PBST solution 0.05% Tween 20 in PBS
  • Example 22 Anti-TIGIT/CTLA-4 bispecific antibody inhibits the binding activity of human CTLA-4 protein to human CD80 protein
  • High-binding clear polystyrene 96-well plates were coated with 2 ⁇ g/mL human CTLA-4-his (Acro Biosystem, Cat. No. CT4-H5229) in phosphate-buffered saline PBS at 50 ⁇ L/well incubated overnight at 4°C. Plates were then washed once on an automatic plate washer using wash buffer PBST (0.05% Tween 20 in PBS). Add 200 ⁇ L of blocking buffer (PBS solution containing 1% BSA) to each well and incubate at room temperature for 1 hour.
  • PBST 0.05% Tween 20 in PBS
  • Ipilimumab analog, THC4, CT2KH and CT1KH could block the binding of CTLA-4 and CD80.
  • Example 23 Binding activity of anti-TIGIT/CTLA-4 bispecific antibody to human TIGIT cells

Abstract

L'invention concerne un anticorps monoclonal qui se lie de manière spécifique à TIGIT, et un anticorps bispécifique construit sur la base de l'anticorps monoclonal anti-TIGIT. L'invention concerne également une molécule d'acide nucléique codant pour ledit anticorps, un vecteur d'expression utilisé pour exprimer l'anticorps, une cellule hôte et un procédé de préparation. L'invention concerne en outre un procédé d'utilisation dudit anticorps pour traiter une maladie.
PCT/CN2022/143967 2021-12-31 2022-12-30 Anticorps à domaine unique anti-tigit et anticorps bispécifique basé sur celui-ci WO2023125941A1 (fr)

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