WO2023125941A1 - Tigit single-domain antibody and bispecific antibody based thereon - Google Patents

Tigit single-domain antibody and bispecific antibody based thereon 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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French (fr)
Chinese (zh)
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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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Publication of WO2023125941A1 publication Critical patent/WO2023125941A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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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    • Y02A50/30—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

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Abstract

Provided are a monoclonal antibody that specifically binds to TIGIT, and a bispecific antibody constructed on the basis of the TIGIT monoclonal antibody. Also provided are a nucleic acid molecule encoding said antibody, an expression vector used for expressing the antibody, a host cell, and a preparation method. Also provided is a method for using said antibody to treat disease.

Description

TIGIT单域抗体以及基于其的双特异性抗体TIGIT single domain antibody and bispecific antibody based on it
本发明提供了一种特异性结合TIGIT的单克隆抗体以及基于该TIGIT单克隆抗体构建的双特异性抗体。还提供了编码所述抗体的核酸分子、用于表达所述抗体的表达载体、宿主细胞和制备方法。本发明还提供了使用本发明的抗体的治疗方法。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.
背景技术Background technique
TIGIT(具有Ig及ITIM结构域的T细胞免疫受体T-cell immunoreceptor with Ig and ITIM domains)属于免疫球蛋白超家族成员，又称为VSTM3、WUCAM或VSIG9，是由一个细胞外免疫球蛋白V样结构域(IgV结构域)、1个I型跨膜结构域和1个基于酪氨酸的免疫受体抑制性基序(ITIM)及免疫球蛋白酪氨酸尾部(ITT)样基序组成的(Jinah Yeo，Minkyung Ko，et al.(2021).″TIGIT/CD226 Axis Regulates Anti-Tumor Immunity″Pharmaceuticals 14(3)：200.)。TIGIT主要表达于效应CD4+T细胞、滤泡辅助CD4+T细胞、调节性T细胞(Treg)、效应CD8+T以及NK细胞，已成为癌症免疫治疗的热门靶点。TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is a member of the immunoglobulin superfamily, also known as VSTM3, WUCAM or VSIG9, is composed of an extracellular immunoglobulin V IgV-like domain (IgV domain), a type I transmembrane domain, a tyrosine-based immunoreceptor inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT)-like motif (Jinah Yeo, Minkyung Ko, et al. (2021). "TIGIT/CD226 Axis Regulates Anti-Tumor Immunity" Pharmaceuticals 14(3):200.). 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.
目前已经发现TIGIT有多个配体，包括：PVR(Necl-5或CD155)、Nectin2(CD112)、Nectin3(CD113)和Nectin4(PVRL4)，但是TIGIT与CD155的相互作用最强，已有报道亲和力约1nM，与Nectin2和3亲和力非常低，与Nectin4的亲和力与CD155的亲和力接近(Reches A.，Ophir Y.，et al.(2020).″Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity″J.Immunother.Cancer.8.)。这些配体主要在APC细胞和多种恶性肿瘤细胞上(比如结直肠癌、黑色素瘤等)过表达。CD155通过与TIGIT、CD226、CD96相互作用发挥免疫调节作用，由于CD155对TIGIT的亲和力远远高于CD226和CD96，因此TIGIT与CD155会优先结合，激活TIGIT的细胞质尾部中存在的两个基序所介导的抑制性信号：基于酪氨酸的免疫受体抑制性基序(ITIM)及免疫球蛋白酪氨酸尾部(ITT)样基序。肿瘤细胞表面配体通过与NK细胞和T细胞表面TIGIT结合，抑制NK细胞毒性和T细胞活性，从而介导肿瘤细胞的免疫逃逸机制。Karsten Mahnke et al表明由于这条抑制信号通路存在于经典的PD1/PD-L1共抑制途径之外，因此，双特异性抗体对两种信号通路的阻断会导致黑色素瘤特异性细胞毒性T细胞的效应功能大大增强。PD1/PD-L1信号通路轴在黑色素瘤中已被确认，以TIGIT/CD155相互作用为特征的第二种抑制途径在黑色素中也是存在的(Karsten Mahnke and Alexander H.Enk(2015).″TIGIT-CD155Interactions in Melanoma：A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention″Journal of Investigative Dermatology 136(1)：9-11.)。It has been found that 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."). These ligands are mainly overexpressed on APC cells and various malignant tumor cells (such as colorectal cancer, melanoma, etc.). 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. 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.).
鉴于TIGIT的抑制信号通路在不同的免疫细胞亚群中发挥强大的抑制作用，并且其配体CD155在多种实体瘤中广泛表达，因此靶向TIGIT是非常有前景的治疗策略。Given that 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.
由于TIGIT既高表达于T细胞表面又高表达于NK细胞表面，而其他免疫检查点如：PD1仅表达于T细胞表面，这就决定了TIGIT作为治疗靶点具有更大的优势，因此本领域存在基于TIGIT抗体开发双特异性抗体药物的需要。Since 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.
发明内容Contents of the invention
本发明提供了一种抗TIGIT单域抗体(sdAb，single domain antibody)，以及应用其构建的双特异性抗体。在一些实施方案中，本发明的抗TIGIT单域抗体与人TIGIT的亲和力很高，能够识别人和食蟹猴TIGIT。The invention provides an anti-TIGIT single domain antibody (sdAb, single domain antibody) and a bispecific antibody constructed using the same. In some embodiments, the anti-TIGIT single domain antibody of the present invention has high affinity to human TIGIT and can recognize human and cynomolgus TIGIT.
在一些实施方案中，抗TIGIT单域抗体可以有效阻断TIGIT与PVR蛋白结合，阻断活性显著优于对照抗体Tiragolumab。In some embodiments, 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.
本发明TIGIT单域抗体也可以有效激活T细胞释放细胞因子。The TIGIT single domain antibody of the present invention can also effectively activate T cells to release cytokines.
本发明中的抗PD1/TIGIT双特异性抗体对TIGIT/CD155和PD1/PD-L1均具有阻断活性，在PD1/PD-L1以及TIGIT/CD155均存在的适应症中具有较光明的治疗前景。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 .
本发明的抗TIGIT/抗CTLA4双特异性抗体具有以下活性/功能中的一种或多种：第一，能够很好地与TIGIT和CTLA4结合，并且与TIGIT亲和力比CTLA4亲和力高(例如高一个数量级)，进而导致抗TIGIT和抗CTLA4双功能抗体能够定位至肿瘤部位，减少双抗在外周系统的停留，从而降低CTLA4抗体端的外周毒性，提高CTLA4抗体的用药剂量；第二，TIGIT和CTLA4在肿瘤内的Treg细胞上较高表达，本发明的双功能抗体可以通过Fc效应清除肿瘤内免疫抑制性的Treg细胞；第三，本发明的双功能抗体能够特异地解除效应T细胞上TIGIT和CTLA4的免疫抑制，激活T细胞，从而发挥抑制肿瘤作用，具有良好的应用前景。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.
在一个方面，本发明涉及以下具体实施方案：In one aspect, the invention relates to the following specific embodiments:
1、特异性结合TIGIT的VHH抗体，其包含1. A VHH antibody specifically binding to TIGIT, comprising
SEQ ID NO：1、6、9、14、16、18和21中任一项所示的VH中所含的三个互补决定区域(CDR)，The three complementarity determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21,
优选地，所述CDR序列根据IMGT定义。Preferably, said CDR sequences are defined according to IMGT.
2、实施方案1的VHH抗体，其包含互补决定区域(CDR)VHH CDR1、VHH CDR2和VHH CDR3，其中2. The VHH antibody of embodiment 1, comprising the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein
(i)VHH CDR1包含SEQ ID NO：3所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(i) 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
(ii)VHH CDR1包含SEQ ID NO：8所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(ii) 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, and VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
(iii)VHH CDR1包含SEQ ID NO：11所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：12所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：13所示的氨基酸序列或由其组成。(iii) 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.
3、实施方案1的VHH抗体，其包含重链可变区或由其组成，所述重链可变区3. The VHH antibody of embodiment 1, comprising or consisting of a heavy chain variable region, said heavy chain variable region
(i)包含与选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 90%, 91%, 92%, 93%, 94%, Amino acid sequences that are 95%, 96%, 97%, 98% or 99% identical or consist of; or
(ii)包含选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列或由其组成；或者(ii) comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21; or
(iii)包含与选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优 选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。(iii) 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.
4、特异性结合TIGIT的重链抗体，其包含实施方案1-3中任一项所述的VHH抗体。4. A heavy chain antibody specifically binding to TIGIT, comprising the VHH antibody of any one of embodiments 1-3.
5、实施方案4的重链抗体，其包含与抗体恒定区或Fc区连接的实施方案1-3中任一项所述的VHH抗体，优选地，所述抗体恒定区或Fc区来自人IgG1、人IgG2、人IgG3或人IgG4，任选地，所述VHH抗体与所述Fc区通过铰链区或其部分连接，优选地，所述铰链区部分的氨基酸序列为EPKSS(SEQ ID NO：43)。5. 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 ).
6、实施方案4的重链抗体，其包含与抗体Fc区连接的实施方案1-3中任一项所述的VHH抗体，其中所述Fc区为来自人IgG1或IgG4的Fc区，优选地，所述Fc区6. 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
(i)包含与SEQ ID NO：40或42中所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more of the amino acid sequence shown in SEQ ID NO: 40 or 42 99% identical amino acid sequences or consisting thereof; or
(ii)包含SEQ ID NO：40或42所示的氨基酸序列或由其组成；或者(ii) comprise or consist of the amino acid sequence shown in SEQ ID NO: 40 or 42; or
(iii)包含与SEQ ID NO：40或42所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列。(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) 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.
7、实施方案5或6的重链抗体，其中所述Fc区包含改善Fc区效应子功能的突变，例如提高ADCC的突变，优选地，所述突变为如下突变组合：S239D、A330L和I332E(EU编号)，优选地，其包含与SEQ ID NO：41所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成，且包含如下突变组合：S239D、A330L和I332E(EU编号)。7. The heavy chain antibody according to embodiment 5 or 6, wherein the Fc region comprises a mutation that improves the effector function of the Fc region, such as a mutation that increases ADCC, preferably, 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 An amino acid sequence of % or 99% identity or consisting thereof, and comprising the following combination of mutations: S239D, A330L and I332E (EU numbering).
8、实施方案4的重链抗体，其8. The heavy chain antibody of embodiment 4, which
(i)包含与选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist thereof; or
(ii)包含选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列或由其组成；或者(ii) comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 2, 7, 10, 15, 17, 19, 20 or 22; or
(iii)包含与选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。(iii) 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.
9、实施方案1-3中任一项的VHH抗体，或实施方案4-8中任一项的重链抗体，其中所述抗体是嵌合抗体或人源化抗体。9. The VHH antibody of any one of embodiments 1-3, or the heavy chain antibody of any one of embodiments 4-8, wherein said antibody is a chimeric antibody or a humanized antibody.
10、双特异性抗体，其包含第一抗原结合区和第二抗原结合区，其中所述第一抗原结合区特异性结合TIGIT，且包含实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体。10. 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.
11、实施方案10的双特异性抗体，其中第二抗原结合区特异性结合PD-1、PD-L1或PD-L2或CTLA-4，优选地，所述第二抗原结合区特异性结合PD-1，其包含来自WO2019219064A的PD1抗体或其抗原结合片段，所述抗原结合片段例如所述抗PD-1抗体 的单链Fv、Fab、Fab′、(Fab)2、单域抗体、VHH或重链抗体；优选地，所述第二抗原结合区特异性结合CTLA-4，其包含来自Ipilimumab抗体或其抗原结合片段，所述抗原结合片段例如所述抗CTLA-4抗体的单链Fv、Fab、Fab′、(Fab)2、单域抗体、VHH或重链抗体。11. The bispecific antibody of embodiment 10, wherein 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.
12、实施方案10或11的双特异性抗体，其中VHH抗体连接到第二抗原结合区Fc片段的C末端，或连接在第二抗原结合区重链VH片段的N末端，或者可以插入第二抗原结合区的Fab片段(Fab片段重链)和Fc片段之间，即与Fab片段重链的C末端和Fc片段的N末端连接，任选地，第一和第二抗原结合区通过接头连接，优选地，接头包含(GGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。12. The bispecific antibody according to embodiment 10 or 11, wherein the VHH antibody is linked to the C-terminus of the Fc fragment of the second antigen-binding region, or to the N-terminus of the heavy chain VH fragment of the second antigen-binding region, or can be inserted into the second between the Fab fragment (Fab fragment heavy chain) of the antigen-binding region and the Fc fragment, that is, the C-terminal of the Fab fragment heavy chain and the N-terminal of the Fc fragment, optionally, the first and second antigen-binding regions are connected through a linker , preferably, the linker comprises (GGS)n amino acid sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
13、实施方案12的双特异性抗体，其中所述双特异性抗体具有以下结构：13. The bispecific antibody of embodiment 12, wherein said bispecific antibody has the following structure:
重链：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc-抗TIGIT VHH；或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
从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-抗TIGIT VHH-重链恒定区Fc；或From N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-anti-TIGIT VHH-heavy chain constant region Fc of the second antigen antibody; or
从N端至C端，抗TIGIT VHH-第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL；Light chain: from N-terminal to C-terminal, the light chain variable region of the second antigen antibody-light chain constant region CL;
优选地，所述第二抗原选自PD-1或CTLA-4。Preferably, the second antigen is selected from PD-1 or CTLA-4.
14、实施方案10或11的双特异性抗体，其中所述双特异性抗体具有以下结构：14. The bispecific antibody of embodiment 10 or 11, wherein said bispecific antibody has the following structure:
重链1：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
重链2：1个或多个(例如2个)串联的抗TIGIT VHH-重链恒定区FcHeavy chain 2: 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL；Light chain: from N-terminal to C-terminal, the light chain variable region of the second antigen antibody-light chain constant region CL;
优选地，所述第二抗原选自PD-1或CTLA-4，例如CTLA-4。Preferably, the second antigen is selected from PD-1 or CTLA-4, such as CTLA-4.
15、实施方案14的双特异性抗体，其中所述抗TIGIT-VHH通过连接肽与重链恒定区Fc的N末端连接，例如所述连接肽是来自人IgG1、2、3或4的铰链区或其部分，包括天然或突变的铰链区或其部分，例如来自人IgG1铰链区，例如所述连接肽是EPKSS(SEQ ID NO：43)。15. The bispecific antibody of embodiment 14, wherein said anti-TIGIT-VHH is linked to the N-terminus of the heavy chain constant region Fc via a linker peptide, for example said 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).
16、实施方案14或15的双特异性抗体，其中当重链2包含多个串联的抗TIGIT单域抗体VHH时，各个串联的VHH之间可以通过接头连接，优选地，接头包含(GGGGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。16. The bispecific antibody according to embodiment 14 or 15, wherein when the heavy chain 2 comprises multiple tandem anti-TIGIT single domain antibody VHHs, each tandem VHH can be connected by a linker, preferably, the linker comprises (GGGGS) n amino acid sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
17、实施方案10-16中任一项的双特异性抗体，其中第二抗原抗体的重链恒定区CH1来自IgG，例如IgG1、IgG2、IgG3或IgG4；优选的，所述重链恒定区CH1来自IgG1或IgG4，更优选地，所述重链恒定区CH1包含SEQ ID NO：28或31所述的氨基酸序列或由其组成。17. The bispecific antibody according to any one of embodiments 10-16, wherein 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.
18、实施方案10-17中任一项的双特异性抗体，其中第二重链恒定区Fc如实施方案5-7中任一项所定义。18. The bispecific antibody according to any one of embodiments 10-17, wherein the second heavy chain constant region Fc is as defined in any one of embodiments 5-7.
19、实施方案14的双特异性抗体，其中重链1的Fc区与重链2的Fc区不同。19. The bispecific antibody of embodiment 14, wherein the Fc region of heavy chain 1 is different from the Fc region of heavy chain 2.
20、实施方案19的双特异性抗体，其中重链1的Fc区与重链2的Fc区中分别引入结(knob)突变和扣(Hole)突变。20. The bispecific antibody according to embodiment 19, wherein the Fc region of heavy chain 1 and the Fc region of heavy chain 2 are respectively introduced with a knob mutation and a button mutation.
21、实施方案21的双特异性抗体，其中21. The bispecific antibody of embodiment 21, wherein
第一Fc区包含结突变，其The first Fc region contains a junction mutation, which
(i)包含氨基酸序列SEQ ID NO：78或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成；或or
(ii)包含与SEQ ID NO：78具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含S239D、A330L和I332E突变和结突变(例如S354C和T366W)；且(ii) comprising an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity to SEQ ID NO: 78 and comprising S239D, A330L and I332E mutations and knot mutations ( such as S354C and T366W); and
第二Fc区包含扣突变，其The second Fc region contains a button mutation, which
(i)包含氨基酸序列SEQ ID NO：77与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成；或(i) 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;
(ii)包含与SEQ ID NO：77具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含S239D、A330L和I332E突变和扣突变。(ii) comprising an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity to SEQ ID NO: 77 and comprising S239D, A330L and I332E mutations and buckle mutations.
22、实施方案13的双特异性抗体，其包含22. The bispecific antibody of embodiment 13, comprising
重链：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc-抗TIGIT VHH；且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; and
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中所述第二抗原为PD-1；wherein the second antigen is PD-1;
其中重链heavy chain
(i)包含SEQ ID NO：30或33的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 30 or 33, or
(ii)包含与所述SEQ ID NO：30、32或33所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or
(iii)由SEQ ID NO：30或33所述的氨基酸组成；(iii) consist of amino acids described in SEQ ID NO: 30 or 33;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：39的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 39, or
(ii)包含与所述SEQ ID NO：39所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 39 the amino acid sequence of
(iii)由SEQ ID NO：39所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:39.
23、实施方案13的双特异性抗体，其包含23. The bispecific antibody of embodiment 13, comprising
重链：Heavy chain:
从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-抗TIGIT VHH-重链恒定区FcFrom N-terminal to C-terminal, the heavy chain variable region of the second antigen antibody VH-heavy chain constant region CH1-anti-TIGIT VHH-heavy chain constant region Fc
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中所述第二抗原为PD-1；wherein the second antigen is PD-1;
其中重链heavy chain
(i)包含SEQ ID NO：32的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 32, or
(ii)包含与所述SEQ ID NO：32所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 32 the amino acid sequence of
(iii)由SEQ ID NO：32所述的氨基酸组成；(iii) consist of the amino acid described in SEQ ID NO: 32;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：39的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 39, or
(ii)包含与所述SEQ ID NO：39所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 39 the amino acid sequence of
(iii)由SEQ ID NO：39所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:39.
24、实施方案13的双特异性抗体，其包含24. The bispecific antibody of embodiment 13, comprising
重链：从N端至C端，抗TIGIT VHH-第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中第二抗原为CTLA-4，Wherein the second antigen is CTLA-4,
其中重链heavy chain
(i)包含SEQ ID NO：69的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 69, or
(ii)包含与所述SEQ ID NO：69所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 69 the amino acid sequence of
(iii)由SEQ ID NO：69所述的氨基酸组成；(iii) consist of the amino acid described in SEQ ID NO: 69;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：68的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 68, or
(ii)包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 68 the amino acid sequence of
(iii)由SEQ ID NO：68所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:68.
25、实施方案14的双特异性抗体，其包含25. The bispecific antibody of embodiment 14, comprising
重链1：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
重链2：1个或多个(例如2个)串联的抗TIGIT VHH-重链恒定区FcHeavy chain 2: 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中重链1of which heavy chain 1
(i)包含SEQ ID NO：71的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 71, or
(ii)包含与所述SEQ ID NO：71所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO:71 the amino acid sequence of
(iii)由SEQ ID NO：71所述的氨基酸组成；(iii) consist of the amino acid described in SEQ ID NO: 71;
和/或and / or
重链2 heavy chain 2
(i)包含SEQ ID NO：72或74的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 72 or 74, or
(ii)包含与所述SEQ ID NO：72或74所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence described in said SEQ ID NO: 72 or 74 the amino acid sequence of identity, or
(iii)由SEQ ID NO：72或74所述的氨基酸组成；(iii) consist of amino acids described in SEQ ID NO: 72 or 74;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：68的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 68, or
(ii)包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 68 the amino acid sequence of
(iii)由SEQ ID NO：68所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:68.
26、核酸分子，其编码实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体，或实施方案10-25中任一项的双特异性抗体中的重链和/或轻链，或由所述核酸序列组成。26. 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.
27、表达载体，其包含实施方案26的核酸分子，优选地，所述表达载体为pCDNA，例如pCDNA3.1。27. An expression vector comprising the nucleic acid molecule according to embodiment 26, preferably, the expression vector is pCDNA, such as pCDNA3.1.
28、宿主细胞，其包含实施方案26所述的核酸分子或实施方案27所述的表达载体，优选地，所述宿主细胞是原核的或真核的，例如293细胞或CHO细胞，例如293FT细胞或CHO-S细胞。28. 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.
29、制备实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体，或实施方案10-25中任一项的双特异性抗体的方法，所述方法包括，在适合所述VHH抗体或重链抗体或双特异性抗体的链表达的条件下，培养实施方案包含编码VHH抗体或重链抗体的核酸、或编码双特异性抗体的各条链的核酸或包含所述核酸的表达载体的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述VHH抗体或重链抗体或双特异性抗体。29. 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 , said method 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 A host cell of the nucleic acid of the chain or an expression vector comprising the nucleic acid, and optionally recovering the VHH antibody or heavy chain antibody or bispecific antibody from the host cell (or host cell culture medium).
30、免疫缀合物，其包含实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体，或实施方案10-25中任一项的双特异性抗体。30. 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.
31、药物组合物或药物或制剂，其包含实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体，或实施方案10-25中任一项的双特异性抗体，或实施方案30的免疫缀合物以及任选地药用辅料。31. 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.
32、药物组合产品，其包含实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体，或实施方案10-25中任一项的双特异性抗体，或实施方案30的免疫缀合物，以及其它治疗剂。32. 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.
33、在受试者中预防或治疗癌症的方法，包括向受试者施用有效量的实施方案1-3和9中任一项的VHH抗体，或实施方案4-9中任一项的重链抗体，或实施方案10-25中任一项的双特异性抗体，或实施方案30的免疫缀合物，或实施方案31的药物组合物或制剂；或实施方案32的药物组合产品。33. 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.
34、实施方案33的方法，其中所述癌症是表征为具有升高的PD-1、PD-L1或PD-L2的蛋白质水平和/或核酸水平(例如表达升高)和/或具有升高的TIGIT的的蛋白质水平和/或核酸水平(例如表达升高)的癌症。34. The method of embodiment 33, wherein the cancer is characterized by having elevated protein levels and/or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, or PD-L2 and/or having elevated Cancers at the protein level and/or nucleic acid level (eg, elevated expression) of TIGIT.
35、实施方案34的方法，其中所述方法还包括与其它疗法例如治疗方式和/或其它治疗剂组合施用。35. The method of embodiment 34, wherein said method further comprises administration in combination with other therapies such as treatment modalities and/or other therapeutic agents.
附图说明Description of drawings
图1显示抗人TIGIT的重链抗体阻断人TIGIT蛋白与CD155结合(EC50，nM)。Figure 1 shows that anti-human TIGIT heavy chain antibody blocks the binding of human TIGIT protein to CD155 (EC50, nM).
图2显示抗人TIGIT的重链抗体与HEK293-人TIGIT细胞的结合活性(EC50，nM)。Fig. 2 shows the binding activity (EC50, nM) of heavy chain antibody against human TIGIT to HEK293-human TIGIT cells.
图3显示抗人TIGIT的重链抗体与HEK293-食蟹猴TIGIT细胞的结合活性(EC50，nM)。Fig. 3 shows the binding activity (EC50, nM) of heavy chain antibody against human TIGIT to HEK293-cynomolgus monkey TIGIT cells.
图4显示抗人TIGIT的重链抗体激活CD8+T细胞释放IFNγ因子。Figure 4 shows that anti-human TIGIT heavy chain antibody activates CD8+ T cells to release IFNγ factor.
图5显示序列优化及Fc改造后的抗人TIGIT的重链抗体阻断人TIGIT蛋白与CD155结合(IC50，nM)。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).
图6显示本发明抗PD1/TIGIT双特异性抗体的结构示意图，图6A为E4示意图，图6B为D1和D4的示意图。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, and Fig. 6B is a schematic diagram of D1 and D4.
图7显示抗PD1/TIGIT双特异性抗体与人TIGIT蛋白结合活性(EC50，nM)。Figure 7 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to human TIGIT protein.
图8显示抗PD1/TIGIT双特异性抗体与人PD1蛋白结合活性(EC50，nM)。Figure 8 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to human PD1 protein.
图9显示抗PD1/TIGIT双特异性抗体阻断人TIGIT蛋白与CD155结合的活性(IC50，nM)。Figure 9 shows the activity of anti-PD1/TIGIT bispecific antibody blocking the binding of human TIGIT protein to CD155 (IC50, nM).
图10显示抗PD1/TIGIT双特异性抗体阻断人PD1蛋白与PD-L1结合的活性(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).
图11显示抗PD1/TIGIT双特异性抗体与HEK293-人TIGIT细胞的结合活性(EC50，nM)。Figure 11 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to HEK293-human TIGIT cells.
图12显示抗PD1/TIGIT双特异性抗体与Jurkat-NFAT-人PD1细胞的结合活性(EC50，nM)。Figure 12 shows the binding activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody to Jurkat-NFAT-human PD1 cells.
图13显示抗PD1/TIGIT双特异性抗体对激活后CD4+T或CD8+T细胞的ADCC杀伤活性(EC50，nM)。Figure 13 shows the ADCC killing activity (EC50, nM) of anti-PD1/TIGIT bispecific antibody on activated CD4+T or CD8+T cells.
图14显示本发明抗TIGIT/CTLA-4双特异性抗体的结构示意图，图14A为THC4示意图，图14B为CT1KH示意图，图14C为CT2KH示意图。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, and Figure 14C is a schematic diagram of CT2KH.
图15显示抗TIGIT/CTLA-4双特异性抗体阻断人TIGIT蛋白与CD155结合的活性。Figure 15 shows the activity of anti-TIGIT/CTLA-4 bispecific antibody blocking the binding of human TIGIT protein to CD155.
图16显示抗TIGIT/CTLA-4双特异性抗体阻断人CTLA-4蛋白与CD80结合的活性。Figure 16 shows the activity of anti-TIGIT/CTLA-4 bispecific antibody blocking the binding of human CTLA-4 protein to CD80.
图17显示抗TIGIT/CTLA-4双特异性抗体与HEK293-人TIGIT细胞的结合活性Figure 17 shows the binding activity of anti-TIGIT/CTLA-4 bispecific antibody to HEK293-human TIGIT cells
发明详述Detailed description of the invention
一、结合TIGIT的单域抗体(VHH抗体)、重链抗体以及包含其的双特异性抗体1. TIGIT-binding single domain antibody (VHH antibody), heavy chain antibody and bispecific antibody containing it
本发明在第一个方面涉及一种结合TIGIT的抗体。在一些实施方案中，本发明的抗体或其抗原结合片段结合哺乳动物TIGIT，例如人TIGIT或食蟹猴TIGIT。In a first aspect the invention relates to an antibody that binds TIGIT. In some embodiments, an antibody or antigen-binding fragment thereof of the invention binds mammalian TIGIT, eg, human TIGIT or cynomolgus TIGIT.
Figure PCTCN2022143967-appb-000001
单域抗体
Figure PCTCN2022143967-appb-000001
single domain antibody
在一些实施方案中，本发明的抗TIGIT抗体是单域抗体，特别是VHH抗体。In some embodiments, the anti-TIGIT antibodies of the invention are single domain antibodies, particularly VHH antibodies.
单域抗体或VHH抗体具有人IgG分子大约十分之一的分子量，以及仅几纳米的物理直径。由于小的分子尺寸，单域单抗相比常规的四链抗体具有如下优势：高稳定性和溶解性，以及能够识别隐藏抗原性位点。此外，单域抗体在制备上也比常规的四链抗体更为便宜。除了作为单独分子应用外，单域抗体也是构建多特异性分子的适宜组件。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.
在一些实施方案中，本发明的单域抗体是包含重链可变区或由所述重链可变区组成的VHH抗体，所述重链可变区通常具有以下结构：FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4，其中FR1至FR4指构架区1至4；VHH CDR1至VHH CDR3指互补决定区1-3。VHH可变区中的CDR序列可以按照“定义”部分描述的任何CDR定义方案进行确定，优选可以通过IMGT来定义VHH序列中的三个CDR的边界。In some embodiments, 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.
在一些实施方案中，本发明的抗TIGIT的VHH抗体包含In some embodiments, the anti-TIGIT VHH antibodies of the invention comprise
(i)SEQ ID NO：1、6、9、14、16、18和21中任一项所示的VH中所含的三个互补决定区域(CDR)，或(i) 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
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；(ii) relative to the sequence of (i), a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions;
优选地，所述CDR序列根据IMGT定义。Preferably, said CDR sequences are defined according to IMGT.
在一些实施方案中，本发明的抗TIGIT的VHH抗体包含重链可变区或由其组成，所述重链可变区包含In some embodiments, an anti-TIGIT VHH antibody of the invention comprises or consists of a heavy chain variable region comprising
(i)SEQ ID NO：1、6、9、14、16、18和21中任一项所示的VH中所含的三个互补决定区域(CDR)，或(i) 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
(ii)相对于(i)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列；(ii) relative to the sequence of (i), a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions;
优选地，所述CDR序列根据IMGT定义。Preferably, said CDR sequences are defined according to IMGT.
在一些实施方案中，本发明的抗TIGIT的VHH抗体包含互补决定区域(CDR)VHH CDR1、VHH CDR2和VHH CDR3。在一些实施方案中，本发明的抗TIGIT的VHH包含重链可变区或由其组成，所述重链可变区包含互补决定区域(CDR)VHH CDR1、VHH CDR2和VHH CDR3。In some embodiments, the anti-TIGIT VHH antibodies of the invention comprise the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, 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.
在一些实施方案中，VHH CDR1包含选自SEQ ID NO：3、8或11的氨基酸序列，或由所述氨基酸序列组成，或者VHH CDR1包含与选自SEQ ID NO：3、8或11的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, 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.
在一些实施方案中，VHH CDR2包含SEQ ID NO：4、12或23的氨基酸序列，或由所述氨基酸序列组成，或者VHH CDR2包含与SEQ ID NO：4、12或23的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, 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.
在一些实施方案中，VHH CDR3包含选自SEQ ID NO：5或13的氨基酸序列或由所述氨基酸序列组成，或者VHH CDR3包含与选自SEQ ID NO：5或13的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。In some embodiments, 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.
在一些实施方案中，本发明的抗TIGIT的VHH抗体包含重链可变区或由其组成，所述重链可变区包含In some embodiments, an anti-TIGIT VHH antibody of the invention comprises or consists of a heavy chain variable region comprising
互补决定区域(CDR)HCDR1、HCDR2和HCDR3，其中HCDR3包含选自SEQ ID NO：5或13的氨基酸序列或由所述氨基酸序列组成，或者HCDR3包含与选自SEQ ID NO：5或13的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。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) compared to the sequence.
在一些实施方案中，本发明的抗TIGIT的VHH抗体的VHH CDR2包含如下氨基酸序列或由所述氨基酸序列组成：In some embodiments, the VHH CDR2 of the anti-TIGIT VHH antibody of the present invention comprises or consists of the following amino acid sequence:
ITTSXSA，优选地，X选自D或S(SEQ ID NO：57)。ITTSXSA, preferably, X is selected from D or S (SEQ ID NO: 57).
在一个实施方案中，本发明的抗TIGIT的VHH抗体包含互补决定区域(CDR)VHH CDR1、VHH CDR2和VHH CDR3，其中In one embodiment, the anti-TIGIT VHH antibody of the present invention comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein
(i)VHH CDR1包含SEQ ID NO：3所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(i) 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
(ii)VHH CDR1包含SEQ ID NO：8所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(ii) 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, and VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
(iii)VHH CDR1包含SEQ ID NO：11所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：12所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：13所示的氨基酸序列或由其组成。(iii) 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.
在一个实施方案中，本发明的抗TIGIT VHH抗体包含重链可变区或由其组成，其中所述重链可变区包含互补决定区域(CDR)VHHCDR1、VHHCDR2和VHHCDR3，其中In one embodiment, 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
(i)VHH CDR1包含SEQ ID NO：3所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(i) 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
(ii)VHH CDR1包含SEQ ID NO：8所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(ii) 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, and VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
(iii)VHH CDR1包含SEQ ID NO：11所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：12所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：13所示的氨基酸序列或由其组成。(iii) 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.
在一些实施方案中，本发明的抗TIGIT的VHH抗体包含重链可变区或由其组成，所述重链可变区In some embodiments, the anti-TIGIT VHH antibody of the present invention comprises or consists of a heavy chain variable region, said heavy chain variable region
(i)包含与选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 90%, 91%, 92%, 93%, 94%, Amino acid sequences that are 95%, 96%, 97%, 98% or 99% identical or consist of; or
(ii)包含选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列或由其组成；或者(ii) comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21; or
(iii)包含与选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。(iii) 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.
在一些实施方案中，本发明的抗TIGIT的VHH抗体包含选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的VHH抗体包含源自通过免疫驼科动物(例如羊驼)产生的驼科重链抗体的CDR氨基酸序列和/或构架(FR)氨基酸序列。在一些实施方案中，源自驼科重链抗体的本发明VHH单抗可以进行工程化，例如，以包含源自人氨基酸序列(即，人抗体)或其他非驼科哺乳动物物种的构架区序列。在一个实施方案中，为进一步改善工程化抗体的性质(例如亲和力)，可以在工程化抗体中，在一个或多个位置(例如构架区)，通过回复突变，引入在亲本驼源抗体中位于相应位置的驼源氨基酸残基。In some embodiments, 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). In some embodiments, 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. In one embodiment, in order to further improve the properties (such as affinity) of the engineered antibody, in the engineered antibody, at one or more positions (such as the framework region), through back mutation, introduce the position located in the parent camel antibody Camel derived amino acid residues at corresponding positions.
在一个实施方案中，本发明的VHH抗体是嵌合抗体。In one embodiment, the VHH antibodies of the invention are chimeric antibodies.
在一个实施方案中，本发明的VHH抗体是人源化抗体。人源化可以通过如下方式实现：将非人源的天然VHH序列(例如来自驼科动物或羊驼免疫的VHH序列)中的一个或多个氨基酸残基，尤其是构架区序列，置换成来自人的常规抗体的重链VH相应位置的残基。人源化VHH的方法在本领域中是熟知的，例如实施例3中所述的方法。通常，人源化置换以保持单域抗体的有利结合性质的方式进行。本领域熟知用于确定人源化单域抗体的生物学性质，例如结合亲和力等的试验，以确定和选择适宜的人源化残基突变或突变组合。In one embodiment, 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. Typically, 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.
在一些实施方案中，可以通过包括如下步骤的方法，获得本发明人源化的单域抗体：In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:
①确定亲本单域抗体(例如来自噬菌体展示文库筛选的驼源VHH抗体)的CDR环结构；① Determine the CDR loop structure of the parental single-domain antibody (such as camel-derived VHH antibody from phage display library screening);
②在人种系序列数据库为每个V/J区域找到最接近的同源序列作为模板，例如通过比对IMGT人类抗体重链可变区种系基因数据库(http：//www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)，挑选与VHH抗体同源性高的重链可变区种系基因作为模板；② Find the closest homologous sequence for each V/J region in the human germline sequence database as a template, for example, by comparing the IMGT human antibody heavy chain variable region germline gene database (http://www.imgt.org /3Dstructure-DB/cgi/DomainGapAlign.cgi), select the heavy chain variable region germline gene with high homology with the VHH antibody as a template;
③将VHH抗体的CDR分别移植到挑选的相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CR3-FR4的可变区序列，优选，用于替代的构架序列，与待人源化抗体的构架序列，具有结构相似性，例如，具有的序列同一性至少为80％，85％，90％，或95％、96％、97％、98％、99％以上；③ Transplant the CDRs of the VHH antibody into the selected corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CR3-FR4, preferably, a framework sequence for replacement, and The framework sequence of the antibody to be humanized has structural similarity, for example, the sequence identity is at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more;
④根据需要，将FR区中关键氨基酸回复突变为纳米抗体(VHH抗体)对应的氨基酸，以保证原有的亲和力，即得到人源化抗TIGIT VHH抗体，任选地测序VHH抗体。④ As needed, 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.
在一些实施方案中，回复突变位点选自T28、L78、W103、R83、V37、G44、L45、W47中的一个或多个。在一些实施方案中，回复突变选自T28P；L78V；W103K；R83K；V37F；G44E；L45R；W47F中的一个或多个。在一些实施方案中，人源化VHH抗体中的回复突变的位点组合选自：In some embodiments, 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:
(1)T28、L78和W103；(1) T28, L78 and W103;
(2)L78、R83和W103；或(2) L78, R83 and W103; or
(3)V37、G44、I45和W47。(3) V37, G44, I45 and W47.
在一些实施方案中，人源化VHH抗体中的回复突变组合选自：In some embodiments, the combination of back mutations in the humanized VHH antibody is selected from:
(1)T28P、L78V和W103K(例如针对重链模板IGHJ4*01)；(1) T28P, L78V and W103K (eg for heavy chain template IGHJ4*01);
(2)L78V、R83K和W103K(例如针对重链模板IGHV3-48*01)；或(2) L78V, R83K and W103K (eg for heavy chain template IGHV3-48*01); or
(3)V37F、G44E、L45R和W47F(例如针对重链模板IGHV3-23*01)。(3) V37F, G44E, L45R and W47F (eg for heavy chain template IGHV3-23*01).
在一些实施方案中，适用于本发明VHH抗体人源化的重链可变区种系基因选自IGHJ4*01、IGHV3-48*01或IGHV3-23*01。In some embodiments, 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.
在一些实施方案中，本发明也提供本发明单域抗体(特别是VHH抗体)的功能性变体。所述功能性变体可以采用本发明熟知的方法，例如通过随机或定点诱变，将突变引入本发明的示例性单域抗体的编码核酸序列中，例如引入CDR序列和/或FR序列中，并随后筛选(例如通过噬菌体展示文库筛选)保持期望性质的变体，来获得功能性变体。通常，功能性变体保持与亲本单域抗体(或VHH)显著的序列的同一性。优选地，功能性变体保持亲本单域抗体(或VHH)的期望生物学性质，例如，相对于亲本的生物活性，变体具有相当(例如，至少50％，60％，70％，80％，优选地90％以上)的生物活性，或改善的生物活性(例如110-150％或更高)。所述的期望生物学性质包括例如，但不限于，目的抗原(例如CD155)结合亲和力(如通过KD值量度)，阻断目的抗原与受体结合的活性(例如通过IC50值量度)，在体外或体内实验中激活T细胞的活性(例如通过释放的细胞因子量度)，在体外或体内实验中抑制肿瘤生长/存活。In some embodiments, 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. Typically, functional variants retain significant sequence identity to the parental single domain antibody (or VHH). Preferably, 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.
在一些实施方案中，本发明提供本发明VHH多肽的亲和力变体。优选地，所述亲和 力变体表现出相对于其来源亲本单域抗体在氨基酸序列上的一个或多个氨基酸变化，其中亲和力变体相比亲本抗体具有改变的对目的抗原的结合亲和力。In some embodiments, the invention provides affinity variants of the VHH polypeptides of the invention. Preferably, 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.
在一些实施方案中，还可以针对抗体的稳定性进行工程化，例如为消除脱酰胺作用而突变抗体中的天冬酰胺。在一个实施实施方案中，本发明的VHH抗体的CDR2中包含D63S突变，以消除脱酰胺作用。In some embodiments, antibody stability can also be engineered, for example, by mutating the asparagine in the antibody to eliminate deamidation. In one embodiment, the VHH antibody of the invention comprises a D63S mutation in CDR2 to eliminate deamidation.
Figure PCTCN2022143967-appb-000002
重链抗体
Figure PCTCN2022143967-appb-000002
heavy chain antibody
在本发明的另一方面中，本发明也提供了包含本发明的VHH抗体重链可变区的重链抗体。In another aspect of the present invention, the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.
在一些实施方案中，可以将本发明的单域抗体或VHH(例如驼源VHH或其人源化形式)，与人抗体的恒定区或其一部分例如Fc区连接，以产生包含VHH-恒定区或VHH-CH1-Fc或VHH-Fc的重链抗体。在一个实施方案中，所述重链抗体包含本发明的VHH抗体和位于其C端的Fc区。在一些实施方案中，通过铰链区或其一部分，例如来自IgG的铰链区(例如IgG1、2、3或4的铰链区)或其部分连接VHH与Fc。In some embodiments, a single domain antibody or VHH of the invention (e.g., a camel VHH or a humanized form thereof) 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. Or heavy chain antibody of VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, 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.
在一些实施方案中，本发明的抗TIGIT重链抗体包含如本文定义的VHH或其中的重链可变区，以及重链恒定区或重链恒定区的Fc区。在一些实施方案中，在VHH或其重链可变区与重链恒定区或Fc区之间包含连接肽，例如抗体铰链区或其部分，例如来自IgG的铰链区或其部分(包括天然或突变的IgG铰链区或其部分)。In some embodiments, 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. In some embodiments, 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).
在一些实施方案中，所述连接肽是来自人IgG1、2、3或4的铰链区或其部分，包括天然或突变的铰链区或其部分，例如来自人IgG1铰链区，例如所述连接肽是EPKSS(SEQ ID NO：43)。In some embodiments, 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).
在一个实施方案中，所述重链抗体包含来自驼科动物(例如羊驼)的Fc部分。在一个实施方案中，通过免疫所述驼科动物例如羊驼产生并分离所述的重链抗体。本领域已知多种方式可用于免疫驼科动物和分离产生的针对目的抗原的VHH抗体或重链抗体。In one embodiment, the heavy chain antibody comprises an Fc portion from a camelid (eg, alpaca). In one embodiment, 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.
在一些实施方案中，所述重链抗体包含来自人或非人灵长类动物(例如食蟹猴)抗体的恒定区，例如来自人IgG1、人IgG2、人IgG3或人IgG4的恒定区。In some embodiments, 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.
在一些实施方案中，所述重链抗体包含来自人或非人灵长类动物(例如食蟹猴)的Fc部分。在再一实施方案中，所述重链抗体包含人IgG Fc区，例如人IgG1、人IgG2、人IgG3或人IgG4 Fc区，优选地人IgG1或人IgG4Fc区，如人IgG1 Fc区。In some embodiments, the heavy chain antibody comprises an Fc portion from a human or non-human primate (eg, cynomolgus monkey). In yet another embodiment, 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.
在一个实施方案中，根据本发明的重链抗体可以通过Fc区，与包含Fc区的另一多肽链(例如相同或不同的另一重链抗体)二聚化。因此，在一个实施方案中，本发明也提供了包含本发明重链抗体的同源或异源多聚体蛋白质。在一个优选的实施方案中，优选所述蛋白包括两条相同重链抗体链配对形成的重链抗体。In one embodiment, 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. Thus, in one embodiment, the invention also provides a homo- or heteromultimeric protein comprising a heavy chain antibody of the invention. In a preferred embodiment, it is preferred that the protein comprises a heavy chain antibody formed by the pairing of two identical heavy chain antibody chains.
可以将本发明的Fc区进行突变以获得所需的特性。本领域已知对Fc区的突变。在一个实施方案中，在Fc区的效应子功能的特性上修饰Fc区。在一个实施方案中，所述效应子功能相对于野生同种型Fc区已经被提高。在一个实施方案中，通过对Fc区进行突变来改善Fc区的效应子功能，例如ADCC，例如通过在以下一个或多个位点进行突变S239、A330和I332(根据EU编号)。在一个实施方案中，通过以下位点组合的突变来改善ADCC：S239、A330和I332(根据EU编号)。在一个实施方案中，所述突变选自 S239D、A330L和I332E中的1、2或3个。在一个实施方案中，所述改变效应子功能的突变为如下突变组合：S239D、A330L和I332E(参考文献“Engineered antibody Fc variants with enhanced effector function.Proc Natl Acad Sci USA.2006 Mar 14；103(11)：4005-10.”)。The Fc region of the invention can be mutated to obtain desired properties. Mutations to the Fc region are known in the art. In one embodiment, 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. In one embodiment, the effector function of the Fc region, eg 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. In one embodiment, ADCC is improved by mutation of the following combination of sites: S239, A330 and I332 (according to EU numbering). In one embodiment, the mutation is selected from 1, 2 or 3 of S239D, A330L and I332E. In one embodiment, 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.").
在一些实施方案中，所述Fc区是来自IgG1的Fc区，其含有突变S239D、A330L和I332E(根据EU编号)。In some embodiments, the Fc region is an Fc region from IgGl containing mutations S239D, A330L, and I332E (according to EU numbering).
在一些实施方案中，所述Fc区：In some embodiments, the Fc region:
(i)包含与SEQ ID NO：40、41或42中所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 40, 41 or 42 % or 99% identical to or consisting of amino acid sequences; or
(ii)包含SEQ ID NO：40、41或42所示的氨基酸序列或由其组成；或者(ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 40, 41 or 42; or
(iii)包含与SEQ ID NO：40、41或42所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列。(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) compared with the amino acid sequence shown in SEQ ID NO: 40, 41 or 42 Amino acid sequence with amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions).
在一些实施方案中，所述Fc区来自IgG1，其包含与SEQ ID NO：40或41所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，所述Fc区来自IgG1，其包含与SEQ ID NO：41所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成，且包含如下突变组合：S239D、A330L和I332E(EU编号)。In some embodiments, 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).
在一些实施方案中，所述Fc区来自IgG4，其包含与SEQ ID NO：42所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗体TIGIT抗体或其抗原结合片段包含重链，所述重链包含重链可变区、Fc区，以及连接重链可变区和Fc区的连接肽。优选地，连接肽包含SEQ ID NO：43所示的氨基酸序列，或由所述氨基酸序列组成。In some embodiments, 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. Preferably, the connecting peptide comprises the amino acid sequence shown in SEQ ID NO: 43, or consists of said amino acid sequence.
在一些实施方案中，本发明的抗TIGIT抗体或其抗原结合片段包含重链或由其组成，所述重链包含本发明VHH的重链可变区、连接肽和Fc区或由其组成，其中所述重链In some embodiments, 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
(i)包含与选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist thereof; or
(ii)包含选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列或由其组成；或者(ii) comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 2, 7, 10, 15, 17, 19, 20 or 22; or
(iii)包含与选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。(iii) 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.
Figure PCTCN2022143967-appb-000003
双特异性抗体或多特异性抗体
Figure PCTCN2022143967-appb-000003
bispecific antibody or multispecific antibody
在某些实施方案中，本发明还涵盖包含本发明的VHH或重链抗体或其片段的多特异性分子，例如双特异性抗体。多特异性抗体分子例如可以是三特异性抗体分子，其包含针对TIGIT的第一结合特异性和针对一种或多种的分子的第二及第三结合特异性。In certain embodiments, 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.
因此，本发明的另一个方面涉及一种双特异性抗体，其包含Therefore, another aspect of the invention relates to a bispecific antibody comprising
第一抗原结合区和第二抗原结合区，其中第一抗原结合区特异性结合TIGIT，第二抗原结合区特异性结合肿瘤相关抗原或免疫检查点分子，例如PD-1、PD-L1或PD-L2或CTLA4。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.
在一些实施方案中，第一抗原结合区包含本文所述的抗TIGTI VHH抗体或重链抗体，特别是VHH抗体。优选地，第一抗原结合区包含或由本发明的抗TIGIT VHH组成，更优选地所述VHH是人源化的VHH。In some embodiments, the first antigen binding region comprises an anti-TIGTI VHH antibody or heavy chain antibody, particularly a VHH antibody, described herein. Preferably, the first antigen binding region comprises or consists of an anti-TIGIT VHH of the invention, more preferably said VHH is a humanized VHH.
在一些实施方案中，第二抗原结合区包含来自WO2019219064A的PD1抗体或其抗原结合片段或结构域，例如包含WO2019219064A的PD1抗体的片段或结构域。适用于本发明的双特异性抗体的第二抗原结合区可以包含抗PD-1全长抗体或其抗原结合片段，或由其组成，只要其能够特异性结合PD-1即可，包括但不限于，例如特异性结合PD-1的全长抗体、单链Fv、Fab、Fab′、(Fab)2、单域抗体、VHH或重链抗体等。In some embodiments, 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.
在一些实施方案中，第二抗原结合区包含来自Ipilimumab抗体或其抗原结合片段或结构域，例如包含Ipilimumab抗体的片段或结构域。适用于本发明的双特异性抗体的第二抗原结合区可以包含抗CTLA-4全长抗体或其抗原结合片段，或由其组成，只要其能够特异性结合CTLA-4即可，包括但不限于，例如特异性结合CTLA-4的全长抗体、单链Fv、Fab、Fab′、(Fab)2、单域抗体、VHH或重链抗体等。In some embodiments, 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.
在根据本发明的双特异性抗体中，本发明的抗TIGIT VHH作为第一抗原结合区，可以连接在第二抗原结合区的N末端或C末端，例如连接在第二抗原结合区Fc片段的C末端，或连接在第二抗原结合区重链VH片段的N末端，或者可以插入第二抗原结合区的Fab片段(Fab片段重链)和Fc片段之间，即与Fab片段重链的C末端和Fc片段的N末端连接。在一些是实施方案中，第一和第二抗原结合区通过接头连接(例如在抗TIGIT VHH插入第二抗原抗体的Fab片段和Fc片段之间时)。在一个实施方案中，接头是大约3个至大约20个氨基酸长度的肽。优选地，接头包含(GGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。In the bispecific antibody according to the present invention, 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 C-terminal, or connected to the N-terminal of the heavy chain VH fragment of the second antigen-binding region, or can be inserted between the Fab fragment (Fab fragment heavy chain) and the Fc fragment of the second antigen-binding region, that is, with the C of the Fab fragment heavy chain The end is linked to the N-terminus of the Fc fragment. In some embodiments, 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). In one embodiment, the linker is a peptide of about 3 to about 20 amino acids in length. Preferably, the linker comprises (GGS)n amino acid sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
在一些实施方案中，本发明的双特异性抗体具有以下结构：In some embodiments, bispecific antibodies of the invention have the following structure:
重链：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc-抗TIGIT VHH；或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
从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-抗TIGIT VHH-重链恒定区Fc；或From N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-anti-TIGIT VHH-heavy chain constant region Fc of the second antigen antibody; or
从N端至C端，抗TIGIT VHH-第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL。Light chain: from N-terminal to C-terminal, light chain variable region-light chain constant region CL of the second antigen antibody.
在一些实施方案中，第二抗原是PD-1，例如人PD-1。在一些实施方案中，第二抗原 是CTLA-4，例如人CTLA-4。In some embodiments, the second antigen is PD-1, such as human PD-1. In some embodiments, the second antigen is CTLA-4, such as human CTLA-4.
在一些实施方案中，本发明的双特异性抗体具有两条重链和两条轻链，优选地相同的两条重链和两条轻链。In some embodiments, the bispecific antibodies of the invention have two heavy chains and two light chains, preferably the same two heavy chains and two light chains.
优选地，所述双特异性抗体的结构如图6A或图6B或图14A所示。Preferably, the structure of the bispecific antibody is as shown in Figure 6A or Figure 6B or Figure 14A.
在另一些实施方案中，抗TIGIT单域抗体VHH通过接头与重链恒定区Fc部分在N端或C端连接。在一个实施方案中，接头是大约3个至大约20个氨基酸长度的肽。优选地，接头包含(GGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。In some other embodiments, 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. In one embodiment, the linker is a peptide of about 3 to about 20 amino acids in length. Preferably, the linker comprises (GGS)n amino acid sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
在根据本发明的双特异性抗体中，本发明的抗TIGIT VHH作为第一抗原结合区，可以与Fc组合成重链抗体，与第二抗原结合区异二聚化形成双特异性抗体。In the bispecific antibody according to the present invention, 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.
在一些实施方案中，本发明的双特异性抗体具有以下结构：In some embodiments, bispecific antibodies of the invention have the following structure:
重链1：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
重链2：1个或多个(例如2个)串联的抗TIGIT VHH-重链恒定区FcHeavy chain 2: 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL。Light chain: from N-terminal to C-terminal, light chain variable region-light chain constant region CL of the second antigen antibody.
在一些实施方案中，本发明的双特异性抗体具有1条重链1、1条重链2和1条轻链。In some embodiments, the bispecific antibody of the invention has 1 heavy chain 1 , 1 heavy chain 2 and 1 light chain.
优选地，所述双特异性抗体的结构如图14B或14C所示。Preferably, the structure of the bispecific antibody is shown in Figure 14B or 14C.
在一些实施方案中，抗TIGIT单域抗体VHH通过连接肽与重链恒定区Fc的N末端连接。在一些实施方案中，所述连接肽是来自人IgG1、2、3或4的铰链区或其部分，包括天然或突变的铰链区或其部分，例如来自人IgG1铰链区，例如所述连接肽是EPKSS(SEQ ID NO：43)。In some embodiments, the anti-TIGIT single domain antibody VHH is linked to the N-terminal of the heavy chain constant region Fc via a linker peptide. In some embodiments, 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).
在一些实施方案中，当重链2包含多个串联的抗TIGIT单域抗体VHH时，各个串联的VHH之间可以通过接头连接。在一个实施方案中，接头是大约3个至大约20个氨基酸长度的肽。优选地，接头包含(GGGGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。In some embodiments, when the heavy chain 2 comprises multiple tandem anti-TIGIT single domain antibody VHHs, each tandem VHH can be connected by a linker. In one embodiment, the linker is a peptide of about 3 to about 20 amino acids in length. Preferably, the linker comprises (GGGGS)n amino acid sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
在一些实施方案中，第二抗原是CTLA-4，例如人CTLA-4。In some embodiments, the second antigen is CTLA-4, eg, human CTLA-4.
在本发明的一个实施方案中，本发明的抗TIGIT VHH如上文所述定义。In one embodiment of the invention, the anti-TIGIT VHH of the invention is as defined above.
在本发明的一个实施方案中，本发明的双特异性抗体中的抗PD1抗体的重链可变区和/或轻链可变区来自WO2019219064A的PD1抗体。In one embodiment of the present invention, 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.
在一些实施方案中，本发明的抗PD1抗体的重链可变区VH包含3个互补决定区VHCDR1、VHCDR2和VHCDR3。在一些实施方案中，本发明的抗PD1抗体的轻链可变区包含3个互补决定区VLCDR1、VLCDR2和VLCDR3。In some embodiments, 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.
在一些实施方案中，本发明的3个重链可变区的互补决定区VHCDR1、VHCDR2和VHCDR3来自包含如SEQ ID NO：24所示的氨基酸序列或由所述氨基酸序列组成的重链可变区VH，优选地，所述CDR通过Kabat定义。In some embodiments, 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.
在一些实施方案中，本发明的3个轻链可变区的互补决定区VLCDR1、VLCDH2和VLCDR3来自包含如SEQ ID NO：34所示的氨基酸序列或由所述氨基酸序列组成的轻链可变区VL，优选地，所述CDR通过Kabat定义。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的互补决定区VHCDR1包含SEQ ID NO：25的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的互补决定区VHCDR2包含SEQ ID NO：26的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的互补决定区VHCDR3包含SEQ ID NO：27的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的互补决定区VLCDR1包含SEQ ID NO：35的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的互补决定区VLCDR2包含SEQ ID NO：36的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的互补决定区VLCDR3包含SEQ ID NO：37的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，抗PD1抗体的重链可变区VH包含VHCDR1，VHCDR2和VHCDR3，其中VHCDR1包含SEQ ID NO：25所示的序列或由其组成；VHCDR2包含SEQ ID NO：26所示的序列或由其组成；和/或VHCDR3包含SEQ ID NO：27所示的序列或由其组成。In some embodiments, 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.
在一些实施方案中，抗PD1抗体的轻链可变区VL包含VLCDR1，VLCDR2和VLCDR3，其中VLCDR1包含SEQ ID NO：35所示的序列或由其组成；VLCDR2包含SEQ ID NO：36所示的序列或由其组成；和/或VLCDR3包含SEQ ID NO：37所示的序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗PD1抗体的重链可变区VH包含SEQ ID NO：24所述的氨基酸序列，或包含与所述SEQ ID NO：24所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：24所述的氨基酸组成。在一些实施方案中，所述重链可变区VH包含与SEQ ID NO：24所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如VHCDR1、VHCDR2或VHCDR3中。In some embodiments, 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. In some embodiments, 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. In some preferred embodiments, the mutation is absent in a CDR, eg, VHCDR1, VHCDR2 or VHCDR3.
在一些实施方案中，本发明的抗PD1抗体的轻链可变区VL包含SEQ ID NO：34所述的氨基酸序列，或包含与所述SEQ ID NO：34所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：34所述的氨基酸组成。在一些实施方案中，所述轻链可变区VL包含与SEQ ID NO：34所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如VLCDR1、VLCDR2或VLCDR3中。In some embodiments, 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. In some embodiments, 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. In some preferred embodiments, the mutation is absent in a CDR, eg, VLCDR1, VLCDR2 or VLCDR3.
在本发明的一个实施方案中，本发明的双特异性抗体中的抗CTLA-4抗体的重链可变区和/或轻链可变区来自Ipilimumab。In one embodiment of the present invention, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的重链可变区VH包含3个互补决定区VHCDR1、VHCDR2和VHCDR3。在一些实施方案中，本发明的抗CTLA-4抗体的轻链可变区包含3个互补决定区VLCDR1、VLCDR2和VLCDR3。In some embodiments, 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. In some embodiments, the light chain variable region of an anti-CTLA-4 antibody of the invention comprises three complementarity determining regions, VLCDR1, VLCDR2 and VLCDR3.
在一些实施方案中，本发明的3个重链可变区的互补决定区VHCDR1、VHCDR2和VHCDR3来自包含如SEQ ID NO：62所示的氨基酸序列或由所述氨基酸序列组成的重链可变区VH，优选地，所述CDR通过Kabat定义。In some embodiments, 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.
在一些实施方案中，本发明的3个轻链可变区的互补决定区VLCDR1、VLCDR2和VLCDR3来自包含如SEQ ID NO：67所示的氨基酸序列或由所述氨基酸序列组成的轻链可变区VL，优选地，所述CDR通过Kabat定义。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的互补决定区VHCDR1包含SEQ ID NO：59的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的互补决定区VHCDR2包含SEQ ID NO：60的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的互补决定区VHCDR3包含SEQ ID NO：61的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的互补决定区VLCDR1包含SEQ ID NO：64的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的互补决定区VLCDR2包含SEQ ID NO：65的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的互补决定区VLCDR3包含SEQ ID NO：66的氨基酸序列或由其组成。In some embodiments, 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.
在一些实施方案中，抗CTLA-4抗体的重链可变区VH包含VHCDR1，VHCDR2和VHCDR3，其中VHCDR1包含SEQ ID NO：59所示的序列或由其组成；VHCDR2包含SEQ ID NO：60所示的序列或由其组成；和/或VHCDR3包含SEQ ID NO：61所示的序列或由其组成。In some embodiments, 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.
在一些实施方案中，抗CTLA-4抗体的轻链可变区VL包含VLCDR1，VLCDR2和VLCDR3，其中VLCDR1包含SEQ ID NO：64所示的序列或由其组成；VLCDR2包含SEQ ID NO：65所示的序列或由其组成；和/或VLCDR3包含SEQ ID NO：66所示的序列或由其组成。In some embodiments, 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.
在一些实施方案中，本发明的抗CTLA-4抗体的重链可变区VH包含SEQ ID NO：62所述的氨基酸序列，或包含与所述SEQ ID NO：62所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：62所述的氨基酸组成。在一些实施方案中，所述重链可变区VH包含与SEQ ID NO：62所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置 换。在一些优选的实施方案中，所述突变不存在于CDR，例如VHCDR1、VHCDR2或VHCDR3中。In some embodiments, 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. In some embodiments, 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. In some preferred embodiments, the mutation is absent in a CDR, eg, VHCDR1, VHCDR2 or VHCDR3.
在一些实施方案中，本发明的抗CTLA-4抗体的轻链可变区VL包含SEQ ID NO：67所述的氨基酸序列，或包含与所述SEQ ID NO：67所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO：67所述的氨基酸组成。在一些实施方案中，所述轻链可变区VL包含与SEQ ID NO：67所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如VLCDR1、VLCDR2或VLCDR3中。In some embodiments, 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. In some embodiments, 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. In some preferred embodiments, the mutation is absent in a CDR, eg, VLCDR1, VLCDR2 or VLCDR3.
在一些实施方案中，本发明的双特异性抗体中的第二抗原抗体的重链恒定区CH1来自IgG，例如IgG1、IgG2、IgG3或IgG4。优选的，所述重链恒定区CH1来自IgG1或IgG4。In some embodiments, 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. Preferably, the heavy chain constant region CH1 is from IgG1 or IgG4.
在一些实施方案中，所述重链恒定区CH1In some embodiments, the heavy chain constant region CH1
(i)包含SEQ ID NO：28或31所述的氨基酸序列，或(i) comprising the amino acid sequence set forth in SEQ ID NO: 28 or 31, or
(ii)其来自IgG1且包含与所述SEQ ID NO：28所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或其来自IgG4且包含与所述SEQ ID NO：31所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) it is from IgG1 and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or An amino acid sequence with 99% identity, or it is derived from IgG4 and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences, or
(iii)由SEQ ID NO：28或31所述的氨基酸组成；或(iii) consist of the amino acid set forth in SEQ ID NO: 28 or 31; or
(iv)与SEQ ID NO：28或31所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。(iv) One or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared with the amino acid sequence shown in SEQ ID NO: 28 or 31 Amino acid sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution.
在一些实施方案中，本发明的双特异性抗体中的重链恒定区CH1和Fc区来自相同的IgG，例如IgG1、IgG2、IgG3或IgG4，优选地，都来自IgG1或IgG4。In some embodiments, 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.
在一些实施方案中，本发明的双特异性抗体轻链恒定区CL为Lambda或Kappa轻链恒定区，优选的Kappa轻链恒定区。在一些实施方案中，所述轻链恒定区CLIn some embodiments, 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
(i)包含与选自SEQ ID NO：38的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 38 A specific amino acid sequence or consists of said amino acid sequence;
(ii)包含选自SEQ ID NO：38的氨基酸序列或由所述氨基酸序列组成；或者(ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38; or
(iii)包含与选自SEQ ID NO：38的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。(iii) 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 (preferably amino acid substitution, more preferably amino acid conservative substitution) is or consists of said amino acid sequence.
在一些实施方案中，适用于本发明的重链抗体的Fc区同样适用于本发明的双特异性抗体。In some embodiments, the Fc region applicable to the heavy chain antibody of the invention is also applicable to the bispecific antibody of the invention.
在一些实施方案中，本发明的双特异性抗体中的重链恒定区Fc来自IgG，例如 IgG1、IgG2、IgG3或IgG4。在一些实施方案中，所述Fc区来自IgG1或来自IgG4。In some embodiments, 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.
在一个实施方案中，本发明双特异性抗体中的两个Fc区二聚化形成二聚Fc。In one embodiment, the two Fc regions in the bispecific antibody of the invention dimerize to form a dimeric Fc.
在一些实施方案中，例如在双特异性抗体包含相同的重链时，第一和第二Fc区相同。在另一些实施方案中，例如在双特异性抗体包含不同重链时，第一Fc区和第二Fc区不同，两者配对并异二聚化。In some embodiments, eg where the bispecific antibody comprises the same heavy chain, 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.
适用于本发明抗体分子的Fc区片段可以是任何抗体Fc区。Fc区可以包括天然序列Fc区和变体Fc区。天然序列Fc结构域涵盖天然存在的各种免疫球蛋白Fc序列，例如各种Ig亚型以及其同种异型的Fc区(Gestur Vidarsson等，IgG subclasses and allotypes：from structure to effector functions，20 October 2014，doi：10.3389/fimmu.2014.00520.)。例如，本发明抗体的Fc区可以包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。在一些实施方案中，抗体Fc区还可以在N端带有IgG铰链区或部分IgG铰链区，例如，IgG1铰链区或部分IgG1铰链区。在所述铰链区中可以含有突变。在一些实施方案中，铰链区可以是EPKSS或EPKSC。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.). For example, 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. In some embodiments, 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. In some embodiments, the hinge region can be EPKSS or EPKSC.
优选地，本发明抗体的Fc区从N端到C端包含：CH2-CH3，或从N端到C端包含：铰链区-CH2-CH3。在一些实施方案中，适用于本发明抗体或双特异性抗体的Fc区为人的IgG Fc，例如，人IgG1 Fc，人IgG2 Fc，人IgG3或人IgG4 Fc。在一些实施方案中，所述Fc区：Preferably, 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. In some embodiments, 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. In some embodiments, the Fc region:
(i)包含与SEQ ID NO：40或42中所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more of the amino acid sequence shown in SEQ ID NO: 40 or 42 99% identical amino acid sequences or consisting thereof; or
(ii)包含SEQ ID NO：40或42所示的氨基酸序列或由其组成；或者(ii) comprise or consist of the amino acid sequence shown in SEQ ID NO: 40 or 42; or
(iii)包含与SEQ ID NO：40或42所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列。(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) 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.
可以将本发明的Fc区进行突变以获得所需的特性。本领域已知对Fc区的突变。在一个实施方案中，在Fc区的效应子功能的特性上修饰Fc区。在一个实施方案中，所述效应子功能相对于野生同种型Fc区已经被提高。在一个实施方案中，通过对Fc区进行突变来改善Fc区的效应子功能，例如ADCC，例如通过在以下一个或多个位点进行突变S239、A330和I332(根据EU编号)。在一个实施方案中，通过以下位点组合的突变来改善ADCC：S239、A330和I332(根据EU编号)。在一个实施方案中，所述突变选自S239D、A330L和I332E中的1、2或3个。在一个实施方案中，所述改变效应子功能的突变为如下突变组合：S239D、A330L和I332E。The Fc region of the invention can be mutated to obtain desired properties. Mutations to the Fc region are known in the art. In one embodiment, 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. In one embodiment, the effector function of the Fc region, eg 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. In one embodiment, ADCC is improved by mutation of the following combination of sites: S239, A330 and I332 (according to EU numbering). In one embodiment, 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.
如本领域技术人员理解的，为了促进本发明的双特异性抗体作为异二聚体的形成，本发明双特异性抗体所包含的Fc区可以包含利于异二聚化的突变。在一个实施方案中，在两个Fc区的CH3区中引入突变。As understood by those skilled in the art, in order to facilitate the formation of the bispecific antibody of the present invention as a heterodimer, the Fc region comprised by the bispecific antibody of the present invention may contain mutations that favor heterodimerization. In one embodiment, mutations are introduced in the CH3 region of both Fc regions.
本领域已知促进Fc区异二聚化的方法。例如，第一Fc区的CH3区和第二Fc区的CH3区以互补方式进行工程化改造使得每个CH3区(或包含它的重链)不再能与其自身同二聚化但被迫与互补工程化改造的其它CH3区异二聚化(使得第一和第二CH3区异二聚化且两个第一CH3区或两个第二CH3区之间不形成同二聚体)。Methods of promoting heterodimerization of Fc regions are known in the art. For example, 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).
优选地，基于结入扣(Knob-in-Hole)技术，在第一单体Fc区和第二单体Fc区中分别引入相应的结(knob)突变和扣(Hole)突变。此技术参见例如Merchant，A.M.，et al.(1998).″An efficient route to human bispecific IgG.″Nat Biotechnol 16(7)：677-681。Preferably, based on the Knob-in-Hole technique, corresponding 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.
在一个特定的实施方案中，在一个Fc区的CH3区中，第366位的苏氨酸残基用色氨酸残基替换(T366W)(结突变)；而在另一个Fc区的CH3区中，第407位的酪氨酸残基用缬氨酸残基替换(Y407V)(扣突变)，任选地366位的苏氨酸残基用丝氨酸残基替换(T366S)且第368位的亮氨酸残基用丙氨酸残基替换(L368A)(编号方式依照EU索引)。In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knot mutation); while in the CH3 region of another Fc region In, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (deduction mutation), optionally 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).
在又一个实施方案中，在一个Fc区的CH3区中，结突变包括或由下述组成：第366位的苏氨酸残基用色氨酸残基替换(T366W)且第354位的丝氨酸残基用半胱氨酸残基替换(S354C)或第356位的谷氨酸残基用半胱氨酸残基替换(E356C)(特别是，第354位的丝氨酸残基用半胱氨酸残基替换)；而在另一个Fc区的CH3区中，扣突变包括或由下述组成：第407位的酪氨酸残基用缬氨酸残基替换(Y407V)，任选地366位的苏氨酸残基用丝氨酸残基替换(T366S)且第368位的亮氨酸残基用丙氨酸残基替换(L368A)(编号方式依照EU索引)，任选地第349位的酪氨酸残基用半胱氨酸残基替换(Y349C)(编号方式依照EU索引)。In yet another embodiment, in the CH3 region of an Fc region, 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 Amino acid residues were replaced with cysteine residues (Y349C) (numbering according to EU index).
在一个具体的实施方案中，一个Fc区包含氨基酸替代S354C和T366W(结突变)，且另一个Fc区包含氨基酸替代Y349C，T366S，L368A和Y407V(扣突变)(编号方式依照EU索引)。In a specific embodiment, 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).
因此，在一个具体的实施方案中，本发明的双特异性抗体包含的两个Fc区异二聚化，其中Thus, in a specific embodiment, the bispecific antibody of the invention comprises two Fc regions that are heterodimerized, wherein
a)一个Fc-区多肽包含突变T366W，而另一个Fc-区多肽包含突变T366S、L368A和Y407V，或a) one Fc-region polypeptide comprises mutation T366W and the other Fc-region polypeptide comprises mutations T366S, L368A and Y407V, or
b)一个Fc-区多肽包含突变T366W和Y349C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和S354C，或b) one Fc-region polypeptide comprises mutations T366W and Y349C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or
c)一个Fc-区多肽包含突变T366W和S354C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和Y349C。c) One Fc-region polypeptide comprises mutations T366W and S354C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and Y349C.
在一些实施方案中，Fc区还包含其他利于异二聚体纯化的突变。例如，可以将H435R突变(Eric J.Smith，，Scientific Reports|5：17943|DOI：10.1038/srep17943)引入异二聚体的Fc区之一中(例如，带Hole突变的Fc区)，以利于使用蛋白A纯化异二聚体。在另一些实施方案中，对于包含铰链区的异二聚体单体，也可以在铰链区中引入突变，例如C220S，以利于异二聚体的形成。In some embodiments, the Fc region further comprises other mutations that facilitate heterodimer purification. For example, the H435R mutation (Eric J. Smith, Scientific Reports | 5:17943 | DOI: 10.1038/srep17943) 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. In other embodiments, for heterodimer monomers comprising a hinge region, mutations such as C220S can also be introduced in the hinge region to facilitate the formation of heterodimers.
在一个具体的实施方案中，本发明的双特异性抗体的两个Fc区异二聚化，其中In a specific embodiment, the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein
第一Fc区包含结突变，其包含氨基酸序列SEQ ID NO：54或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成；在一些实施方案中，所述Fc区包含与SEQ ID NO：54具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含结突变(例如S354C和T366W)；在一些实施方案中，所述Fc区包含或不包含铰链区EPKSS或EPKSC；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 In some embodiments, 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;
第二Fc区包含扣突变，其包含氨基酸序列SEQ ID NO：75与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成。在一些实施方案 中，所述Fc区包含与SEQ ID NO：75具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含扣突变；在一些实施方案中，所述Fc区包含或不包含铰链区EPKSS或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 . In some embodiments, 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; In some embodiments, the Fc region may or may not comprise a hinge region EPKSS or EPKSC.
在一个具体的实施方案中，本发明的双特异性抗体的两个Fc区异二聚化，其中In a specific embodiment, the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein
第一Fc区包含结突变，其包含氨基酸序列SEQ ID NO：78或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成；在一些实施方案中，所述Fc区包含与SEQ ID NO：78具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含S239D、A330L和I332E突变和结突变(例如S354C和T366W)；在一些实施方案中，所述Fc区包含或不包含铰链区EPKSS或EPKSC；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; In some embodiments, 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;
第二Fc区包含扣突变，其包含氨基酸序列SEQ ID NO：77与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成。在一些实施方案中，所述Fc区包含与SEQ ID NO：77具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含S239D、A330L和I332E突变和扣突变；在一些实施方案中，所述Fc区包含或不包含铰链区EPKSS或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 . In some embodiments, 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.
在一些实施方案中，本发明的双特异性抗体中的抗TIGIT单域抗体VHH包含HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3来自SEQ ID NO：18或21所示的VHH。在一些实施方案中，所述HCDR1包含SEQ ID NO：3所示的氨基酸序列或由其组成，HCDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，HCDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成。在一些实施方案中，本发明的双特异性抗体中的抗TIGIT单域抗体VHH包含重链可变区VH或由重链可变区VH组成，所述重链可变区VH包含SEQ ID NO：18或21所示的氨基酸序列或由所述氨基酸序列组成。In some embodiments, 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. In some embodiments, 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, and HCDR3 comprises SEQ ID NO: The amino acid sequence shown in 5 or consists of it. In some embodiments, 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.
在一些实施方案中，本发明的双特异性抗体包含In some embodiments, the bispecific antibodies of the invention comprise
重链：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc-抗TIGIT VHH；或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
从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-抗TIGIT VHH-重链恒定区FcFrom N-terminal to C-terminal, the heavy chain variable region of the second antigen antibody VH-heavy chain constant region CH1-anti-TIGIT VHH-heavy chain constant region Fc
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中所述第二抗原为PD-1；wherein the second antigen is PD-1;
其中重链heavy chain
(i)包含SEQ ID NO：30、32或33的氨基酸序列，或(i) comprises the amino acid sequence of SEQ ID NO: 30, 32 or 33, or
(ii)包含与SEQ ID NO：30、32或33所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence described in SEQ ID NO: 30, 32 or 33 the amino acid sequence of identity, or
(iii)由SEQ ID NO：30、32或33所述的氨基酸组成，或(iii) consists of the amino acid set forth in SEQ ID NO: 30, 32 or 33, or
(iv)包含与SEQ ID NO：30、32或33所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失 或添加，优选地置换，例如保守置换，优选地，所述突变不存在于抗TIGIT VHH的CDR中，或不存在于抗TIGIT的VHH中，或不存在于抗PD1抗体的重链可变区CDR中，或优选不存在于重链可变区中；(iv) comprising one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) amino acid sequences compared with the amino acid sequence shown in SEQ ID NO: 30, 32 or 33 ) 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;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：39的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 39, or
(ii)包含与所述SEQ ID NO：39所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 39 the amino acid sequence of
(iii)由SEQ ID NO：39所述的氨基酸组成，或(iii) consists of the amino acid set forth in SEQ ID NO: 39, or
(iv)包含与SEQ ID NO：39所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地，所述突变不存在于抗PD1抗体的轻链可变区CDR中，或优选不存在于轻链可变区中。(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: 39 Sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution, preferably, said mutation does not exist in the light chain variable region CDR of anti-PD1 antibody, or preferably does not exist in the light chain variable region in the district.
在一些实施方案中，本发明的双特异性抗体包含In some embodiments, the bispecific antibodies of the invention comprise
(1)重链，其包含SEQ ID NO：30、32或33的氨基酸序列，或包含与所述SEQ ID NO：30、32或33所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述序列组成；和(1) 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; and
(2)轻链，其包含SEQ ID NO：39的氨基酸序列，或包含与所述SEQ ID NO：39所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。(2) 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.
在一些实施方案中，本发明的双特异性抗体包含In some embodiments, the bispecific antibodies of the invention comprise
重链：从N端至C端，抗TIGIT VHH-第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中第二抗原为CTLA-4，Wherein the second antigen is CTLA-4,
其中重链heavy chain
(i)包含SEQ ID NO：69的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 69, or
(ii)包含与SEQ ID NO：69所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising an amino acid having 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: 69 sequence, or
(iii)由SEQ ID NO：69所述的氨基酸组成，或(iii) consists of the amino acid set forth in SEQ ID NO: 69, or
(iv)包含与SEQ ID NO：69所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地，所述突变不存在于抗TIGIT VHH的CDR中，或不 存在于抗TIGIT的VHH中，或不存在于抗CTLA-4抗体的重链可变区CDR中，或优选不存在于重链可变区中；(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;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：68的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 68, or
(ii)包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 68 the amino acid sequence of
(iii)由SEQ ID NO：68所述的氨基酸组成，或(iii) consists of the amino acid set forth in SEQ ID NO: 68, or
(iv)包含与SEQ ID NO：68所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地，所述突变不存在于抗CTLA-4抗体的轻链可变区CDR中，或优选不存在于轻链可变区中。(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: 68 Sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution, preferably, said mutation does not exist in the light chain variable region CDR of anti-CTLA-4 antibody, or preferably does not exist in the light chain in the variable region.
在一些实施方案中，本发明的双特异性抗体包含In some embodiments, the bispecific antibodies of the invention comprise
(1)重链，其包含SEQ ID NO：69的氨基酸序列，或包含与所述SEQ ID NO：69所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述序列组成；和(1) 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; and
(2)轻链，其包含SEQ ID NO：68的氨基酸序列，或包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。(2) 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.
在一些实施方案中，本发明的双特异性抗体包含In some embodiments, the bispecific antibodies of the invention comprise
重链1：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
重链2：1个或多个(例如2个)串联的抗TIGIT VHH-重链恒定区FcHeavy chain 2: 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
其中第二抗原为CTLA-4，Wherein the second antigen is CTLA-4,
其中重链1of which heavy chain 1
(i)包含SEQ ID NO：71的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 71, or
(ii)包含与SEQ ID NO：71所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising an amino acid having 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:71 sequence, or
(iii)由SEQ ID NO：71所述的氨基酸组成；或(iii) consists of the amino acid set forth in SEQ ID NO: 71; or
(iv)包含与SEQ ID NO：71所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地，所述突变不存在于抗CTLA-4抗体的重链可变区CDR中，或优选不存在于重链可变区中；(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: 71 Sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution, preferably, said mutation does not exist in the heavy chain variable region CDR of anti-CTLA-4 antibody, or preferably does not exist in the heavy chain in the variable region;
和/或and / or
重链2 heavy chain 2
(i)包含SEQ ID NO：72或74的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 72 or 74, or
(ii)包含与SEQ ID NO：72或74所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(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
(iii)由SEQ ID NO：72或74所述的氨基酸组成；或(iii) consist of the amino acid set forth in SEQ ID NO: 72 or 74; or
(iv)包含与SEQ ID NO：72或74所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换；优选地，所述突变不存在于抗TIGIT VHH的CDR中，或不存在于抗TIGIT的VHH中；(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;
和/或and / or
轻链light chain
(i)包含SEQ ID NO：68的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 68, or
(ii)包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 68 the amino acid sequence of
(iii)由SEQ ID NO：68所述的氨基酸组成；或(iii) consist of the amino acid set forth in SEQ ID NO: 68; or
(iv)包含与SEQ ID NO：68所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地，所述突变不存在于抗CTLA-4抗体的轻链可变区CDR中，或优选不存在于轻链可变区中。(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: 68 Sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution, preferably, said mutation does not exist in the light chain variable region CDR of anti-CTLA-4 antibody, or preferably does not exist in the light chain in the variable region.
在一些实施方案中，本发明的双特异性抗体包含In some embodiments, the bispecific antibodies of the invention comprise
(1)重链1，其包含SEQ ID NO：71的氨基酸序列，或包含与所述SEQ ID NO：71所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述序列组成；和(1) 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; and
(2)重链2，其包含SEQ ID NO：72或74的氨基酸序列，或包含与所述SEQ ID NO：72或74所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述序列组成；和(2) 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; and
(2)轻链，其包含SEQ ID NO：68的氨基酸序列，或包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。(2) 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.
在本发明的一个实施方案中，本文所述的VHH或重链抗体或双特异性抗体包含一个或多个氨基酸突变。在一些实施方案中，氨基酸突变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。In one embodiment of the invention, the VHH or heavy chain antibody or bispecific antibody described herein comprises one or more amino acid mutations. In some embodiments, amino acid mutations include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
在优选的实施方案中，本发明所述的氨基酸突变发生在CDR外的区域(例如在FR中)。在一些实施方案中，本发明所述的氨基酸突变发生在抗体重链恒定区，例如Fc区 上，在优选的实施方案中，所述Fc区上的氨基酸突变增强了抗体的ADCC作用。In preferred embodiments, 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.
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸突变，以此产生Fc区变体，以改变抗体的一种或多种功能特性，例如血清半衰期、补体结合、补体依赖性细胞毒性、Fc受体结合和/或抗体依赖性细胞毒性。Fc区变体可包括在一或多个氨基酸位置处包含氨基酸突变(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4 Fc区)。In certain embodiments, 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.
在某些实施方案中，可能需要对抗体的可变区进行突变以防止脱酰胺作用，例如将可变区，例如CDR中的1个或2个易于脱酰胺的氨基酸(例如天冬氨酸)突变。In certain embodiments, it 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.
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合所述衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。In certain embodiments, 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 mixtures thereof.
Figure PCTCN2022143967-appb-000004
本发明VHH抗体或重链抗体或双特异性抗体具有以下一种或多种性质。
Figure PCTCN2022143967-appb-000004
The VHH antibody or heavy chain antibody or bispecific antibody of the present invention has one or more of the following properties.
在一些实施方案中，本发明抗TIGIT VHH或重链抗体具有一种或多种以下特性In some embodiments, anti-TIGIT VHH or heavy chain antibodies of the invention have one or more of the following properties
(i)能够特异性结合TIGIT，例如人TIGIT或食蟹猴TIGIT；(i) capable of specifically binding to TIGIT, such as human TIGIT or cynomolgus TIGIT;
(ii)能够特异性结合人TIGIT，例如以高亲和力，例如，如Fortebio检测系统所测定的，例如如实施例4所描述的方法所测定的，K D小于1nM，例如小于0.9nM或0.8nM，还例如小于0.7、0.6、0.5、0.4、0.3、0.2或0.1nM，还例如小于5×10 -12M、4×10 -12M、3×10 -12M、2×10 -12M、或1×10 -12M； (ii) capable of specifically binding to human 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 1 nM, e.g., less than 0.9 nM or 0.8 nM , also for example less than 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1nM, also for example less than 5×10 -12 M, 4×10 -12 M, 3×10 -12 M, 2×10 -12 M, or 1×10 -12 M;
(iii)能够特异性结合食蟹猴TIGIT，例如以高亲和力，例如，如Fortebio检测系统所测定的，例如如实施例4所描述的方法所测定的，K D小于6、5、4、3、2或1nM，还例如小于0.9nM或0.8nM，还例如小于5×10 -12M、4×10 - 12M、3×10 -12M、2×10 -12M、或1×10 -12M； (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 ;
(iv)抑制TIGIT(例如人TIGIT)与CD155的结合，例如人CD155，阻断活性高于已知的TIGIT抗体，例如WO2017053748A2的Tiragolumab analog；(iv) inhibiting the binding of TIGIT (such as human TIGIT) to CD155, such as human CD155, the blocking activity is higher than that of known TIGIT antibodies, such as Tiragolumab analog of WO2017053748A2;
(v)与表达TIGIT(例如人或食蟹猴TIGIT)的细胞结合，结合活性高于已知的TIGIT抗体，例如WO2017053748A2的Tiragolumab analog；(v) bind to cells expressing TIGIT (such as human or cynomolgus TIGIT), and the binding activity is higher than that of known TIGIT antibodies, such as Tiragolumab analog of WO2017053748A2;
(vi)有效激活T细胞(例如原代T细胞，例如CD8+T细胞)，例如激活T细胞释放细胞因子，例如IFNγ；(vi) effectively activate T cells (such as primary T cells, such as CD8+ T cells), such as activating T cells to release cytokines, such as IFNγ;
(vii)具有较好的抑制肿瘤的效果；(vii) have better effect of inhibiting tumor;
(viii)具有较低的毒性。(viii) have lower toxicity.
在一些实施方案中，本发明的双特异性抗体能够特异性结合TIGIT和PD-1，例如人TIGIT和人PD-1，例如以高亲和力。在一些实施方案中，本发明的双特异性抗体能够特异性结合TIGIT和CTLA-4，例如人CTLA-4和人TIGIT，例如以高亲和力。In some embodiments, 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.
在一些实施方案中，本发明的特异性结合TIGIT和PD-1的双特异性抗体具有一种或多种以下性质：In some embodiments, the bispecific antibody that specifically binds TIGIT and PD-1 of the present invention has one or more of the following properties:
(i)能够特异性结合TIGIT，例如人和/或食蟹猴TIGIT，例如以高亲和力；(i) capable of specifically binding to TIGIT, such as human and/or cynomolgus TIGIT, for example with high affinity;
(ii)能够特异性结合PD1，例如人PD1，例如以高亲和力；(ii) capable of specifically binding to PD1, such as human PD1, for example with high affinity;
(iii)抑制TIGIT(例如人TIGIT)与CD155的结合，例如人CD155，阻断活性高于已知的TIGIT抗体，例如WO2017053748A2的Tiragolumab analog；(iii) inhibit the binding of TIGIT (such as human TIGIT) to CD155, such as human CD155, and the blocking activity is higher than that of known TIGIT antibodies, such as Tiragolumab analog of WO2017053748A2;
(iv)阻断PD1与PD-L1的结合，例如阻断人PD1蛋白与人PD-L1的结合；(iv) blocking the binding of PD1 to PD-L1, for example, blocking the binding of human PD1 protein to human PD-L1;
(v)与表达TIGIT(例如人或食蟹猴TIGIT)的细胞结合(v) Binding to cells expressing TIGIT (e.g. human or cynomolgus TIGIT)
(vi)与表达TIGIT(例如人或食蟹猴TIGIT)的细胞结合，同时与表达PD1(例如人PD1)的细胞结合；(vi) binding to cells expressing TIGIT (e.g., human or cynomolgus TIGIT) while simultaneously binding to cells expressing PD1 (e.g., human PD1);
(vii)具有较好的结构安全性，例如对杀伤T细胞(例如CD4+T和CD8+T细胞)杀伤不明显，优选地不能明显诱导NK细胞杀伤激活后的CD4+T和CD8+T细胞，ADCC杀伤活性与已知的TIGIT抗体，例如WO2017053748A2的Tiragolumab analog相当；(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;
(viii)具有良好的药代动力学特征，成药性(例如稳定性)较好；(viii) have good pharmacokinetic characteristics and good druggability (such as stability);
(ix)具有PD1抗体，例如已知的PD1抗体(例如WO2019219064A公开的PD1抗体)的活性；具有本发明的TIGIT VHH或重链抗体的活性；(ix) have the activity of 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;
(x)具有较好的抑制肿瘤的效果；(x) have better effect of suppressing tumor;
(xi)具有较低的毒性。(xi) has lower toxicity.
在一些实施方案中，本发明的特异性结合TIGIT和CTLA-4的双特异性抗体具有一种或多种以下性质：In some embodiments, the bispecific antibodies of the invention that specifically bind TIGIT and CTLA-4 have one or more of the following properties:
特异性结合TIGIT和CTLA-4的双特异性抗体具有一种或多种以下性质：Bispecific antibodies that specifically bind TIGIT and CTLA-4 have one or more of the following properties:
(i)能够特异性结合TIGIT，例如人和/或食蟹猴TIGIT，例如以高亲和力；(i) capable of specifically binding to TIGIT, such as human and/or cynomolgus TIGIT, for example with high affinity;
(ii)能够特异性结合CTLA-4，例如人CTLA-4，例如以高亲和力；(ii) capable of specifically binding to CTLA-4, such as human CTLA-4, for example with high affinity;
(iii)与TIGIT的结合亲和力高于与CTLA-4的结合亲和力(例如高1个数量级)(iii) Higher binding affinity to TIGIT than to CTLA-4 (eg, 1 order of magnitude higher)
(iv)抑制TIGIT(例如人TIGIT)与CD155的结合，例如人CD155，阻断活性高于已知的TIGIT抗体，例如WO2017053748A2的Tiragolumab analog；(iv) inhibiting the binding of TIGIT (such as human TIGIT) to CD155, such as human CD155, the blocking activity is higher than that of known TIGIT antibodies, such as Tiragolumab analog of WO2017053748A2;
(v)阻断CTLA-4与CD80的结合，例如阻断人CTLA-4与人CD80的结合；(v) blocking the binding of CTLA-4 to CD80, for example blocking the binding of human CTLA-4 to human CD80;
(vi)与表达TIGIT(例如人或食蟹猴TIGIT)的细胞结合(vi) Binding to cells expressing TIGIT (e.g. human or cynomolgus TIGIT)
(vii)具有较好的结构安全性，例如对杀伤T细胞(例如CD4+T和CD8+T细胞)杀伤不明显，优选地不能明显诱导NK细胞杀伤激活后的CD4+T和 CD8+T细胞，ADCC杀伤活性与已知的TIGIT抗体，例如WO2017053748A2的Tiragolumab analog相当；(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;
(viii)具有良好的药代动力学特征，成药性(例如稳定性)较好；(viii) have good pharmacokinetic characteristics and good druggability (such as stability);
(ix)具有抗CTLA-4抗体，例如已知的CTLA-4抗体(例如Ipilimumab)的活性；(ix) have the activity of anti-CTLA-4 antibody, such as known CTLA-4 antibody (such as Ipilimumab);
(x)具有本发明的TIGIT VHH或重链抗体的活性；(x) have the activity of TIGIT VHH or heavy chain antibody of the present invention;
(xi)具有较好的抑制肿瘤的效果；(xi) have better effect of suppressing tumor;
(xii)具有较低的毒性。(xii) has lower toxicity.
二、编码抗体的核酸以及包含其的宿主细胞2. Antibody-encoding nucleic acid and host cells containing it
在一方面，本发明提供了编码以上任何VHH或重链抗体或双特异性抗体或其任一条链的核酸。In one aspect, the invention provides a nucleic acid encoding any of the above VHH or heavy chain antibodies or bispecific antibodies or any chain thereof.
例如，本发明的核酸包含编码选自SEQ ID NO：1、2、6、7、8、9、10、14-22、30、32、33、69、71、72或74中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO：1、2、6、7、8、9、10、14-22、30、32、33、69、71、72或74中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。For example, 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.
如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。编码本发明分子的核酸序列可以采用本领域熟知的方法，例如通过从头固相DNA合成，或通过PCR扩增而产生。As will be apparent to those skilled in the art, 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.
在一方面，本发明提供编码以上任何单域抗体或VHH的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示人TIGTI抗原(和/或食蟹猴)结合能力。在一些实施方案中，所述核酸在读框中与编码另一肽/多肽的核酸可操作连接，从而当从适宜的表达载体表达时，产生包含所述单域抗体或VHH与另一肽/多肽的融合蛋白或嵌合多肽。例如，在一些实施方案中，所述核酸在读框中与编码Fc区(例如人Fc区)的核酸可操作连接，从而当从适宜的表达载体表达时，产生包含所述单域抗体或VHH与Fc区的重链抗体。In one aspect, 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. In some embodiments, 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. For example, in some embodiments, 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.
为了便于生产和纯化，单域抗体或VHH可以在N端融合分泌性信号肽，和/或利于纯化的标签肽，如六组氨酸标签或生物素标记或hFc标记。For ease of production and purification, 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.
在再一方面，本发明提供编码以上任何双特异性抗体的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示人TIGIT和第二抗原(例如人PD-1或人CTLA-4)结合能力。在一个实施方案中，编码双特异性抗体的重链和轻链的核酸可以在相同的载体中或在不同的载体中。在再一实施方案中，编码双特异性抗体的重链和轻链的核酸可以引入相同或不同的宿主细胞以表达。因此，在一些实施方案中，本发明的双特异性抗体的生产方法包括步骤：在适于表达所述分子的重链和轻链的条件下，培养包含编码所述重链和轻链的核酸的宿主细胞，产生本发明双特异性抗体。In a further aspect, the invention provides a nucleic acid encoding any of the above bispecific antibodies. 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. In one embodiment, the nucleic acids encoding the heavy and light chains of the bispecific antibody may be in the same vector or in different vectors. In yet another embodiment, 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. Accordingly, in some embodiments, 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.
在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是例如pcDNA载体，例如pcDNA3.1。In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, 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). In one embodiment, the vector is eg a pcDNA vector, eg pcDNA3.1.
在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞，例如用于克隆或表达编码VHH或重链抗体或双特异性抗体的载体。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞(例如CHO-S)或293细胞(例如293F或HEK293细胞))或适用于制备抗体或其片段的其它细胞。在一个实施方案中，宿主细胞是原核的，例如是细菌，例如大肠杆菌。In one embodiment, 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. In one embodiment, the host cell is eukaryotic. In another embodiment, 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. In one embodiment, the host cell is prokaryotic, such as a bacterium, such as E. coli.
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293、293F或293T细胞)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞、CHO-S细胞、ExpiCHO等；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。本领域已知适合产生抗体的哺乳动物宿主细胞系。In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, 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. For example, mammalian cell lines adapted for growth in suspension can be used. Other examples of 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.
三、本发明的VHH抗体或重链抗体或双特异性抗体的生产和纯化3. Production and purification of the VHH antibody or heavy chain antibody or bispecific antibody of the present invention
在一个实施方案中，提供了制备本发明VHH抗体或重链抗体或双特异性抗体的方法，其中所述方法包括，在适合VHH抗体或重链抗体或双特异性抗体或其链表达的条件下，培养包含编码所述VHH或重链抗体或双特异性抗体(例如任意一条多肽链和/或多条多肽链)的核酸或包含所述核酸的表达载体的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述VHH或重链抗体或双特异性抗体。In one embodiment, there is provided a method for preparing a VHH antibody or a heavy chain antibody or a bispecific antibody of the present invention, wherein the method 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).
可以将编码本发明VHH抗体或重链抗体或双特异性抗体的多肽链的多核苷酸插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。可以使用本领域技术人员熟知的方法来构建表达载体。一旦已经制备了用于表达的包含本发明的一种或多种核酸分子的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。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.
如本文所述制备的VHH抗体或重链抗体或双特异性抗体可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、尺寸排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。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.
可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括尺寸排阻层析、凝胶电泳、高效液相色谱等。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.
四、对VHH抗体或重链抗体或双特异性抗体的测定法。4. Assays for VHH antibodies or heavy chain antibodies or bispecific antibodies.
可以通过本领域中已知的多种测定法对本文中提供的VHH或重链抗体或双特异性抗体进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。The 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.
一方面，对本发明的VHH或重链抗体或双特异性抗体测试其靶标(例如抗原)结合活性，例如通过已知的方法诸如生物膜薄层干涉技术、ELISA，等来进行。可使用本领域已知方法来测定对TIGIT和/或PD1的结合，本文中公开了例示性方法。在一些实施方案中，使用放射性免疫测定(RIA)或生物膜薄层干涉测定法(BLI)或电化学发光(ECL)或表面等离子体共振法(SPR)或流式细胞术(FACS)测量。In one aspect, 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.
本发明还提供了用于鉴定VHH或重链抗体或双特异性抗体的生物学活性的测定法。生物学活性选自本发明的VHH或重链抗体或双特异性抗体的性质。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.
例如，对于本发明的抗体分子对表达TIGIT和/或第二抗原(例如PD-1或CTLA-4)的细胞的结合活性，可以通过本领域已知的方法，例如荧光报道分子和流式细胞术，或本文实施例公开的示例性方法测定，例如通过如实施例6所示的方法测定本发明的抗体分子与细胞上表达的TIGIT和/或PD-1的结合。For example, the binding activity of the antibody molecule of the present invention to cells expressing TIGIT and/or a second antigen (such as PD-1 or CTLA-4) 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.
例如，对于本发明抗体分子对TIGIT和/或第二抗原(例如PD-1或CTLA-4)的抑制活性，可以通过本领域已知的方法，例如ELISA阻断试验，受体荧光报道分子激活试验，细胞增殖试验，或本文实施例公开的例示性方法来测定。例如，可以使用ELISA阻断试验，例如实施例5或14所示的方法，测定分子阻断人TIGIT和/或人PD-1与其相关受体结合的阻断活性，例如实施例21或22所示的方法，测定分子阻断人TIGIT和/或人CTLA-4与其相关受体结合的阻断活性For example, for the inhibitory activity of the antibody molecule of the present invention on TIGIT and/or a second antigen (such as PD-1 or CTLA-4), 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. For example, the blocking activity of molecules that block the binding of human TIGIT and/or human PD-1 to their associated receptors, such as those described in Example 21 or 22, can be determined using an ELISA blocking assay, such as the method described in Example 5 or 14. The method shown for determining the blocking activity of molecules that block the binding of human TIGIT and/or human CTLA-4 to their associated receptors
例如，对于本发明的抗体分子对T细胞的激活活性，可以通过本领域已知的方法，例如T细胞激活试验系统，例如原代T细胞激活试验系统测定，例如通过实施7所示的方法，通过检测T细胞(例如CD8+T细胞)释放的细胞因子(例如IFNγ)的量。For example, 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 IFNγ) released by T cells (such as CD8+ T cells).
例如，对于本发明抗体分子对细胞的杀伤活性或结构安全性，可以通过本领域已知的方法，例如细胞毒性试验系统，例如NK细胞依赖的细胞毒性试验系统，例如通过实施例17所示的方法来测定。For example, 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. method to measure.
供任何上述体外测定法使用的细胞为原代细胞或细胞系，包括天然表达或过表达TIGIT(例如人或食蟹猴)或CD155或第二抗原(PD1或PDL1(例如人或食蟹猴PD1或PDL1)或CTLA-4(例如人CTLA-4)的细胞，例如过表达TIGIT和/或CD155和/或PD1和/或PDL1的细胞，例如293细胞或CHO细胞或Jurkat细胞，例如HEK293或CHO-K1或Jurkat/NFAT-Luc细胞。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.
本发明还提供了用于检测本发明的抗体分子的成药性的方法，例如通过检测抗体分子的药代动力学特征，可以通过本领域已知的方法，例如实施例18所示的方法获得动物模型(例如大鼠模型)中抗体分子的药代动力学参数。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).
可以理解的是，能够使用本发明的抗体和别的活性剂的组合来进行任何上述测定法。It will be appreciated that any of the above assays can be performed using combinations of antibodies of the invention and additional active agents.
五、本发明VHH或重链抗体或双特异性抗体的融合蛋白或免疫缀合物、药物组合物、药物联合和试剂盒5. Fusion protein or immunoconjugate, pharmaceutical composition, drug combination and kit of VHH or heavy chain antibody or bispecific antibody of the present invention
在一些实施方案中，本发明还提供了包含本文所述的任何VHH或重链抗体或双特异性抗体的融合蛋白，例如其包含本发明的VHH或重链抗体或双特异性抗体，以及与其连接的其他分子(例如其他蛋白，例如用于治疗的蛋白质)。In some embodiments, 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).
在一些实施方案，本发明提供了包含本文所述的任何VHH或重链抗体或双特异性抗体的免疫缀合物。优选地，所述免疫缀合物包含一种或多种其他治疗剂(例如细胞毒素或小分子化合物)或标记物。In some embodiments, the invention provides immunoconjugates comprising any of the VHH or heavy chain antibodies or bispecific antibodies described herein. Preferably, the immunoconjugate comprises one or more additional therapeutic agents (eg, cytotoxins or small molecule compounds) or markers.
在一些实施方案中，本发明提供包含本文所述的任何VHH或重链抗体或双特异性抗体的组合物或药物或制剂，优选地组合物为药物组合物。In some embodiments, 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.
在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的VHH或重链抗体或双特异性抗体，以及一种或多种其它治疗剂的组合。In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, 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.
本发明的所述组合物或药物或制剂还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。The 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.
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”，第八版，R.C.Rowe，P.J.Seskey和S.C.Owen，Pharmaceutical Press，London，Chicago。For the use and use of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.
本发明的组合物或药物或制剂可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，注射液或滴眼液)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。The 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. The preferred form depends on the intended mode of administration and therapeutic use.
可以通过将具有所需纯度的本发明的VHH或重链抗体或双特异性抗体与一种或多种任选的药用辅料混合来制备包含本文所述的VHH或重链抗体或双特异性抗体的药物或制剂，例如以冻干制剂或水溶液的形式。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.
本发明的组合物或药物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂。The 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.
本发明还提供了药物组合或药物组合产品，其包含本发明的VHH抗体或重链抗体或双特异性抗体，以及一种或多种其它治疗剂。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.
本发明还提供了包含所述药物组合的成套药盒，例如所述成套药盒在同一包装内包含：The present invention also provides a complete kit containing the drug combination, for example, the complete kit contains in the same package:
-含有包含本发明的VHH抗体或重链抗体或双特异性抗体的药物组合物的第一容器；- a first container containing a pharmaceutical composition comprising a VHH antibody or heavy chain antibody or bispecific antibody of the invention;
-含有包含其它治疗剂的药物组合物的第二容器。- a second container containing a pharmaceutical composition comprising an additional therapeutic agent.
六、VHH或重链抗体或双特异性抗体的用途和应用其的方法。6. Uses of VHH or heavy chain antibodies or bispecific antibodies and methods of using them.
本发明一方面提供了在受试者中预防或治疗疾病的方法，包括向受试者施用本发明的抗TIGIT VHH或重链抗体或双特异性抗体，或包含其的免疫缀合物、组合物或药物或制剂。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.
在一些实施方案中，所述疾病例如肿瘤，例如癌症。癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，癌症可以是实体肿瘤或血液肿瘤。在一些实施方案中，所述肿瘤是对已知药物，例如已知抗PD-1抗体或抗CTLA-4抗体具有耐受性的肿瘤或癌症，例如难治性肿瘤或癌症。In some embodiments, the disease is eg a tumor, eg cancer. Cancer can be early, middle or advanced or metastatic. In some embodiments, the cancer can be a solid tumor or a hematological tumor. In some embodiments, 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.
在一些实施方案中，所述患者中具有(例如升高水平的，例如核酸或蛋白质水平的)PD1或PDL1或PDL2或CTLA-4，和/或TIGIT(例如与健康个体的相同组织相比，或与患者相邻的健康组织相比)。In some embodiments, 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).
在一些实施方案中，所述疾病治疗将受益于抑制核酸或蛋白质水平的PD1或PDL1或PDL2或CTLA-4，和/或TIGIT。In some embodiments, 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.
在一些实施方案中，所述癌症是表征为具有升高的PD-1、PD-L1或PD-L2或CTLA-4的蛋白质水平和/或核酸水平(例如表达升高)和/或具有升高的TIGIT的的蛋白质水平和/或核酸水平(例如表达升高)的癌症，例如，所述癌症的肿瘤细胞中具有升高的PD-1、PD-L1和/或PD-L2或CTLA-4的蛋白质水平和/或核酸水平(例如表达升高)，和/或升高的TIGIT的蛋白质水平和/或核酸水平(例如表达升高)(例如与健康个体的相同组织相比，或与患者相邻的健康组织相比)。In some embodiments, 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).
本发明的抗TIGIT VHH或重链抗体或双特异性抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外注射或输注包括肌内、静脉内、动脉内、腹膜内或皮下注射或输注。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。Anti-TIGIT VHH or heavy chain antibodies or bispecific antibodies of the invention (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) 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.
为了预防或治疗疾病，本发明的抗TIGIT VHH或重链抗体或双特异性抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等)的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。在其他方面，本发明提供本发明的抗TIGIT VHH或重链抗体或双特异性抗体或包含其的免疫缀合物或组合物在生产或制备药物中的用途，所述药物用于本文所述的用途，例如用于预防或治疗本文提及的相关疾病或病症。For the prevention or treatment of diseases, 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. In other aspects, 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.
在一些实施方案中，抗TIGIT VHH或重链抗体或双特异性抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂等)还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的用途，例如用于预防和/或治疗本文提及的相关疾病或病症。In some embodiments, an anti-TIGIT VHH or heavy chain antibody or bispecific antibody (and immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) 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.
七、诊断和检测7. Diagnosis and testing
在一个方面，本发明还涉及对VHH或重链抗体或双特异性抗体的用于诊断和检测的方法和包含其的用于诊断和检测的组合物。In one aspect, 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.
在某些实施方案中，本文中提供的抗TIGIT VHH或重链抗体抗体可以用于检测TIGIT在生物样品中的存在。在某些实施方案中，本文中提供的抗双特异性抗体可以用于 检测TIGIT和/或PD1在生物样品中的存在。在某些实施方案中，本文中提供的抗双特异性抗体可以用于检测TIGIT和/或CTLA-4在生物样品中的存在。In certain embodiments, the anti-TIGIT VHH or heavy chain antibody antibodies provided herein can be used to detect the presence of TIGIT in a biological sample. In certain embodiments, the anti-bispecific antibodies provided herein can be used to detect the presence of TIGIT and/or PD1 in a biological sample. In certain embodiments, the anti-bispecific antibodies provided herein can be used to detect the presence of TIGIT and/or CTLA-4 in a biological sample.
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是体液，例如血液、血清或血浆。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). In certain embodiments, the biological sample is a bodily fluid, such as blood, serum or plasma.
在某些实施方案中，所述方法包括将生物样品与如本文所述的VHH或重链抗体在允许其与TIGIT结合的条件下接触，并检测在该VHH或重链抗体和TIGIT之间是否形成复合物。复合物的形成表示存在TIGIT。该方法可以是体外或体内方法。在一个实施方案中，本发明的抗体用于选择适合利用本发明的VHH或重链抗体治疗的受试者，例如其中TIGIT是用于选择所述受试者的生物标记物。In certain embodiments, 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. In one embodiment, 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.
在某些实施方案中，所述方法包括将生物样品与如本文所述的双特异性抗体在允许其与TIGIT和/或PD-1结合的条件下接触，并检测在该抗体与TIGIT和/或PD-1之间是否形成复合物。复合物的形成表示存在TIGIT和/或PD-1。该方法可以是体外或体内方法。在一个实施方案中，本发明的抗体用于选择适合利用本发明的双特异性抗体治疗的受试者，例如其中TIGIT和/或PD-1是用于选择所述受试者的生物标记物。In certain embodiments, 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. In one embodiment, 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 .
在某些实施方案中，所述方法包括将生物样品与如本文所述的双特异性抗体在允许其与TIGIT和/或CTLA-4结合的条件下接触，并检测在该抗体与TIGIT和/或CTLA-4之间是否形成复合物。复合物的形成表示存在TIGIT和/或CTLA-4。该方法可以是体外或体内方法。在一个实施方案中，本发明的抗体用于选择适合利用本发明的双特异性抗体治疗的受试者，例如其中TIGIT和/或CTLA-4是用于选择所述受试者的生物标记物。In certain embodiments, 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. In one embodiment, 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 .
在某些实施方案中，提供标记的VHH或重链抗体或双特异性抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。在一些实施方案中，所述标记例如生物素或hFc等标记物。In certain embodiments, labeled VHH or heavy chain antibodies or bispecific antibodies are provided. Labels 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. In some embodiments, the label is, for example, a label such as biotin or hFc.
在本文中提供的一些实施方案中，样品是在用本发明的VHH或重链抗体或双特异性抗体治疗之前获得的。在一些实施方案中，样品是在用其他疗法之前获得的。在一些实施方案中，样品是在用其他疗法治疗过程中，或者用其他疗法治疗后获得的。In some embodiments provided herein, 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.
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测TIGIT和/或PD1。在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测TIGIT和/或CTLA-4。In some embodiments, TIGIT and/or PD1 are measured prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval. In some embodiments, 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.
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验TIGIT的存在，因而确定TIGIT值，将TIGIT值与对照值(例如正常个体中的值)比较，并且如果TIGIT值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的本发明所述的VHH抗体或重链抗体，因而治疗所述疾病。In some embodiments, there is provided a method of treating a disease of the present invention, the method 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.
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验TIGIT和/或PD1的存在，因而确定TIGIT和/或PD1值，将TIGIT和/或PD1值与对照值(例如正常个体中的值)比较，并且如果TIGIT 和/或PD1值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的本发明所述的双特异性抗体，因而治疗所述疾病。In some embodiments, there is provided a method of treating a disease of the invention, the method 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.
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验TIGIT和/或CTLA-4的存在，因而确定TIGIT和/或PD1值，将TIGIT和/或CTLA-4值与对照值(例如正常个体中的值)比较，并且如果TIGIT和/或CTLA-4值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的本发明所述的双特异性抗体，因而治疗所述疾病。In some embodiments, there is provided a method of treating a disease of the invention, the method 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.
八、发明定义8. Definition of Invention
应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。It is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。In order to explain this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated numerical value.
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项或全部。As used herein, the term "and/or" means any one of the alternatives or two or more or all of the alternatives.
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps. For example, when referring to 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.
如本文中使用的，术语“TIGIT”或“具有Ig和ITIM域的T细胞免疫受体”指来自任何脊椎动物来源，包括哺乳动物诸如灵长类(例如人或食蟹猴)和啮齿类(例如小鼠和大鼠)的任何天然TIGIT，除非另有说明。TIGIT在本领域也称作含有V集和免疫球蛋白域的蛋白9，含有V集和跨膜域的蛋白3，VSIG9，VSTM3，和WUCAM。该术语涵盖“全长”、未加工的TIGIT以及因细胞中的加工所致的任何形式的TIGIT。该术语还涵盖TIGIT的天然发生变体，例如剪接变体或等位变体。一种例示性的人TIGIT的氨基酸序列可以见UniProt登录号Q495A1。在本发明的一些实施方案中，人TIGIT包含如SEQ ID NO：46所示的氨基酸序列，或由所述序列组成。在本发明的一些实施方案中，食蟹猴TIGIT包含如SEQ ID NO：48所示的氨基酸序列，或由所述序列组成。As used herein, the term "TIGIT" or "T cell immune receptor with Ig and ITIM domains" 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. The term also encompasses naturally occurring variants of TIGIT, such as splice variants or allelic variants. The amino acid sequence of an exemplary human TIGIT can be found at UniProt Accession No. Q495A1. In some embodiments of the present invention, human TIGIT comprises the amino acid sequence shown in SEQ ID NO: 46, or consists of said sequence. In some embodiments of the present invention, cynomolgus monkey TIGIT comprises the amino acid sequence shown in SEQ ID NO: 48, or consists of said sequence.
术语″PD-1″是指程序性细胞死亡蛋白1。术语″PD-1″包含变体、同种型、同源物、直系同源物和旁系同源物。举例来说，在某些情况下，特异性针对人PD-1蛋白的抗体可与来自除人以外的物种(例如食蟹猴)的PD-1蛋白交叉反应。在其它实施方案中，特异性针对人PD-1蛋白的抗体可完全特异性针对人PD-1蛋白且不展现与其它物种或其它类型的交叉反应性，或可与来自某些其它物种但不是所有其它物种的PD-1交叉反应。The term "PD-1" refers to programmed cell death protein 1. The term "PD-1" includes variants, isoforms, homologs, orthologs and paralogs. For example, in certain instances, an antibody specific for human PD-1 protein may cross-react with PD-1 protein from a species other than human (eg, cynomolgus monkey). In other embodiments, 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.
术语″人PD-1″指人PD-1序列，如具有Genbank登录号.NP_005009.2的人PD-1的完整氨基酸序列。The term "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.
术语“细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte associated protein 4)”或“CTLA-4”是在T细胞上上调的抑制性受体(A1egre等人，2001，《自然·免疫学综述》(Nat Rev Immunol)1：220-8)。CTLA-4以几种方式抑制免疫反应：它与T细胞共刺激受体CD28竞争其配体CD80和CD86，并由此阻断共刺激；它发出负信号以抑制T细胞活化；并且它还可以通过反式内吞作用从相对的细胞中捕获CD80和CD86，导致通过CD28的T细胞共刺激减弱。它在Treg细胞上组成型表达，只在激活后的传统T细胞上表达，CTLA4是T细胞的关键负调节因子，与CD28有相同的B7配体(CD80、CD86)，但是CTLA4与B7分子亲和力更高，因此CTLA4与T细胞上的CD28竞争性地与APC细胞上的B7分子结合，从而抑制T细胞的激活(Shunsuke Chikuma.(2017).″CTLA-4，an Essential Immune-Checkpoint for T-Cell Activation″Curr Top Microbiol Immunol 410：99-126.)。CTLA4也可以介导Treg细胞对APC细胞表面的B7分子进行反式内吞，从而降低APC细胞表面B7分子的表达来减少对T细胞上CD28的激活。在肿瘤微环境中，抑制CTLA4可以通过两种独立却互补的机制来恢复抗肿瘤免疫应答，第一是促进肿瘤浸润T细胞的增殖和活化，第二是减弱免疫抑制性Treg细胞的功能。伊匹木单抗可阻断细胞毒性T淋巴细胞相关抗原4(CTLA4)，但是临床上有很多治疗相关的不良事件，特别是在较高剂量下的剂量限制毒性阻止了其最大的抗肿瘤活性潜力。目前的研究报道表明伊匹木单抗产生irAE可能的机制包括：阻断CTLA4会激活对自身抗原有反应的T细胞克隆，导致自身免疫性疾病样症状；伊匹木单抗的Fc效应会导致组织驻留Treg细胞的耗竭从而阻碍外周耐受性，使患者在CTLA4抗体治疗后更容易发生irAE；CTLA4抗体治疗会导致CTLA4受体内吞降解，伊匹木单抗会显著下调细胞表面的CTLA4受体。The term "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. In the tumor microenvironment, inhibition of 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. Current research reports indicate that the possible mechanisms of 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.
“单域抗体”(single domain antibody，sdAb)在本文中用于指，通过单个可变抗体结构域，例如单个VH或单个VL，来识别并结合目的抗原的抗体多肽。单域抗体的单个可变抗体结构域无需与另一抗体可变结构域配对，即能够识别和结合目的抗原。在本文中，包含重链可变结构域的单域抗体也称作VHH。"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. Herein, a single domain antibody comprising a heavy chain variable domain is also referred to as VHH.
术语“VHH”或“VHH抗体”在本文中可以互换使用，通常指这样的抗体，其仅包含一个重链可变区或由其组成，具有与抗原结合的活性。VHH通常包含三个CDR与高度保守的4个构架区中，并且通常具有下式的结构：FR1-CDR-FR2-CDR2-FR3-CDR3-FR4，其中FR1至FR4指构架区1至4；CDR1至CDR3指互补决定区1-3。VHH可变区中的CDR序列可以按照“定义”部分描述的任何CDR定义方案进行确定，优选可以通过IMGT来定义可变区序列中的三个CDR的边界。VHH通常仅包括从缺乏轻链的重链抗体衍生的重链可变结构域，也称作纳米抗体。本发明中使用的VHH优选来自骆驼科动物，例如羊驼，或是其人源化形式或序列优化形式(例如，以增加结合亲和力的亲和力成熟形式)。在一些实施方案中，本发明的VHH为由或基本上由单个重链可变区(例如重链抗体的重链可变区)组成的单价单特异性多肽分子。The terms "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). In some embodiments, 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).
本发明的单域抗体或VHH也可以包含在更大的多肽/蛋白中。包含本发明VHH的多肽/蛋白例子包括但不限于，重链抗体(HcAb)或双特异性抗体或融合蛋白。本发明所述的“重链抗体”是指不具有轻链的抗体，例如其从N段到C段可以包含VH-Fc或VH-CH2-CH3或VH-铰链区-CH2-CH3，或可以包含VH-CH1-CH2-CH3；可以涵盖同型二聚体，例如不具有轻链的重链二聚体抗体。本发明的重链抗体中可以包含来自标准抗体的VH或者来自单域抗体的VH。例如，本发明的重链抗体中的VH可以就是VHH。在一些实施方案中，本发明的重链抗体可以是具有源自骆驼科动物(美洲驼、骆驼，尤其是羊驼)的构架区和/或重链恒定区的重链抗体，其人源化形式或其序列优化形式(亲和力成熟形式)、或其片段(例如包含至少部分恒定区的片段)。本发明的重链抗体还涵盖将重链可变区或 VHH与Fc区(例如人IgG Fc区，例如人IgG1或IgG4Fc区)融合后形成的抗体。当在重链抗体或双特异性抗体或融合蛋白的背景下提及“VHH”时，应当理解，其为双特异性抗体中的一部分，而不作为一个单独的分子。The single domain antibodies or VHHs of the invention may also be contained within larger polypeptides/proteins. Examples of 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. 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. For example, the VH in the heavy chain antibody of the invention can be VHH. In some embodiments, 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). When referring to "VHH" in the context of a heavy chain antibody or bispecific antibody or fusion protein, it is to be understood that it is part of the bispecific antibody and not as a separate molecule.
如本文所用，术语“单特异性”指，具有一个或多个抗原结合位点的多肽/蛋白分子，所述位点中的每一个位点与相同抗原的相同表位结合。As used herein, 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.
如本文所用，术语“多特异性”抗体指具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。在一个实施方案中，本文提供了这样的双特异性抗体，其具有针对第一抗原和第二抗原的结合特异性。As used herein, the term "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.
术语“多特异性结合分子”是指至少是双特异性的多特异性结合分子，例如双特异性结合分子，即所述分子包含至少第一靶标结合区和第二靶标结合区，其中所述第一靶标结合区结合一种靶标或抗原且所述第二靶标结合区结合另一抗原或靶标。因此，根据本发明的分子包含对于至少两种不同的抗原或靶标的特异性。根据本发明的分子也涵盖包含多个靶标结合区/结合位点的多特异性分子，诸如三特异性结合分子。在一些实施方案中，本发明的双特异性结合分子是双特异性抗体。The term "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. Thus, 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. In some embodiments, bispecific binding molecules of the invention are bispecific antibodies.
如本文所用的术语“接头”是指使得能够直接连接双特异性结合分子的不同部分的任何分子。在不同分子部分之间建立共价连接的接头的实例包括肽接头和非蛋白质聚合物，包括但不限于聚乙二醇(PEG)、聚丙二醇、聚氧化烯或聚乙二醇、聚丙二醇的共聚物。在一些实施方案中，接头是肽接头(又称为“连接肽”)，其是指是指由氨基酸组成的短氨基酸序列，例如单独或组合使用的甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T)，或来自免疫球蛋白的铰链区，用于将结合分子的第一部分的氨基酸序列连接至结合分子的第二部分。例如，肽接头可以将结合分子的第一靶标结合区连接至第二靶标结合区。例如，肽接头也可以将抗体的一部分连接至抗体的另一部分，诸如将轻链可变区连接至重链可变区。优选地，所述肽接头具有这样的长度，其足以连接两个实体，其方式使得它们维持它们相对于彼此的构象，使得不妨碍期望的活性。在一个实施方案中，连接肽具有5-50个氨基酸长度，例如，10，15，20，25，30个氨基酸长度。在一个实施方案中，连接肽包含氨基酸序列(GS)n、(GGS)n、(GSGGS)n、(GGGGS)n、(GGGS)n和(GGGGS)nG，其中n是等于或大于1的整数，例如，n是2、3、4、5、6、7、8、9、10的整数。有用的接头还包括甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其他柔性接头。在一些实施方案中，所述肽接头是(GGS)n，其中n＝1、2、3或4，例如SEQ ID NO：44所示的序列。The term "linker" as used herein refers to any molecule that enables the direct linking of different parts of a bispecific binding molecule. Examples of 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. In some embodiments, 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. and/or a threonine residue (T), or a hinge region from an immunoglobulin, for linking the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule. For example, a peptide linker can link a first target-binding region of a binding molecule to a second target-binding region. For example, 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. Preferably, 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. In one embodiment, the connecting peptide is 5-50 amino acids in length, eg, 10, 15, 20, 25, 30 amino acids in length. In one embodiment, 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. In some embodiments, the peptide linker is (GGS)n, where n=1, 2, 3 or 4, such as the sequence shown in SEQ ID NO:44.
在再一实施方案中，连接肽为来自免疫球蛋白的铰链区或铰链区部分，包括天然的铰链区或其部分，或者突变的铰链区或其部分。在一个实施方案中，连接肽例如是免疫球蛋白(例如IgG，例如IgG1、IgG2、IgG3或IgG4)的铰链区或其部分(例如EPKSC)或是突变的铰链区或其部分，例如EPKSS。或者，可以使用计算机程序模拟蛋白和肽的三维结构，或通过噬菌体展示方法，来合理地设计合适的柔性连接肽。In yet another embodiment, 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. In one embodiment, 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. Alternatively, 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.
如本文所用的术语“靶标结合区”是指多特异性结合分子，例如双特异性结合分子的结合特定靶标或抗原的任何部分。靶标结合区可以是例如抗体或免疫球蛋白本身或抗体片段。这种靶标结合区可以具有或可以不具有独立于BsAB的剩余部分的三级结构，并且可以作为单独实体结合或不结合其靶标。靶标结合区还可以是受体或配体，或受体的能够结合配体的结构域。在多特异性抗体或双特异性抗体时，也将“靶标结合区”称为“抗原结合区”。The term "target binding region" as used herein 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. In the context of multispecific antibodies or bispecific antibodies, the "target-binding region" is also referred to as "antigen-binding region".
术语“全抗体”或“全长抗体”在本文中可互换使用，是指具有天然免疫球蛋白分子结构的抗体分子。在常规四链IgG抗体的情况下，全长抗体包含由二硫键相互连接的两条重链(H)和两条轻链(L)。在仅具有重链而缺乏轻链的重链抗体情况下，全长抗体包含由二硫键相互连接的两条重链(H)。对于常规四链IgG抗体，全长抗体重链通常由重链可变区(本文中缩写为VH)和重链恒定区组成，其中重链恒定区至少包含3个结构域CH1、CH2和CH3。全长抗体轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成，其中轻链恒定区由一个结构域CL组成。每个重链可变区VH和每个轻链可变区都由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。术语“抗体片段”包括完整抗体的一部分。在优选的实施方案中，抗体片段为抗原结合片段。The terms "whole antibody" or "full-length antibody" are used interchangeably herein to refer to an antibody molecule having the molecular structure of a native immunoglobulin. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) inter-connected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, 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. 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.
术语抗体的“抗原结合片段”是与全长抗体不同的分子，其包含全长抗体的一部分，但其能结合全长抗体的抗原或与全长抗体(即与抗原结合片段所来源的全长抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、双体抗体(diabody)、单域抗体(sdAb)、纳米抗体。例如，通过木瓜蛋白酶消化全长抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab′)2，其为Fab’的二聚体，是二价的抗体片段。F(ab′)2可以在中性条件下通过破坏铰链区中的二硫键而被还原，由此将F(ab′)2二聚体转化为Fab′单体。Fab′单体基本上是具有铰链区的Fab片段。Fv片段由抗体单臂的VL和VH结构域组成。Fv片段的两个结构域VL和VH可以由独立的基因编码，但是也可以使用重组方法，使用合成性连接肽连接这两个结构域以使其作为单条蛋白链产生，并且在所述单条蛋白链中VL区和VH区配对以形成单链Fv(scFv)。The term "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. For example, 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).
“Fab片段”或“Fab”在本文中可互换使用，用于指，由两条多肽链组成的、包含免疫球蛋白重链可变结构域VH、重链恒定结构域CH1、轻链可变结构域VL和轻链恒定结构域CL的免疫球蛋白片段，其中，一条多肽链从N端到C端包含VH和选自CH1和CL的一个恒定区，另一条多肽链从N端到C端包含VL和选自CL和CH1的另一恒定区，其中所述VH结构域和VL结构域配对形成抗原结合位点。在本文中，包含重链恒定区CH1的Fab多肽链也称作“Fab重链”；相应地，包含轻链恒定区CL的Fab多肽链也称作“Fab轻链”。"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. Herein, 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".
术语“靶标”是指结合分子所针对的被结合物。靶标可以是抗原，也可以是配体或受体。The term "target" refers to a bound substance to which a binding molecule is directed. Targets can be antigens, ligands or receptors.
术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。如本文所用，术语“表位”指抗原中与抗体分子特异性相互作用的部分。“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派方案的任一种或其组合确定，所述指派方案包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883，A1-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological  Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinitypropagation clustering)的North CDR定义。The term "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. The skilled artisan will appreciate that any macromolecule, including essentially any protein or peptide, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" 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. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of 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. Department of Health and Human Services, National Institutes of Health (1987) ), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr/), and affinity propagation clustering based on exploiting a large number of crystal structures The North CDR definition.
以下为采用kabat、AbM、Chothia、Contact和IMGT方案定义的CDR的区域范围。The following are the regions of CDRs defined using the kabat, AbM, Chothia, Contact and IMGT schemes.
Figure PCTCN2022143967-appb-000005
Figure PCTCN2022143967-appb-000005
除非另有说明，否则在本发明中，术语“CDR，”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))的编号位置。Unless otherwise stated, in the present invention, the term "CDR," or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways. 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). Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it is meant according to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
在一本实施方案中，本发明的VHH或重链抗体中的CDR按照IMGT确定。本发明的抗PD1抗体中的VHCDR和VLCDR按照Kabat确定。In one embodiment, 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.
应该注意，基于不同的指派方案获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派方案下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派方案规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。It should be noted that 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. Thus, where reference is made to defining antibodies with a particular CDR sequence as defined in the present invention, 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.
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR(在同一指派方案下)。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量 的氨基酸位置直接参与抗原结合。使用Kabat，Chothia，AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs (under the same assignment scheme). However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the methods of Kabat, Chothia, AbM, Contact and North, the region of minimum overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. A minimal binding unit may be a subsection of a CDR. As will be apparent to those skilled in the art, 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.
术语“Fc结构域”或“Fc区”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”或“Fc区”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如Edelman，G.M.et a1.，Proc.Natl.Acad.USA，63，78-85(1969)(https：//pubmed.ncbi.nlm.nih.gov/5257969/)中所述的EU编号体系(也称作EU索引)进行编号，还参见http：//www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html。在本文中，术语“Fc区”、“Fc部分”和“Fc片段”不包括免疫球蛋白的重链可变区VH和轻链可变区VL以及重链恒定区CH1和轻链恒定区CL，但在一些情况下可以包括在重链恒定区N端的铰链区。The terms "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. For example, in native antibodies, 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. Unless otherwise stated herein, 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. USA, 63, 78-85 (1969) (https:// pubmed.ncbi.nlm.nih.gov/5257969/) for numbering according to the EU numbering system (also known as the EU Index), see also http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html. Herein, the terms "Fc region", "Fc part" and "Fc fragment" exclude 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.
术语“嵌合抗体”是这样的抗体分子，其中(a)将恒定区或其部分进行改变、替换或交换，从而抗原结合位点与具有不同的或改变的类别、效应子功能和/或物种来源的恒定区连接、或与赋予嵌合抗体新性能的完全不同的分子(例如，酶、毒素、激素、生长因子、药物)等连接；或(b)将可变区或其部分用具有不同或改变的抗原特异性的可变区改变、替换或交换。例如，驼源重链抗体可以通过将其恒定区更换为来自人免疫球蛋白的恒定区进行修饰。由于更换为人类恒定区，该嵌合抗体可以保留其在识别抗原方面的特异性，同时与原始驼源抗体相比，具有在人类中降低的抗原性。The term "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. For example, 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.
如本文所用，“人源化抗体”是一种保留非人类抗体(例如羊驼单克隆抗体)的抗原特异反应性，同时作为治疗药对人施用时免疫原性较低的抗体。这可以例如通过保留非人抗原结合位点并将抗体的剩余部分替换成它们的人类相应部分(即，将恒定区以及可变区中不参与结合的部分替换为人类抗体的相应部分)来实现。As used herein, 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) .
如本文所用，术语“抗”、“结合”或“特异性结合”意指结合作用对靶标或抗原是选择性的并且可以与不想要的或非特异的相互作用区别。结合位点与特定靶标或抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。As used herein, 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.
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。The term "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.
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数或改善可度量参数至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。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%.
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。A "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.
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer 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.
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。在一些实施方案中，标记是hFc或生物素。As used herein, the term "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. In some embodiments, the label is hFc or biotin.
“个体”或“受试者”可以互换的使用，包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。"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.
“分离的”抗体或分子是这样的抗体或分子，其已经与其天然环境的组分分离。在一些实施方案中，将抗体或分子纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, 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).
氨基酸序列的“同一性百分数(％)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。在一些实施方案中，本发明考虑本发明抗体分子的变体，所述变体相对于在本文中具体公开的抗体分子及其序列而言具有相当程度的同一性，例如同一性为至少80％，85％，90％，95％，97％，98％或99％或更高。所述变体可以包含保守性改变。"Percent identity (%)" of an amino acid sequence 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. In some embodiments, 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.
对于多肽序列，“保守性改变”包括对多肽序列的置换、缺失或添加，但不实质性改变多肽序列的期望功能活性。例如，保守性置换常常导致某个氨基酸置换为化学上相似的氨基酸。提供功能上相似氨基酸的保守性置换表是本领域熟知的。以下列出了8组含有互为保守替换的氨基酸：1)丙氨酸(A)、甘氨酸(G)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；7)丝氨酸(S)、苏氨酸(T)；和8)半胱氨酸(C)、甲硫氨酸(M)。在一些实施方案中，术语“保守序列改变”用于指不显著影响或改变含有氨基酸序列的本发明抗体分子或结合蛋白分子的目的抗原结合特征的氨基酸修饰。例如保守修改变体相对于亲本抗体或结合蛋白保持对目的抗原至少80％，85％，90％，95％，98％，99％或更高，例如100-110％或更高的结合亲和力。With respect to a polypeptide sequence, "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). In some embodiments, 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. For example 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.
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。The term "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.
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有 效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical 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.
术语“药物组合或组合产品”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的免疫缀合物、以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "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. The term "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.
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。The term "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. Such 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. Alternatively, such 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. Furthermore, such 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.
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。The term "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.
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors. The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth.
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.
如本文中使用的，“肿瘤相关抗原”指靶细胞的表面上呈现的抗原性决定簇，其中靶细胞是肿瘤中的细胞，诸如癌细胞、肿瘤基质的细胞。As used herein, "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.
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转疾病的症状、并发症、或生化指征的发作、缓解症状或阻止或抑制疾病、病状或病症的进一步发展。As used herein, "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.
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "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.
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。术语“表达载体”是指包含重组多核苷酸的载体，其包含有效连接要表达的核苷酸序列的表达控制序列。表达载体包含足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞提供或在体外表达系统中。表达载体包括本领域已知的所有那些，包括被掺入重组多核苷酸的粘粒、质粒(例如，裸的或包含在脂质体中)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。The term "vector" as used herein 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. The term "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).
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样 品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如泪液、玻璃体液、脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。在一些实施方案中，组织样品是肿瘤组织。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。"Subject/patient/individual 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. In some embodiments, 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.
本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the invention. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the appended claims.
具体实施方式Detailed ways
实施例Example
实施例1：羊驼免疫Example 1: Alpaca Immunization
选择健康强壮、精神状态良好、体型适中的A和B两只羊驼(成都阿帕克生物科技有限公司)开始免疫，免疫前各采血10mL，收集阴性血清作为免疫效价检测使用。免疫时，将完全弗氏佐剂与0.5mg抗原hTIGIT-Fc(ACRO，货号TIT-H5254)混合，乳化后皮下多点注射。第一次免疫后21天进行第二次免疫，将0.25mg抗原hTIGIT-Fc(ACRO，货号TIT-H5254)与不完全弗氏佐剂混合，乳化后皮下多点注射。总共进行7次免疫，每次免疫间隔3周，其中第五次和第七次采用纯化的食蟹猴TIGIT-11maFc蛋白(SEQ ID NO：47)与不完全弗氏佐剂混合免疫。Two alpacas A and B (Chengdu Apak Biotechnology Co., Ltd.) who are healthy, strong, in good spirits, and of moderate size were selected to start immunization. Before immunization, 10 mL of blood was collected from each, and negative serum was collected for the detection of immune titer. For immunization, mix complete Freund's adjuvant with 0.5 mg antigen hTIGIT-Fc (ACRO, product number TIT-H5254), emulsify and inject subcutaneously at multiple points. The second immunization was carried out 21 days after the first immunization. 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.
从第二次免疫开始，每次免疫后间隔7天采集10mL外周血，监测免疫反应。多次免疫后获得可用于建库的羊驼PBMC。From the second immunization, 10 mL of peripheral blood was collected 7 days after each immunization to monitor the immune response. Alpaca PBMCs that can be used for bank construction were obtained after multiple immunizations.
实施例2：羊驼免疫文库构建Example 2: Alpaca immune library construction
用Trizol法从羊驼外周血中提取总RNA，将5μg RNA反转录，应用PrimeScript TM II 1st Strand cDNA Synthesis Kit(Takara，货号6210A)，获得cDNA。将反转录获得cDNA进行巢氏PCR扩增，第一轮PCR获得VH和VHH产物，通过琼脂糖凝胶割胶回收750bp左右的VHH片段，再通过第二轮PCR获得大小约500bp的VHH片段，用DNA产物纯化试剂盒纯化VHH片段。将VHH片段利用酶切连接技术(NheI和NotI进行酶切)连接到噬菌体载体中(连接摩尔比例为载体∶VHH＝1∶3)。共进行了10次电击转化，电击之后立即向电击杯中加入1mL 2YT培养基(37℃预热)复苏，吸出电击产物并用2YT培养基洗净电击杯，共计获得10mL复苏产物，37℃，180rpm复苏60min，取100μL梯度稀释至10-3和10-4测定库转化子数目，涂布于90mr的平板上，其余涂布于10块200mm的平板上。第二天测定库容，经检测构建的文库库容为6×107CFU，文库插入率为100％。 Total RNA was extracted from alpaca peripheral blood by Trizol method, 5 μg of RNA was reverse transcribed, and cDNA was obtained by using PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, product number 6210A). The cDNA obtained by reverse transcription was amplified by nested PCR. The VH and VHH products were obtained in the first round of PCR. A VHH fragment of about 750 bp was recovered by agarose gel tapping, and a VHH fragment of about 500 bp in size was obtained by the second round of PCR. Purify the VHH fragments with a DNA product purification kit. The VHH fragments were ligated into the phage vector by enzyme-cut ligation technology (NheI and NotI were cut) (the molar ratio of ligation was vector:VHH=1:3). A total of 10 electric shock transformations were performed. Immediately after electric shock, 1 mL of 2YT medium (preheated at 37°C) was added to the electric shock cup for resuscitation, the electric shock product was sucked out and the electric shock cup was washed with 2YT medium, and a total of 10 mL of resuscitated product was obtained, 37°C, 180rpm After recovering for 60 minutes, take 100 μL of gradient dilution to 10-3 and 10-4 to determine the number of transformants in the library, spread on a 90mr plate, and spread the rest on ten 200mm plates. The library capacity was measured on the second day, and the library capacity of the constructed library was 6×107 CFU, and the insertion rate of the library was 100%.
随后利用噬菌体展示技术进行文库筛选，经3轮液相筛选，第一轮用20μg生物素标记(EZ-Link TM Sulfo-NHS-LC-Biotin试剂盒，Thermo，货号A39257)的cynoTIGIT蛋白(ACRO，货号TIT-C5223)吸附，0.1％PBST洗涤15次，最终再用1mL 100mM的三乙胺进行洗脱，取500μL的1M pH7.4的Tris-Hcl进行中和。取750μL洗脱噬菌体侵染5mL处于对数期的TG1，37℃静置30分钟，然后5000g离心5分钟，取1mL菌液涂布2YT(50 mg/mL羧苄青霉素+2％葡萄糖)固体平板。37℃倒置过夜培养，次日用10mL 2YT液体培养基刮下平板上所有菌落，取1mL菌液加入100mL 2YT(50μg/mL羧苄青霉素+2％葡萄糖)液体培养基中，培养至对数期，加入MOI比20∶1的辅助噬菌体M13K07(NEB，货号：N0315S)，37℃侵染30分钟，然后5000rpm离心10min，弃尽上清，用等体积2YT+Car+K(Car：50μg/mL、Kan：50μg/mL)培养基重悬沉淀，30℃，220rpm过夜。过夜培养物4℃，10000rpm离心20min，收取上清，弃沉淀。更换离心筒，4℃，10000rpm，离心20min，收取上清。Subsequently, the library was screened using phage display technology. After three rounds of liquid phase screening, 20 μg of cynoTIGIT protein (ACRO, Product No. TIT-C5223) adsorption, washed 15 times with 0.1% PBST, and finally eluted with 1mL 100mM triethylamine, and neutralized with 500μL 1M Tris-HCl pH7.4. Take 750 μL of the eluted phage to infect 5 mL of TG1 in the logarithmic phase, let stand at 37°C for 30 minutes, then centrifuge at 5000 g for 5 minutes, take 1 mL of the bacterial liquid and coat 2YT (50 mg/mL carbenicillin + 2% glucose) solid plate . Cultivate overnight at 37°C, scrape off all the colonies on the plate with 10mL 2YT liquid medium the next day, take 1mL of bacterial liquid and add it to 100mL 2YT (50μg/mL carbenicillin + 2% glucose) liquid medium, and cultivate to the logarithmic phase , add helper phage M13K07 (NEB, product number: N0315S) at an MOI ratio of 20:1, infect at 37°C for 30 minutes, then centrifuge at 5000rpm for 10 minutes, discard the supernatant, and use an equal volume of 2YT+Car+K (Car: 50μg/mL , Kan: 50 μg/mL) medium to resuspend the pellet, 30° C., 220 rpm overnight. The overnight culture was centrifuged at 10,000 rpm for 20 min at 4°C, the supernatant was collected, and the precipitate was discarded. Replace the centrifuge cylinder, centrifuge at 4°C, 10,000rpm for 20min, and collect the supernatant.
按上清体积的1/5加入PEG8000/NaCl，混匀，冰浴沉淀2小时以上。4℃，10000rpm，离心20min，弃上清，空离一次除尽上清。1mL 1×PBS悬起沉淀，加入1/5体积的PEG8000/NaCl二次沉淀1h。4℃，12000rpm，离心10min弃上清，空离一次除尽上清。根据沉淀的量，加入1×PBS重悬沉淀。取10μL库噬菌体用2YT梯度稀释，从10-8和10-9管中取10μL加到90μL的TG1菌液中，轻柔混匀。37℃静置30min，分别涂布羧苄抗性平板，过夜培养。次日数平板上克隆，计算噬菌体文库滴度。第二轮筛选采用10μg生物素标记的huTIGIT蛋白吸附，0.1％PBST洗涤15次，最终再用1mL 100mM的三乙胺进行洗脱，取500μL的1M pH7.4的Tris-Hcl进行中和。此后再按第一轮的方法进行扩增，获得第三轮筛选所需的噬菌体亚库，第三轮筛选采用10μg生物素标记的cynoTIGIT蛋白吸附，0.1％PBST洗涤15次，最终再用1mL 100mM的三乙胺进行洗脱，取500μL的1M pH7.4的Tris-Hcl进行中和。Add PEG8000/NaCl according to 1/5 of the volume of the supernatant, mix well, and precipitate in ice bath for more than 2 hours. Centrifuge at 10,000 rpm at 4°C for 20 minutes, discard the supernatant, and centrifuge once to remove the supernatant. Suspend the precipitate in 1mL 1×PBS, add 1/5 volume of PEG8000/NaCl for secondary precipitation for 1h. Centrifuge at 12,000 rpm at 4°C for 10 minutes to discard the supernatant, and centrifuge once to remove the supernatant. According to the amount of pellet, add 1×PBS to resuspend the pellet. Take 10 μL of library phage and dilute it with 2YT gradient, take 10 μL from 10-8 and 10-9 tubes and add it to 90 μL of TG1 bacterial solution, and mix gently. Let stand at 37°C for 30 minutes, coat carbenzyl-resistant plates respectively, and culture overnight. The next day, count the clones on the plate and calculate the titer of the phage library. In the second round of screening, 10 μg of biotin-labeled huTIGIT protein was adsorbed, washed 15 times with 0.1% PBST, and finally eluted with 1 mL of 100 mM triethylamine, and neutralized with 500 μL of 1M Tris-HCl at pH 7.4. Afterwards, amplify according to the method of the first round to obtain the phage sub-library required for the third round of screening. The third round of screening uses 10 μg 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 μL of 1M Tris-HCl at pH 7.4 was used for neutralization.
三轮筛选结束后，将洗脱的噬菌体稀释之后侵染处于对数期的TG1，涂布2YT(50μg/mL羧苄青霉素+2％葡萄糖)平板，37℃，过夜培养，次日，挑取平板上单克隆，进入含有400μL的2YT(50μg/mL羧苄青霉素)液体培养基的深孔板中培养，37℃，220rpm培养至对数期，加入1mM的IPTG，30℃，低温诱导过夜。第二日，将深孔板500g，离心5分钟，取上清待ELISA检测。After the three rounds of selection, the eluted phages were diluted and infected TG1 in the logarithmic phase, spread on 2YT (50 μg/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 μL of 2YT (50 μg/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. On the second day, centrifuge the deep well plate at 500 g for 5 minutes, and take the supernatant for ELISA detection.
用pH7.4的PBS缓冲液将huTIGIT-his(ACRO，货号TIT-H52H3)，cynoTIGIT-his(ACRO，货号TIT-C5223)稀释至4μg/mL浓度，以50μL/孔的体积加入96孔酶标板中，于4℃放置过夜，次日，弃去液体后，加入用PBS稀释的1％脱脂牛奶封闭液200μL/孔，37℃孵育箱孵育1小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.005％tween-20)洗板2次后，加入50μL/孔诱导上清，置37℃孵育箱孵育1小时，孵育结束后，弃去酶标板中的反应液，用PBST洗板2次后，加入50ul/孔稀释过的HRP标记的鼠抗HA标签的二抗(Sinobiological，货号100028-MM10)，7℃孵育1小时，用PBST洗板2次后，加入50μL/孔1M H2SO4终止反应，用酶标仪在波长450nm处读取吸收值，计算对huTIGIT，cynoTIGIT结合的克隆个数。将获得的阳性克隆全部进行测序鉴定，所有序列不同的抗体均作为候选对象，获得1G3-VHH、6F6-VHH、5H2-VHH核苷酸序列。Dilute huTIGIT-his (ACRO, Cat. No. TIT-H52H3) and cynoTIGIT-his (ACRO, Cat. No. TIT-C5223) with PBS buffer at pH 7.4 to a concentration of 4 μg/mL, and add 96-well enzyme labels at a volume of 50 μL/well Place the plate overnight at 4°C, and the next day, after discarding the liquid, add 200 μL/well of 1% skimmed milk blocking solution diluted with PBS, and incubate in a 37°C incubator for 1 hour to block. After blocking, discard the blocking solution, wash the plate twice with PBST buffer (pH7.4 PBS containing 0.005% tween-20), add 50 μL/well induction supernatant, and incubate in a 37°C incubator for 1 hour. Afterwards, discard the reaction solution in the ELISA plate, wash the plate twice with PBST, add 50ul/well diluted HRP-labeled mouse anti-HA-labeled secondary antibody (Sinobiological, Cat. No. 100028-MM10), and incubate at 7°C for 1 After washing the plate twice with PBST, 50 μL/well 1M H2SO4 was added to terminate the reaction, and the absorbance value was read at a wavelength of 450 nm with a microplate reader, and the number of clones bound to huTIGIT and cynoTIGIT was calculated. All the obtained positive clones were sequenced and identified, all antibodies with different sequences were used as candidates, and the nucleotide sequences of 1G3-VHH, 6F6-VHH, and 5H2-VHH were obtained.
将编码1G3-VHH、6F6-VHH、5H2-VHH和EPKSS连接肽分别插入含有人IgG1重链恒定区Fc序列(SEQ ID NO：40)的编码基因的表达载体pcDNA3.1(+)中，以获得表达抗TIGIT重链抗体(1G3、6F6、5H2)的质粒。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.
抗TIGIT的重链抗体1G3氨基酸序列如下(SEQ ID NO：7)，其中连接肽以粗体加下划线表示，恒定区Fc以斜体表示。The 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.
抗TIGIT的重链抗体6F6氨基酸序列如下(SEQ ID NO：2)，其中连接肽以粗体加下划线表示，恒定区Fc以斜体表示。The 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.
抗TIGIT的重链抗体5H2氨基酸序列如下(SEQ ID NO：10)，其中连接肽以粗体加下划线表示，恒定区Fc以斜体表示。The 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.
实施例3：抗TIGIT抗体的人源化和表达纯化Example 3: Humanization and expression purification of anti-TIGIT antibody
通过比对IMGT人类抗体重链可变区种系基因数据库(http：//www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)，分别挑选与实施例2中筛选的单域抗体1G3、6F6和5H2同源性高的重链可变区种系基因作为模板，将单域抗体的CDR分别移植到相应的人源模板中，形成次序为FRI-CDR1-FR2-CDR2-FR3-CR3-FR4的可变区序列。根据需要，将FR区中关键氨基酸回复突变为纳米抗体(VHH抗体)对应的氨基酸，以保证原有的亲和力，即得到人源化抗TIGIT VHH抗体。其中CDR区氨基酸残基的确定由IMGT编号系统确定并注释。By comparing the IMGT human antibody heavy chain variable region germline gene database (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi), 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. As required, 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.
抗体1G3人源化重链模板为IGHJ4*01，经人源化后得到人源化抗体1G3-H1，人源化可变区序列如下：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:
表1列出所述序列，并列出了相应的回复突变位点。Table 1 lists the sequences and lists the corresponding back mutation sites.
Figure PCTCN2022143967-appb-000006
Figure PCTCN2022143967-appb-000006
抗体6F6人源化重链模板为IGHV3-48*01，经人源化后得到人源化抗体6F6-H1，人源化可变区序列如下：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:
表2列出所述序列，并列出了相应的回复突变位点。Table 2 lists the sequences and lists the corresponding back mutation sites.
Figure PCTCN2022143967-appb-000007
Figure PCTCN2022143967-appb-000007
抗体5H2人源化重链模板为IGHV3-23*01，经人源化后得到人源化抗体5H2-H1V2，人源化可变区序列如下：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:
表3列出所述序列，并列出了相应的回复突变位点。Table 3 lists the sequences and lists the corresponding back mutation sites.
Figure PCTCN2022143967-appb-000008
Figure PCTCN2022143967-appb-000008
将编码1G3-H1-VH、6F6-H1-VH、5H2-H1V2-VH加EPKSS连接肽的核酸分别插入含有人IgG1重链恒定区Fc序列的编码基因的表达载体pcDNA3.1(+)中，以获得表达抗 TIGIT全长人源化重链抗体(1G3-H1、6F6-H1、5H2-H1V2)的质粒。依据制造商产品手册说明，使用ExpiCHO TM表达系统(ThermoFisher，货号A29133) 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)
将实施例2和3上文获得的编码1G3；6F6；5H2；1G3-H1；6F6-H1；5H2-H1V2的质粒转染ExpiCHO-S细胞以表达TIGIT抗体。转染后培养细胞10-12天，当细胞存活率下降到60％至70％时，收集上清液，使用MabSelect Sure蛋白A亲和层析系统(GE healthcare)纯化表达在上清液中的抗体，获得抗TIGIT重链抗体的同源二聚体重链抗体。浓缩纯化的抗体，无菌过滤，通过SDS-PAGE和分子排阻检测抗体蛋白的纯度大于95％，结果表明抗体的纯度符合要求，可以用于下一步实验。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.
对照抗体Tiragolumab的序列来自于专利WO2017053748A2，由通用生物系统(安徽)有限公司进行密码子优化和基因合成并克隆到表达载体pcDNA3.1(+)中。之后应用上述表达和纯化技术获得该对照抗体，在本文中后文称为Tiragolumab analog。The sequence of the 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.
实施例4：抗人TIGIT的重链抗体与人TIGIT蛋白、食蟹猴TIGIT蛋白的亲和力实验Example 4: Affinity experiment of anti-human TIGIT heavy chain antibody with human TIGIT protein and cynomolgus monkey TIGIT protein
在本实验中，根据厂商说明，利用ForteBio Octet RED96e检测了抗体与人TIGIT-his(ACRO，货号TIT-H52H3)、食蟹猴TIGIT蛋白(ACRO，货号TIT-C5223)的结合亲和力。In this experiment, according to the manufacturer's instructions, ForteBio Octet RED96e was used to detect the binding affinity of the antibody to human TIGIT-his (ACRO, catalog number TIT-H52H3) and cynomolgus monkey TIGIT protein (ACRO, catalog number TIT-C5223).
简要地，将AHC传感器(ForteBio，货号18-5060)放进Running Buffer(1X PBS Hyclone，货号SH30256.01，包含0.02％Tween20，pH7.0)，在室温条件下进行预平衡10min。在96孔板中，动力学实验方法按照以下步骤进行：a)用Running Buffer平衡基线100s，b)加入用Running Buffer稀释的抗人TIGIT的重链抗体，终浓度5μg/mL，固化200s，c)用Running buffer平衡基线300s，d)向各孔中加入用Running Buffer稀释的100nM人TIGIT蛋白和食蟹猴TIGIT蛋白，结合200s，解离600s。实验数据使用Fortebio Data Ahalysis软件1∶1结合模型进行拟合并计算。Briefly, put the AHC sensor (ForteBio, Cat. No. 18-5060) into Running Buffer (1X PBS Hyclone, Cat. No. SH30256.01, containing 0.02% Tween20, pH7.0), and pre-equilibrate at room temperature for 10 min. In a 96-well plate, 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 μg/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.
表4总结了本发明的抗人TIGIT的重链抗体与人TIGIT蛋白、食蟹猴TIGIT蛋白的结合亲和力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
表4Table 4
Figure PCTCN2022143967-appb-000009
Figure PCTCN2022143967-appb-000009
Figure PCTCN2022143967-appb-000010
Figure PCTCN2022143967-appb-000010
表4表明本申请构建的抗人TIGIT的重链抗体可以特异地结合人TIGIT蛋白和食蟹猴TIGIT蛋白并具有较高的结合活性。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.
实施例5：ELISA方法检测抗人TIGIT的重链抗体抑制人TIGIT蛋白与CD155结合Example 5: ELISA method detects that the heavy chain antibody against human TIGIT inhibits the binding of human TIGIT protein to CD155
将0.5μg/mL的人TIGIT(M22-P141)-Fc蛋白(SEQ ID NO：45)以50μL/孔包被在96孔板上，4℃孵育过夜。用封闭缓冲液(含有1％BSA的PBS溶液)将板在37℃孵育，封闭1h。封闭后，用PBST溶液(含有0.05％吐温20的PBS溶液)将板洗涤三次。用稀释液梯度稀释抗TIGIT抗体(起始终浓度为14nM，3倍稀释，7个浓度点)，并以1∶1的体积比与1.1μg/mL(混合前浓度)CD155-mFc(ACRO，货号CD5-H5254)混合，加入板中，在37℃孵育1h。孵育结束后用PBST溶液洗板。用稀释液稀释二抗(辣根过氧化物酶HRP标记亲和纯化山羊抗小鼠IgG，Fcγ，Jackson Immuno Research，货号115-035-164)，加入板中在37℃孵育1h，孵育结束后再次清洗，TMB显色15分钟，之后用1M H 2SO 4终止，之后于酶标仪中读取OD450m-OD620nm的吸光值，结果见图1。 0.5 μg/mL human TIGIT(M22-P141)-Fc protein (SEQ ID NO: 45) was coated on a 96-well plate at 50 μL/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 the anti-TIGIT antibody with diluent (the initial concentration is 14nM, 3-fold dilution, 7 concentration points), and mix it with 1.1μg/mL (concentration before mixing) CD155-mFc (ACRO, catalog number) at a volume ratio of 1:1. CD5-H5254) were mixed, added to the plate, and incubated 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, Fcγ, 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 OD450m-OD620nm in a microplate reader, the results are shown in Figure 1.
结果显示抗人TIGIT的重链抗体能够阻断人TIGIT蛋白与CD155结合，并且人源化TIGIT抗体阻断活性比阳性对照抗体Tiragolumab analog(来自于专利WO2017053748A2，SEQ ID NO：50，SEQ ID NO：51)更强。The results show that 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.
实施例6：抗TIGIT重链抗体与人、猴TIGIT细胞的结合活性Example 6: Binding activity of anti-TIGIT heavy chain antibody to human and monkey TIGIT cells
(1)通过细胞结合实验来判断本发明中的抗TIGIT重链抗体能否与稳定表达在HEK293细胞(中国科学院典型培养物保藏委员会细胞库，货号GNHu 43)上的人TIGIT蛋白结合。(1) Determine whether the anti-TIGIT heavy chain antibody of the present invention can 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) by cell binding experiments.
本实验采用流式细胞术进行测定。首先采用Lipofectamine TM 2000(Invitrogen，货号11668019)转染试剂，根据厂商说明，将编码人的TIGIT全长蛋白(氨基酸序列SEQ ID NO：46)的核苷酸构建到真核表达载体，然后转染到HEK293胞中，转染后48小时用0.3μg/mL嘌呤霉素(Puromycin Dihydrochloride，Gibco，货号A1113802)筛选3～5天，得到了高表达人TIGIT蛋白的细胞(即HEK293/人TIGIT细胞)。消化离心收集HEK293/人 TIGIT细胞，PBS重悬细胞后接种至96孔板(Corning，货号3799)，每孔1×10 5个细胞，96孔板离心后弃上清，将100μL梯度稀释的待测抗体(实施例3制备的1G3、6F6、5H2、1G3-H1、6F6-H1重链抗体，最高浓度为150nM，4倍稀释，总共8个浓度点)加入到HEK293/人TIGIT细胞中，4℃孵育30分钟后离心(1000rpm，5分钟)弃上清。每孔加入100μLPBS将细胞洗两遍后再加入100μl 1∶200稀释的荧光二抗R-PE-conjugated AffiniPure Goat Anti-Human IgG，FcγFragment Specific(Jackson ImmunoResearch，货号109-116-098)，4℃孵育30分钟。用PBS将细胞洗两遍后每孔加入100μL PBS重悬细胞，最后用Cytoflex(Beckman)流式细胞仪检测荧光信号。采用上文制备的Tiragolumab analog作为阳性对照。 This experiment was carried out by flow cytometry. First, using Lipofectamine TM 2000 (Invitrogen, product number 11668019) transfection reagent, according to the manufacturer's instructions, the nucleotide encoding human TIGIT full-length protein (amino acid sequence SEQ ID NO: 46) was constructed into a eukaryotic expression vector, and then transfected into HEK293 cells, and 48 hours after transfection, they were screened with 0.3 μg/mL puromycin (Puromycin Dihydrochloride, Gibco, Cat. No. A1113802) for 3 to 5 days to obtain cells that highly express human TIGIT protein (ie, HEK293/human TIGIT cells). . 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 ℃ for 30 minutes, centrifuge (1000 rpm, 5 minutes) and discard the supernatant. Add 100 μL PBS to each well to wash the cells twice, then add 100 μl 1:200 diluted fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific (Jackson ImmunoResearch, Cat. No. 109-116-098), and incubate at 4°C 30 minutes. After the cells were washed twice with PBS, 100 μL of PBS was added to each well to resuspend the cells, and finally the fluorescent signal was detected with a Cytoflex (Beckman) flow cytometer. The Tiragolumab analog prepared above was used as a positive control.
从图2所示的结果可知，测试的TIGIT重链抗体都能和HEK293/人TIGIT细胞结合，EC50在0.6386nM-0.7873nM，并且结合活性比阳性对照Tiragolumab更强(EC50为1.605nM)。From the results shown in Figure 2, it can be seen that 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).
(2)通过细胞结合实验来判断本发明中的抗TIGIT重链抗体能否与稳定表达在HEK293细胞(中国科学院典型培养物保藏委员会细胞库，货号GNHu 43)表面的食蟹猴TIGIT蛋白结合。首先采用Lipofectamine TM 2000(Invitrogen，货号11668019)转染试剂，根据厂商说明，将编码食蟹猴的TIGIT蛋白(氨基酸序列SEQ ID NO：48)的核苷酸构建到真核表达载体上，然后转染到HEK293细胞中，转染后48小时用0.3μg/mL嘌呤霉素(Puromycin Dihydrochloride，Gibco，货号A1113802)筛选3～5天，得到了高表达食蟹猴TIGIT蛋白的细胞。流式细胞术参见上文，将梯度稀释的待测抗体(实施例3制备的1G3、6F6、5H2、1G3-H1、6F6-H1重链抗体，初始浓度为150nM，4倍稀释，8个点)加入到细胞中，4℃孵育30分钟。用PBS将细胞洗两遍后加入荧光二抗R-PE-conjugated AffiniPure Goat Anti-Human IgG，FcγFragment Specific，4℃孵育30分钟。用PBS将细胞洗两遍后重悬细胞，最后用Cytoflex(Beckman)流式细胞仪检测荧光信号。采用Tiragolumab analog作为阳性对照。 (2) Determine whether the anti-TIGIT heavy chain antibody of the present invention can bind to the cynomolgus monkey TIGIT protein stably expressed on the surface of HEK293 cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, Cat. No. GNHu 43) by cell binding experiments. First, using Lipofectamine TM 2000 (Invitrogen, product number 11668019) transfection reagent, according to the manufacturer's instructions, 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 μg/mL puromycin (Puromycin Dihydrochloride, Gibco, Cat. No. A1113802) for 3 to 5 days, and cells highly expressing cynomolgus TIGIT protein were obtained. See above for flow cytometry, 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, FcγFragment 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.
从图3所示的结果可知，测试的TIGIT重链抗体可以与HEK293细胞上表达的食蟹猴TIGIT蛋白结合，并且结合活性比阳性对照Tiragolumab更强。From the results shown in Figure 3, it can be seen that 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.
实施例7：抗人TIGIT的重链抗体激活原代T细胞活性Example 7: Anti-human TIGIT heavy chain antibody activates primary T cell activity
为了检测抗TIGIT抗体激活T细胞释放细胞因子的活性，我们建立了原代T细胞激活试验系统，以CHO-K1/OKT3/CD155细胞为靶细胞，人外周血单核细胞PBMCs(上海儒百生物科技有限公司)中分离的CD8+T为效应细胞。In order to detect the activity of anti-TIGIT antibody to activate T cells to release cytokines, we established a primary T cell activation assay system, using CHO-K1/OKT3/CD155 cells as target cells, human peripheral blood mononuclear cells PBMCs (Shanghai Rubai Biotechnology Co., Ltd. CD8+ T cells isolated from Technology Co., Ltd.) are effector cells.
CHO-K1/OKT3/CD155靶细胞构建：采用Lipofectamine TM 2000(Invitrogen，货号11668019)转染试剂，根据厂商说明，将编码人CD155蛋白(氨基酸序列SEQ ID NO：49)的核苷酸构建到真核表达载体上，然后转染到CHO-K1细胞(ATCC，货号CCL-61)中，转染后48小时用4μg/mL嘌呤霉素(Puromycin Dihydrochloride，Gibco，货号A1113802)筛选3～5天，得到了稳定表达CD155蛋白的CHO-K1/CD155稳定细胞株。再将编码人OKT3蛋白的核苷酸(氨基酸序列SEQ ID NO：53)构建到真核表达载体上，采用相同方法转染到已构建的CHO-K1/CD155稳定细胞株上，转染后48小时用500μg/mL G418(Gibco，货号10131027)筛选10～14天，得到了高表达人OKT3蛋白的CHO-K1/CD155稳定细胞株(CHO-K1/OKT3/CD155细胞)。 Construction of 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 μg/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. Then the nucleotide (amino acid sequence SEQ ID NO: 53) encoding human OKT3 protein was constructed on the eukaryotic expression vector, and transfected into the established CHO-K1/CD155 stable cell line by the same method. After transfection, 48 After 10-14 days of screening with 500 μg/mL G418 (Gibco, Cat. No. 10131027), a CHO-K1/CD155 stable cell line (CHO-K1/OKT3/CD155 cells) with high expression of human OKT3 protein was obtained.
复苏PBMC细胞，参考美天旎细胞分选试剂盒说明书分离CD8+T细胞(Miltenyi  Biotec，货号130-096-495)，用含10％FBS(Gibco，货号10099-141C)的1640完全培养基(Gibco，货号22400-071)重悬后，调整细胞密度至1E6/mL，按每孔100μL(即每孔100,000个细胞)铺到96孔板(Corning，货号3599)中。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 μL per well (that is, 100,000 cells per well).
胰蛋白酶消化培养的靶细胞CHO-K1/OKT3/CD155，1000rpm离心5min收集细胞，弃上清，用含10％FBS(Gibco，货号10099-141)的1640完全培养基(Gibco，货号22400-071)重悬，调整靶细胞密度至1×105/ml，按每孔50μL(即每孔5,000个细胞)铺到96孔板(Corning，货号3599)中。准备4倍于最终检测浓度的实施例3制备的待测抗体6F6-H1和对照抗体Tiragolumab analog，待测抗体最终检测浓度为50nM和5nM，每孔50μL加到96孔板中，然后在放细胞培养箱继续孵育72h。孵育结束后，取出实验板，2000rpm离心3min使所有细胞沉到板底，小心吸取100μL上清至新的96孔板，检测上清中的人IFNγ(Cisbio，货号62HIFNGPEH)因子水平。结果见图4，表明抗TIGIT抗体能够激活CD8+T细胞释放细胞因子。Trypsinize the cultured target cells CHO-K1/OKT3/CD155, centrifuge at 1000rpm for 5min to collect the cells, discard the supernatant, and use 1640 complete medium (Gibco, catalog number 22400-071) containing 10% FBS (Gibco, catalog number 10099-141) ) to resuspend, adjust the target cell density to 1×105/ml, and plate 50 μL per well (ie, 5,000 cells per well) into a 96-well plate (Corning, Cat. No. 3599). Prepare the test antibody 6F6-H1 and the control antibody Tiragolumab analog prepared in Example 3 that are 4 times the final detection concentration. The final detection concentrations of the test antibody are 50nM and 5nM, add 50 μL 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 μL of the supernatant into a new 96-well plate, and detect the level of human IFNγ (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.
实施例8：抗人TIGIT重链序列优化及Fc改造Example 8: Sequence optimization and Fc modification of anti-human TIGIT heavy chain
以6F6-H1序列为模板，PCR扩增VHH序列并连接至IgG1-Fc恒定区N端，同时对该Fc区域进行以下位点突变：S239D、A330L、I332E(根据EU编号)，最终构建的载体经真核细胞表达得到抗体6F6-DLE(制备方法如实施例3)，其氨基酸序列如SEC ID NO：20所示。经在线工具Abysis(http：//www.abysis.org/abysis/)分析可知6F6-H1序列CDR2区的D63位天冬酰胺易发生酰胺位点变化，为消除脱酰胺作用影响，进行D63S(根据IMGT编码)突变，将6F6(D63S)的可变区序列按照前述方法构建至IgG1-Fc恒定区的N端，该Fc区域同时含有突变位点S239D、A330L、I332E(根据Kabat编码)，形成的载体经真核细胞表达，得到抗体6F6-DS-DLE，其氨基酸序列为SEC ID NO：22。Using the 6F6-H1 sequence as a template, 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. According to the analysis of the online tool Abysis (http://www.abysis.org/abysis/), the asparagine at the D63 position in the CDR2 region of the 6F6-H1 sequence is prone to amide site changes. In order to eliminate the influence of deamidation, D63S (according to IMGT coding) mutation, the 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.
应用实施例3中的方法表达上述优化后抗体。The method in Example 3 was used to express the above-mentioned optimized antibody.
实施例9：序列优化及Fc改造后的TIGIT重链抗体与人TIGIT蛋白、食蟹猴TIGITExample 9: TIGIT heavy chain antibody after sequence optimization and Fc modification, human TIGIT protein, cynomolgus monkey TIGIT 蛋白的亲和力实验protein affinity test
在本实验中，根据厂商说明，利用ForteBio Octet RED96e检测了抗体与人TIGIT-his(ACRO，货号TIT-H52H3)、食蟹猴TIGIT蛋白(ACRO，货号TIT-C5223)的结合亲和力。In this experiment, according to the manufacturer's instructions, ForteBio Octet RED96e was used to detect the binding affinity of the antibody to human TIGIT-his (ACRO, catalog number TIT-H52H3) and cynomolgus monkey TIGIT protein (ACRO, catalog number TIT-C5223).
简要地，将AHC传感器(ForteBio，货号18-5060)放进Running Buffer(1 X PBS Hyclone，货号SH30256.01，包含0.02％Tween20，pH7.0)，在室温条件下进行预平衡10min。在96孔板中，动力学实验方法按照以下步骤进行：a)用Running Buffer平衡基线180s，b)加入用Running Buffer稀释的抗人TIGIT的重链抗体(6F6-DS-DLE、6F6DLE和6F6-H1)，终浓度5μg/mL，固化200s，c)用Running buffer平衡基线300s，d)向各孔中加入用Running Buffer稀释的100nM人TIGIT蛋白和食蟹猴TIGIT蛋白，结合200s，解离600s。实验数据使用Fortebio Data Analysis软件1∶1结合模型进行拟合并计算。Briefly, 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. In the 96-well plate, 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 μg/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.
表5总结了序列优化及Fc改造后的抗人TIGIT的重链抗体与人TIGIT蛋白、食蟹猴TIGIT蛋白的结合亲和力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
表5table 5
Figure PCTCN2022143967-appb-000011
Figure PCTCN2022143967-appb-000011
表5表明本申请构建的PTM修饰及Fc改造后的抗人TIGIT的VHH抗体可以特异地结合人TIGIT蛋白和食蟹猴TIGIT蛋白，并且活性与改造前无明显差异。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.
实施例10：序列优化及Fc改造后的TIGIT重链抗体抑制人TIGIT蛋白与CD155结合Example 10: TIGIT heavy chain antibody after sequence optimization and Fc modification inhibits the binding of human TIGIT protein to CD155 的ELISA方法ELISA method
将0.5μg/mL的人TIGIT(M22-P141)-Fc(SEQ ID NO：45)以50μL/孔包被在96孔板上，4℃孵育过夜。用封闭缓冲液(含有1％BSA的PBS溶液)将板在37℃孵育，封闭1h。封闭后，用PBST溶液(含有0.05％吐温20的PBS溶液)将板洗涤三次。用稀释液梯度稀释抗TIGIT抗体(起始浓度为14nM，3倍梯度稀释)，并以1∶1的体积比与1.1μg/mL(混合前浓度)CD155-mFc(ACRO，货号CD5-H5254)混合，加入板中，在37℃孵育1h。孵育结束后用PBST溶液洗板。用稀释液稀释二抗(辣根过氧化物酶HRP标记亲和纯化山羊抗小鼠IgG，Fcγ，Jackson Immuno Research，货号115-035-164)，加入板中在37℃孵育1h，孵育结束后再次清洗，TMB显色15分钟，之后用1M H 2SO 4终止，之后于酶标仪中读取OD450nm-OD620nm的吸光值，结果见图5。 0.5 μg/mL human TIGIT(M22-P141)-Fc (SEQ ID NO: 45) was coated on a 96-well plate at 50 μL/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 μg/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, Fcγ, 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.
结果显示PTM修饰及Fc改造后的抗人TIGIT的重链抗体能够阻断人TIGIT蛋白与CD155结合，并且阻断能力与改造前无明显差异。The results showed that the anti-human TIGIT heavy chain antibody after PTM modification and Fc modification could block the binding of human TIGIT protein to CD155, and the blocking ability was not significantly different from that before modification.
实施例11：抗PD1/TIGIT双特异性抗体构建Example 11: Construction of anti-PD1/TIGIT bispecific antibody
本发明构建了如图6所示的两种结构的双特异性抗体，其中各个双特异性抗体的抗TIGIT抗体部分来源于上述人源化的6F6-H1抗体；抗PD1部分来源于专利WO2019219064A中的PD1抗体1B12，双特异性抗体恒定区分为IgG1和IgG4两种形式。这类双特异性抗体在本文中也称“抗PD1/TIGIT双特异性抗体”，在实施例中有时简称“双特异性抗体、双抗”。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.
本申请使用标准构建方法构建如图6所示结构的三种双抗D1、D4和E4，其中This application uses standard construction methods to construct three kinds of double antibodies D1, D4 and E4 with the structures shown in Figure 6, wherein
双特异性抗体D1的重链(D1-H)从N端到C端的顺序为抗PD1抗体重链可变区VH、CH1恒定区、抗TIGIT抗体VHH以及IgG1 Fc恒定区，其氨基酸序列为SEQ ID NO：30；双特异性抗体D1的轻链(D1-L)从N端至C端顺序为抗PD1轻链抗体可变区VL和轻链恒定区CL，其氨基酸序列为SEC ID NO：39；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;
双特异性抗体D4的重链(D4-H)从N端到C端的顺序为抗PD1重链抗体可变区VH、CH1恒定区、抗TIGIT抗体VHH以及IgG4 Fc恒定区，其氨基酸序列为SEQ ID NO：32；双特异性抗体D4的轻链(D4-L)从N端至C端顺序为抗PD1抗体可变区VH和轻链恒定区CL，其氨基酸序列为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;
双特异性抗体E4的重链从N端到C端的顺序为抗PD1抗体可变区、CH1恒定区、IgG4 Fc恒定区以及抗TIGIT抗体VHH，其氨基酸序列为SEC ID NO：33；双特异性抗体E4轻链从N端至C端顺序为抗PD1抗体可变区和轻链恒定区CL，其氨基酸序列为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.
如下方法构建各个双特异性抗体表达载体：Each bispecific antibody expression vector was constructed as follows:
D1-H：以专利申请公开号WO2019219064A中PD1抗体重链编码序列(SEQ ID NO：56，对应于WO2019219064A中的SEQ ID NO：23)为模板，扩增PD1重链可变区和恒定区CH1段的核苷酸序列；以6F6-H1为模板扩增TIGIT抗体可变区VHH的核苷酸序列，并采用重叠PCR技术将以上两段序列通过接头GGS拼接，并直接连接于含有人IgG1恒定区CH2-CH3的表达载体上，表达获得D1-H分子。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 On the expression vector of CH2-CH3 region, D1-H molecule can be obtained by expression.
D1-L：以WO2019219064A中PD1抗体轻链编码的序列(SEQ ID NO：39，对应于WO2019219064A的SEQ ID NO：39)为模板，扩增PD1轻链可变区和恒定区CL段的核苷酸序列，并连接于表达载体上，表达获得D1-L分子。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以WO2019219064A中PD1抗体重链编码序列(SEQ ID NO：55，对应于WO2019219064A中的SEQ ID NO：24)为模板，扩增PD1重链可变区和恒定区CH1段的核苷酸序列；以6F6-H1为模板扩增TIGIT抗体可变区VHH的核苷酸序列，并采用重叠PCR技术将以上两段序列通过连接肽GGS拼接；以人IgG4重链为模板扩增CH2-CH3段恒定区序列，采用重叠PCR技术将以上两段序列连接至表达载体上(两段序列之间没有连接肽)，表达获得D4-H分子。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：以WO2019219064A中PD1抗体轻链编码的序列(SEQ ID NO：39，对应于WO2019219064A的SEQ ID NO：39)为模板，扩增PD1轻链可变区和恒定区CL段的核苷酸序列，并连接于表达载体上，表达获得D4-L分子。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：以专利申请公开号WO2019219064A中PD1抗体重链编码序列(SEQ ID NO：56，对应于WO2019219064A中的SEQ ID NO：23)为模板，扩增PD1重链可变区和恒定区的核苷酸序列；以6F6-H1为模板扩增TIGIT抗体可变区VHH的核苷酸序列，并采用重叠PCR技术将以上两段序列拼接，并直接连接于表达载体上，表达获得E4-H分子。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：以WO2019219064A中PD1抗体轻链编码的序列(SEQ ID NO：39，对应于WO2019219064A的SEQ ID NO：39)为模板，扩增PD1轻链可变区和恒定区CL段的核苷酸序列，并连接于表达载体上，表达获得E4-L分子。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.
将上述各组编码重链和轻链的表达载体分别转染到ExpiCHO-S细胞以表达双抗。转 染后培养细胞10-12天，当细胞存活率下降到60％至70％时，收集上清液，使用MabSelect Sure蛋白A亲和层析系统(GE healthcare)纯化表达在上清液中的抗体，获得D1、D4和E4双抗。浓缩纯化的双抗，无菌过滤，通过SDS-PAGE和分子排阻检测抗体蛋白的纯度大于95％，结果表明抗体的纯度符合要求，可以用于下一步实验。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. 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, 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.
实施例12：抗PD1/TIGIT双特异性抗体与人TIGIT蛋白结合活性实验Example 12: Experiment of binding activity of anti-PD1/TIGIT bispecific antibody to human TIGIT protein
用ELISA的方法测定抗PD1/TIGIT双特异性抗体对人TIGIT蛋白的相对结合活性。The relative binding activity of the anti-PD1/TIGIT bispecific antibody to human TIGIT protein was determined by ELISA.
人TIGIT-his(ACRO，货号TIT-H52H3)用PBS(HyClone，货号SH30256.01)稀释至0.5μg/ml包被于96孔板，50μL/孔，4℃孵育过夜。通过在37℃与包含1％的BSA的PBS一起孵育1小时来封闭非特异性结合位点。封闭结束后，PBST(含0.05％Tween20的PBS)洗板三次。用结合缓冲液(含0.05％Tween20和0.5％BSA的PBS)稀释实施例11制备的抗PD1/TIGIT双特异性抗体和Tiragolumab analog(对照)(起始浓度为1.5nM，3倍梯度稀释，7个浓度点)，并与已包被的蛋白在37℃孵育1小时。孵育结束后PBST洗板三次，将过氧化物酶标记的羊抗人Fc二抗(Jackson Immuno Research，109-035-098)用结合缓冲液稀释至1：25000，37℃孵育1小时，再次洗涤，TMB显色15分钟后用1M H2SO4终止。Human TIGIT-his (ACRO, Cat. No. TIT-H52H3) was diluted with PBS (HyClone, Cat. No. SH30256.01) to 0.5 μg/ml, coated on a 96-well plate, 50 μL/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). 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.
测定了在450nm-620nm的吸光度，抗PD1/TIGIT双特异性抗体与人TIGIT蛋白的结合曲线见图7。The absorbance at 450nm-620nm was measured, and the binding curve of the anti-PD1/TIGIT bispecific antibody to human TIGIT protein is shown in Figure 7.
结果表明所有抗PD1/TIGIT双特异性抗体与人类TIGIT蛋白均有结合，其中D1与对照活性相当。The results showed that all anti-PD1/TIGIT bispecific antibodies bound to human TIGIT protein, and the activity of D1 was comparable to that of the control.
实施例13：抗PD1/TIGIT双特异性抗体与人PD1蛋白结合活性实验Example 13: Experiment on binding activity of anti-PD1/TIGIT bispecific antibody to human PD1 protein
用ELISA的方法测定抗PD1/TIGIT双特异性抗体对人类PD1蛋白的相对结合活性。The relative binding activity of the anti-PD1/TIGIT bispecific antibody to human PD1 protein was determined by ELISA.
人PD1-his蛋白(Sino，货号10377-H08H-100)用PBS(HyClone，SH30256.01)稀释至0.2μg/ml，包被于96孔板，50μl/孔，4℃孵育过夜。通过在37℃与包含1％的BSA的PBS一起孵育1小时来封闭非特异性结合位点。封闭结束后，PBST(含0.05％Tween20的PBS)洗板三次。用结合缓冲液(含0.05％Tween20和0.5％BSA的PBS)稀释实施例11制备的抗PD1/TIGIT双特异性抗体和1B12PD1(IgG1mut)(对照)(WO2019219064A的1B12PD1的IgG1突变体，序列为SEQ ID NO：39，SEQ ID NO：58)(起始浓度为1.5nM，3倍梯度稀释，7个浓度点)，并与已包被的蛋白在37℃孵育1小时。孵育结束后PBST洗板三次，将过氧化物酶标记的羊抗人Fc二抗(Jackson Immuno Research，109-035-098)用结合缓冲液稀释至1：25000，37℃孵育1小时，再次洗涤，TMB显色15分钟后用1M H2SO4终止。Human PD1-his protein (Sino, Cat. No. 10377-H08H-100) was diluted to 0.2 μg/ml with PBS (HyClone, SH30256.01), coated on a 96-well plate, 50 μl/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). 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. 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.
测定了在450nm-620nm的吸光度，抗PD1/TIGIT双特异性抗体与人PD1的结合曲线见图8。The absorbance at 450nm-620nm was measured, and the binding curve of the anti-PD1/TIGIT bispecific antibody to human PD1 is shown in Figure 8.
结果表明所有抗PD1/TIGIT双特异性抗体与人类PD1蛋白均有结合，其中D1与对照活性相当。The results showed that all anti-PD1/TIGIT bispecific antibodies bound to human PD1 protein, and the activity of D1 was comparable to that of the control.
实施例14：抗PD1/TIGIT双特异性抗体抑制人TIGIT蛋白与CD155蛋白结合活性Example 14: Anti-PD1/TIGIT bispecific antibody inhibits the binding activity of human TIGIT protein to CD155 protein
将0.5μg/mL的人TIGIT-人IgG1 Fc蛋白(SEQ ID NO.45)以50μL/孔包被在96孔板上，4℃孵育过夜。用封闭缓冲液(含有1％BSA的PBS溶液)将板在37℃孵育，封闭1h。封闭后，用PBST溶液(含有0.05％吐温20的PBS溶液)将板洗涤三次。用稀释液梯度稀释抗PD1/TIGIT双特异性抗体、对照Tiragolumab ahalog和1B12PD1(IgG1mut)(起始浓度为28nM，3倍梯度稀释，7个浓度点)，并分别与CD155(ACRO，货号CD5-H5254)混合，加入板中，在37℃孵育1h。孵育结束后用PBST溶液洗板。用稀释液稀释二抗(辣根过氧化物酶HRP标记亲和纯化山羊抗小鼠IgG，Fcγ，Jackson Immuno Research，货号115-035-164)，加入板中在37℃孵育1h，孵育结束后再次清洗，TMB显色15分钟，之后用1M H 2SO 4终止，之后于酶标仪中读取OD450nm-OD620nm的吸光值，结果见图9。采用Tiragolumab analog作为阳性对照，PD1单抗1B12PD1(IgG1mut)作为阴性对照。 0.5 μg/mL of human TIGIT-human IgG1 Fc protein (SEQ ID NO.45) was coated on a 96-well plate at 50 μL/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-PD1/TIGIT bispecific antibody, control Tiragolumab ahalog and 1B12PD1 (IgG1mut) with diluent (initial concentration is 28nM, 3-fold serial dilution, 7 concentration points), and respectively mix with CD155 (ACRO, Cat. No. CD5- H5254) were mixed, added to the plate, and incubated 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, Fcγ, 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 9. Tiragolumab analog was used as a positive control, and PD1 monoclonal antibody 1B12PD1 (IgG1mut) was used as a negative control.
结果显示D1、D4和E4均可阻断TIGIT蛋白与CD155蛋白结合，D1和D4阻断能力比阳性对照样品更好。The results showed that D1, D4, and E4 could all block the binding of TIGIT protein to CD155 protein, and the blocking ability of D1 and D4 was better than that of the positive control sample.
实施例15：抗PD1/TIGIT双特异性抗体阻断人类PD1与PD-L1相互作用Example 15: Anti-PD1/TIGIT bispecific antibody blocks the interaction between human PD1 and PD-L1
为了评估抗PD1/TIGIT双特异性抗体阻断人类PD1蛋白与PD-L1结合的能力，用ELISA的方法测定抗PD1/TIGIT双特异性抗体对人类PD1蛋白的相对抑制活性。In order to evaluate the ability of anti-PD1/TIGIT bispecific antibody to block the binding of human PD1 protein to PD-L1, the relative inhibitory activity of anti-PD1/TIGIT bispecific antibody to human PD1 protein was determined by ELISA.
人PD1(M1-V170)-人IgG1 Fc蛋白(SEQ ID NO：52)用PBS(HyClone，SH30256.01)稀释至0.5μg/ml包被于96孔板，50μl/孔，4℃孵育过夜。通过在37℃与包含1％的BSA的PBS一起孵育1小时来封闭非特异性结合位点。封闭结束后，PBST(含0.05％Tween20的PBS)洗板三次。用结合缓冲液(含0.05％Tween20和0.5％BSA的PBS)稀释抗PD1/TIGIT双特异性抗体(其浓度为22.5nM，3倍梯度稀释，7浓度点)，分别与稀释成0.8μg/ml的PD-L1蛋白(Sino，货号10084-H05H)按1∶1混合后与已包被的蛋白在37℃孵育1小时。采用PD1单抗1B12PD1(IgG1mut)作为阳性对照，IgG(SinoBiological货号HG1K)为阴性对照。孵育结束后PBST洗板三次，将过氧化物酶标记的羊抗小鼠Fc二抗(Jackson Immuno Research，115-035-164)用结合缓冲液稀释至1：10000，37℃孵育1小时，再次洗涤，TMB显色15分钟后用1M H2SO4终止。Human PD1 (M1-V170)-human IgG1 Fc protein (SEQ ID NO: 52) was diluted with PBS (HyClone, SH30256.01) to 0.5 μg/ml, coated on a 96-well plate, 50 μl/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). 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 0.8μg/ml respectively 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. After the incubation, 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.
测定了在450nm-620nm的吸光度，抗人类PD1/TIGIT双特异性抗体PD1的抑制曲线，见图10。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 .
结果表明所有抗PD1/TIGIT双特异性抗体均能阻断人类PD1蛋白与PD-L1的相互作用，并且所有抗体抑制活性与对照相当。The results showed that all anti-PD1/TIGIT bispecific antibodies could block the interaction between human PD1 protein and PD-L1, and the inhibitory activity of all antibodies was comparable to that of the control.
实施例16：抗PD1/TIGIT双特异性抗体与人TIGIT、人PD1细胞结合活性Example 16: Binding activity of anti-PD1/TIGIT bispecific antibody to human TIGIT and human PD1 cells
(1)通过细胞结合实验来判断抗PD1/TIGIT双特异性抗体能否与稳定表达在HEK293细胞(中国科学院典型培养物保藏委员会细胞库，货号GNHu 43)上的人TIGIT蛋白结合。实验过程和方法如实施例6(1)，采用Tiragolumab ahalog作为阳性对照，PD1单抗1B12PD1(IgG1 mut)作为阴性对照。(1) 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.
从图11所示的结果可知，测试的PD1/TIGIT双特异性抗体都能和HEK293/人TIGIT 细胞结合。From the results shown in Figure 11, it can be seen that all tested PD1/TIGIT bispecific antibodies can bind to HEK293/human TIGIT cells.
(2)通过细胞结合实验来判断抗PD1/TIGIT双特异性抗体能否与稳定表达在Jurkat/NFAT-Luc细胞(稳定表达NFAT-Luc报告基因元件的Jurkat细胞，构建方法参见WO2019219064A)表面的人PD1蛋白结合。实验过程和方法如实施例6(1)，采用PD1单抗1B12PD1(IgG1 mut)作为阳性对照，Tiragolumab analog作为阴性对照。(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.
从图12所示的结果可知，测试的PD1/TIGIT双特异性抗体可以与Jurkat/NFAT-Luc/hPD1细胞结合。From the results shown in Figure 12, it can be seen that the tested PD1/TIGIT bispecific antibody can bind to Jurkat/NFAT-Luc/hPD1 cells.
实施例17：抗PD1/TIGIT双特异性抗体对激活后CD4+T和CD8+T细胞的ADCC杀Example 17: ADCC killing of activated CD4+T and CD8+T cells by anti-PD1/TIGIT bispecific antibody 伤活性Traumatic activity
为了检测抗PD1/TIGIT IgG1亚型的双特异性抗体是否介导NK细胞杀伤激活后的CD4+T和CD8+T细胞的活性，我们建立了原代NK细胞依赖的细胞毒性试验系统，分别以激活后的CD4+T和CD8+T为靶细胞，人外周血单核细胞PBMCs为效应细胞。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.
靶细胞准备：用预冷的PBS配制含1μg/mL OKT3(Invitrogen，货号16-0037-85)和1μg/mL anti-CD28(Biolegend，货号302934)的包板悬液，取5mL加入至10cm细胞培养皿中，4度孵育过夜。第二天，弃去包板液，用预冷的PBS洗涤一次备用。复苏PBMC细胞，参考美天旎细胞分选试剂盒说明书分别分离CD4+T(Miltenyi Biotec，货号130-096-533)或者CD8+T细胞(Miltenyi Biotec，货号130-096-495)，用含10％FBS(Gibco，货号10099-141C)的1640完全培养基(Gibco，货号22400-071)将CD4+T细胞或CD8+T细胞重悬后，调整细胞密度至5E5/mL，然后加入包被好的10cm细胞培养皿中，放入细胞培养箱培养72小时。Target cell preparation: prepare a plate suspension containing 1 μg/mL OKT3 (Invitrogen, catalog number 16-0037-85) and 1 μg/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. 22400-071) with %FBS (Gibco, Cat. No. 10099-141C) to resuspend CD4+T cells or CD8+T cells, adjust the cell density to 5E5/mL, and then add the well-coated 10cm cell culture dish, placed in a cell culture incubator for 72 hours.
复苏PBMC细胞，用含10％FBS的1640完全培养基调整细胞密度至1～2E6/mL，加入IL2(江苏金丝利药业有限公司)激活过夜(体系中终浓度为100IU/mL)，作为效应细胞悬液。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.
第二天上午分别离心收集激活后的CD4+T和CD8+T靶细胞，1000rpm离心5min，弃上清，用含1％FBS(Gibco，货号10099-141)的MEM-α测试缓冲液(Gibco，货号41061-029)调整靶细胞密度至2×10 5/ml，按每孔50μL(即每孔10,000个细胞)铺到96孔板(Corning，货号3599)中。准备4×最终检测浓度的待测抗体D1和对照Tiragolumab analog，抗体最高检测浓度为50nM，进行5倍梯度系列稀释共获得8个浓度点，每孔加入50μL。 The next morning, 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-α test buffer (Gibco , Cat. No. 41061-029) to adjust the target cell density to 2×10 5 /ml, spread 50 μL per well (that is, 10,000 cells per well) into a 96-well plate (Corning, Cat. No. 3599). Prepare the antibody D1 to be tested and the control Tiragolumab analog at 4×final detection concentration. 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 μL to each well.
实验同时设置靶细胞最大杀伤(靶细胞中加2％Triton100裂解液)、最小杀伤(靶细胞中加测试缓冲液)及自然杀伤(靶细胞中仅加效应细胞悬液)对照。In the experiment, the maximum killing of target cells (adding 2% Triton100 lysate to target cells), minimum killing (adding test buffer to target cells) and natural killing (adding only effector cell suspension to target cells) controls were set.
PBMC效应细胞悬液中含有200IU/mL IL2(江苏金丝利药业有限公司)。在上述各个包含靶细胞的孔中，最后加入100μL的含IL2的PBMC效应细胞悬液，使得最终细胞密度至1×10 6/mL(效应细胞E∶靶细胞T＝10∶1)，然后在细胞培养箱继续孵育24h。孵育结束后，取出实验板，2000rpm离心3min使所有细胞沉到板底，小心吸取50μL上清至新的96孔板，加入50μLLDH检测液(Roche，货号11644793001)室温孵育，颜色变化时在F50酶标仪上读板，检测波长为OD492nm，背景波长为OD650m。结果见图13，表明D1不能特异性诱导NK细胞杀伤激活后的CD4+T和CD8+T细胞，和Tiragolumab analog ADCC杀伤活性相当，提示D1结构安全性较好。 The PBMC effector cell suspension contained 200IU/mL IL2 (Jiangsu Kingsley Pharmaceutical Co., Ltd.). In each of the above-mentioned wells containing target cells, 100 μL of IL2-containing PBMC effector cell suspension was finally added to make the final cell density reach 1×10 6 /mL (effector cell E:target cell T=10:1), and then in The cell incubator continued to incubate for 24h. 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 50 μL of the supernatant into a new 96-well plate, add 50 μL of LDH detection solution (Roche, catalog number 11644793001) and incubate at room temperature. Read the plate on the standard instrument, the detection wavelength is OD492nm, and the background wavelength is OD650m. The results are shown in Figure 13, indicating that D1 cannot specifically induce NK cells to kill activated CD4+T and CD8+T cells, which is equivalent to Tiragolumab analog ADCC killing activity, suggesting that D1 has better structural safety.
实施例18：抗PD1/TIGIT双特异性抗体在大鼠中的药代动力学Example 18: Pharmacokinetics of anti-PD1/TIGIT bispecific antibody in rats
在大鼠模型中评价D1、D4与E4的药代动力学特征。The pharmacokinetic characteristics of D1, D4 and E4 were evaluated in a rat model.
在本研究中，D1、D4与E4分别以10mg/kg的剂量静脉注射到大鼠(澎立生物医药技术)体内，在0～336h(0～14day)的不同时间点(给药前，给药后10min，30min，1h，4h，8h，24h，48h，7day，10day，14day)采集血样。所有样品处理成血浆，在-70～-86℃冰冻保存直至分析。In this study, 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.
人TIGIT-his(ACRO，货号TIT-H52H3)蛋白用PBS(Biosharp，货号BL302A)稀释至0.3μg/mL，50μL/孔加入酶标板中(Costar，货号42592)4℃孵育过夜。然后用含1％牛血清白蛋白(上海生工，货号：A500023-0025g)的PBS溶液中在37℃孵育1小时。封闭结束后，用PBST(含0.05％吐温-20的PBS)清洗3次。Human TIGIT-his (ACRO, Cat. No. TIT-H52H3) protein was diluted to 0.3 μg/mL with PBS (Biosharp, Cat. No. BL302A), 50 μL/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或E4在含血清的稀释缓冲液(含0.05％吐温-20、0.5％牛血清白蛋白、2％v/v大鼠血清)中稀释，起始浓度为50n扣L，2倍倍比稀释6个浓度点，共7个浓度点的抗体溶液为标准曲线。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.
同时用含血清的稀释缓冲液稀释D1、D4或E4至浓度分别为30ng/mL、6ng/mL和1.5ng/mL，作为高、中、低质控。所有大鼠血清均用空白混合大鼠血清和稀释缓冲液(含0.05％吐温-20和0.5％牛血清白蛋白的PBS)稀释，使抗体最终浓度保持在30～1.5ng/mL。At the same time, 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.
将标准曲线、质控和血浆样品加入酶标板中，37℃孵育1h。Add the standard curve, quality control and plasma samples to the microtiter plate and incubate at 37°C for 1h.
然后用PBST洗涤3次，将山羊抗人IgG Fc特异性抗体(Jackson ImmunoReasearch，货号109-035-098)在稀释缓冲液中稀释40000倍或20000倍，加入酶标板中37℃孵育1h，然后用PBST再次洗涤。在酶标板中加入50μL/孔TMB(Thermo，货号34029)，13分钟后，用1M H2SO4终止反应，测定450～620nm的吸光度。并通过软件计算药代动力学参数，列于表6、表7和表8中。Then wash 3 times with PBST, dilute the goat anti-human IgG Fc specific antibody (Jackson ImmunoReasearch, Cat. No. 109-035-098) in the dilution buffer 40000 times or 20000 times, add to the microtiter plate and incubate at 37°C for 1h, then Wash again with PBST. Add 50 μL/well TMB (Thermo, Cat. No. 34029) to the microtiter plate. After 13 minutes, stop the reaction with 1M H2SO4 and measure the absorbance at 450-620 nm. And the pharmacokinetic parameters are calculated by software, which are listed in Table 6, Table 7 and Table 8.
人PD1-mFc蛋白(Sino，货号10377-H05H)用PBS(Biosharp，货号：BL302A)稀释至0.2μg/mL，50μL/孔加入酶标板中(Costar，货号42592)4℃孵育过夜。然后用含1％牛血清白蛋白(上海生工，货号：A500023-0025g)的PBS溶液中在37℃孵育1小时。封闭结束后，用PBST(含0.05％吐温-20的PBS)清洗3次。D1、D4和E4在含血清的稀释缓冲液(含0.05％吐温-20、0.5％牛血清白蛋白、2％v/v大鼠血清)稀释，起始浓度为50ng/mL，2倍倍比稀释6个浓度点，共7个浓度点的抗体溶液为标准曲线。Human PD1-mFc protein (Sino, Cat. No. 10377-H05H) was diluted with PBS (Biosharp, Cat. No.: BL302A) to 0.2 μg/mL, 50 μL/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 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.
同时用含血清的稀释缓冲液稀释D1、D4或E4至浓度分别为30ng/mL、6ng/mL和1.5ng/mL，作为高、中、低质控。所有大鼠血清均用空白混合大鼠血清和稀释缓冲液(含0.05％吐温-20和0.5％牛血清白蛋白的PBS)稀释，使最终浓度保持在30～1.5ng/mL。At the same time, 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.
将标准曲线、质控和血浆样品加入酶标板中，37℃孵育1h。然后用PBST洗涤3次，将驴抗人IgG重轻链特异性抗体(Jackson ImmunoReasearch，货号709-035-149)在稀释缓冲液中稀释10000倍，加入酶标板中37℃孵育1h，然后用PBST再次洗涤。在酶标板中加入50μL/孔TMB(Thermo，货号34029)，13分钟后，用1M H2SO4终止反应，测定450～620nm的吸光度。并通过软件计算药代动力学参数，列于表9、表10和表11中。Add the standard curve, quality control and plasma samples to the microtiter plate and incubate at 37°C for 1h. Then washed 3 times with PBST, the donkey anti-human IgG heavy and light chain specific antibody (Jackson ImmunoReasearch, product number 709-035-149) was diluted 10000 times in the dilution buffer, added to the microtiter plate and incubated at 37 ° C for 1 h, and then used Wash again with PBST. Add 50 μL/well TMB (Thermo, Cat. No. 34029) to the microtiter plate. After 13 minutes, stop the reaction with 1M H2SO4 and measure the absorbance at 450-620 nm. And the pharmacokinetic parameters are calculated by software, which are listed in Table 9, Table 10 and Table 11.
表6 D1 TIGIT药代动力学特性汇总Table 6 Summary of pharmacokinetic properties of D1 TIGIT
Figure PCTCN2022143967-appb-000012
Figure PCTCN2022143967-appb-000012
表7 D4 TIGIT药代动力学特性汇总Table 7 Summary of D4 TIGIT Pharmacokinetic Properties
Figure PCTCN2022143967-appb-000013
Figure PCTCN2022143967-appb-000013
表8 E4 TIGIT药代动力学特性汇总Table 8 Summary of Pharmacokinetic Properties of E4 TIGIT
Figure PCTCN2022143967-appb-000014
Figure PCTCN2022143967-appb-000014
Figure PCTCN2022143967-appb-000015
Figure PCTCN2022143967-appb-000015
表9 D1 PD-1药代动力学特性汇总Table 9 D1 Summary of PD-1 pharmacokinetic properties
Figure PCTCN2022143967-appb-000016
Figure PCTCN2022143967-appb-000016
表10 D4 PD-1药代动力学特性汇总Table 10 Summary of D4 PD-1 pharmacokinetic properties
Figure PCTCN2022143967-appb-000017
Figure PCTCN2022143967-appb-000017
Figure PCTCN2022143967-appb-000018
Figure PCTCN2022143967-appb-000018
表11 E4 PD-1药代动力学特性汇总Table 11 Summary of pharmacokinetic properties of E4 PD-1
Figure PCTCN2022143967-appb-000019
Figure PCTCN2022143967-appb-000019
上述药代动力学实验表明，本发明的抗体在大鼠机体内具有一般抗体的药代动力学特征，稳定性和成药性好，适于成药。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.
实施例19：构建TIGIT/CTLA4双特异性抗体Example 19: Construction of TIGIT/CTLA4 bispecific antibody
本发明构建了如图14所示的三种结构的TIGIT/CTLA4双特异性抗体，其中各个双特异性抗体的抗TIGIT抗体可变区部分来源于6F6-D63S，抗CTLA4抗体可变区部分来源于伊匹单抗(Ipilimumab)。双特异性抗体恒定区为IgG1(DLE)形式。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.
本申请使用标准构建方法构建如图示14结构的三种双抗THC4、CT1KH、CT2KH，其中双特异性抗体THC4的重链(命名为HTC)从N端到C端的顺序为抗TIGIT抗体重链可变区VHH、抗CTLA4抗体重链可变区VH、IgG1恒定区CH1以及IgG1 Fc恒定区，其中Fc恒定区进行(S239D、A330L、I332E)简称“DLE”的突变(参考文献“Engineered antibody Fc variants with enhanced effector function.Proc Natl Acad Sci USA.2006 Mar 14；103(11)：4005-10.”)，重链HTC氨基酸序列为SEC ID NO：69。双特异性抗体THC4的轻链(命名为ipilimumab LC)从N端至C端顺序为抗CTLA4抗体轻链抗体可变区VL和轻链恒定区CL，其氨基酸序列为SEC ID NO：68。This application uses a standard construction method to construct three kinds of double-antibody THC4, CT1KH, and CT2KH, as shown in Figure 14. 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.
双特异性抗体CT1KH的重链1(命名为CK)从N端到C端的顺序为抗CTLA4抗体重链可变区VH、IgG1恒定区CH1以及IgG1 Fc恒定区，其中Fc恒定区进行点突变(S239D、A330L、I332E、T366W、S354C)形成ADCC增强型的IgG1“knob”链(参考文献“Engineered antibody Fc variants with enhanced effector function.Proc Nat1Acad Sci USA.2006 Mar 14；103(11)：4005-10.”和Merchant，A.M.，et al.(1998).″An efficient route to human  bispecific IgG.″Nat Biotechnol 16(7)：677-681.“Engineered antibody Fc variants with enhanced effector function”)，重链CK的氨基酸序列为SEQ ID NO：71；双特异性抗体CT1KH的重链2(命名为TH)从N端到C端的顺序为抗TIGIT抗体的可变区VHH和恒定区IgG1Fc，其中恒定区IgG1 Fc区进行点突变(S239D、A330L、I332E、Y349C、T366S、L368A、Y407V)以形成ADCC增强型IgG1 Fc“hole”链(参考文献“Engineered antibody Fc variants with enhanced effector function.Proc Natl Acad Sci USA.2006Mar 14；103(11)：4005-10.”和Merchant，A.M.，et al.(1998).″An efficient route to human bispecific IgG.″Nat Biotechnol 16(7)：677-681.)，重链TH的氨基酸序列详见SEQ ID NO：72。双特异性抗体CT1KH的轻链(命名为ipilimumab LC)其氨基酸序列为SEQ 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). "An efficient route to human bispecific IgG." Nat Biotechnol 16 (7): 677-681. "Engineered antibody Fc variants with enhanced effector function"), heavy chain CK The amino acid sequence of the amino acid sequence is SEQ ID NO: 71; the heavy chain 2 (named as TH) of the bispecific antibody CT1KH is sequenced from N-terminal to C-terminal as the variable region VHH and constant region IgG1Fc of the anti-TIGIT antibody, wherein the constant region IgG1 Fc Point mutations (S239D, A330L, I332E, Y349C, T366S, L368A, Y407V) were performed in the region to form an ADCC-enhanced IgG1 Fc "hole" chain (reference "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA.2 006Mar 14;103(11):4005-10." and Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.), heavy chain TH The amino acid sequence of is shown in SEQ ID NO: 72. The amino acid sequence of the light chain of the bispecific antibody CT1KH (named ipilimumab LC) is SEQ ID NO: 68.
双特异性抗体CT2KH的重链1(命名为CK)，详细氨基酸序列为SEQ ID NO：71；重链2(命名为2TH)，从N端到C端的顺序为抗TIGIT抗体的可变区VHH、接头(SEQ ID NO：73)、抗TIGIT抗体的可变区VHH、接头(SEQ ID NO：70)和恒定区IgG1Fc，其中恒定区Fc进行点突变(S239D、A330L、I332E、Y349C、T366S、L368A、Y407V)以形成ADCC增强型IgG1 Fc“hole”链(参考文献“Engineered antibody Fc variants with enhanced effector function.Proc Natl Acad Sci USA.2006Mar 14；103(11)：4005-10.”和Merchant，A.M.，et al.(1998).″An efficient route to human bispecific IgG.″Nat Biotechnol 16(7)：677-681.)，2TH氨基酸序列为SEQ ID NO：74。双特异性抗体CT2KH的轻链(命名为ipilimumab LC)其氨基酸序列为SEQ ID NO：68。The heavy chain 1 (named CK) of the bispecific antibody CT2KH, the detailed amino acid sequence is SEQ ID NO: 71; the heavy chain 2 (named 2TH), the sequence from the N-terminal to the C-terminal is the variable region VHH of the anti-TIGIT antibody , linker (SEQ ID NO: 73), variable region VHH of anti-TIGIT antibody, linker (SEQ ID NO: 70) and constant region IgG1Fc, wherein constant region Fc carries out point mutation (S239D, A330L, I332E, Y349C, T366S, L368A, Y407V) to form ADCC enhanced IgG1 Fc "hole" chain (reference "Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006 Mar 14; 103 (11): 4005-10." and Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16 (7): 677-681.), 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.
如下方法构建各个双特异性抗体表达载体：Each bispecific antibody expression vector was constructed as follows:
HTC：以6F6-DS-DLE质粒为模板，扩增6F6-D63S VHH片段，以ipilimumab-HC为模板扩增ipilimumab VH-CH1，采用重叠PCR技术将以上两段序列通过接头GGS拼接，并重组于含Fc的(Fc包含S239D、A330L、I332E点突变)表达载体上，形成HTC完整重链表达载体。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.
CK：以ipilimumab-HC为模板，扩增ipilimumab VHCH1，以ipilimumab-H IgG1wt Rknob(该恒定区含有T366W、S354C点突变形成knob结构)为模板扩增IgG1 Fc恒定区，并将Fc恒定区进行“DLE”点突变(S239D、A330L、I332E)，将以上片段重组于表达载体上，形成重链CK表达载体。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和2TH表达载体为通用生物(安徽)股份有限公司基因合成得到。TH and 2TH expression vectors were synthesized by General Biology (Anhui) Co., Ltd.
实施例20：抗TIGIT/CTLA-4双特异性抗体的结合亲和力Example 20: Binding affinity of anti-TIGIT/CTLA-4 bispecific antibodies
在该实施例中，使用ForteBio Octet RED 96e(生物层干涉测量法)测定抗TIGIT//CTLA-4双特异性抗体与人TIGIT蛋白、人CTLA-4蛋白的结合亲和力(KD)。In this example, ForteBio Octet RED 96e (biolayer interferometry) was used to determine the binding affinity (KD) of anti-TIGIT//CTLA-4 bispecific antibody to human TIGIT protein and human CTLA-4 protein.
简要地，将AHC(IgG Fc捕获)传感器尖端(ForteBio，货号18-5060)在室温下PBST(PBS，0.02％Tween20，pH7.0)中预平衡10分钟。动力学实验在96孔板中进行，按以下步骤进行：a)在PBST中平衡基线180秒，b)分别加载5ug/mL抗TIGIT/CTLA-4双特异性抗体(THC4、CT2KH和CT1KH)和对照单抗(6F6-H1和Ipilimumab)200s停止，c)平衡基线300秒，d)与带有His标签的人TIGIT(SEC ID NO：)或人CTLA-4(Sino Biological，货号11159-H08H)以浓度为100nM分别缔合200秒，和e)在PBST中解离600秒。使用Fortebio Data Analysis软件对数据集进行1∶1局部拟合模型拟合。Briefly, 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 the data set using Fortebio Data Analysis software.
表12总结了抗TIGIT/CTLA-4双特异性抗体(THC4、CT2KH和CT1KH)和对照单抗(6F6-H1和Ipilimumab analog)与人TIGIT蛋白、人CTLA-4蛋白的结合亲和力。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.
表12Table 12
Figure PCTCN2022143967-appb-000020
Figure PCTCN2022143967-appb-000020
结果表明THC4、CT2KH、CT1KH均可与人TIGIT及CTLA-4特异性结合，并且与单抗相比TIGIT和CTLA4结合活性没有明显变化。The results showed that 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.
实施例21：抗TIGIT/CTLA-4双特异性抗体抑制人TIGIT蛋白与人CD155蛋白结合活性Example 21: Anti-TIGIT/CTLA-4 bispecific antibody inhibits the binding activity of human TIGIT protein to human CD155 protein
高结合透明聚苯乙烯96孔板用在磷酸盐缓冲液PBS中的0.5μg/mL人TIGIT(M22-P141)-Fc(SEQ ID NO：45)以50μL/孔在4℃孵育过夜包被。然后使用PBST溶液(含有0.05％吐温20的PBS溶液)在自动洗板机上洗涤板一次。每孔加入200μL封闭缓冲液(含有1％BSA的PBS溶液)，37℃孵育1小时。用稀释缓冲液(PBS+0.5％牛血清白蛋白+0.05％吐温20)制备逐级稀释抗体(6F6-H1、THC4、CT2KH和CT1KH)和阴性对照抗体(Ipilimumab analog)(起始浓度：100nM(混合前浓度)，3倍稀释)并以1∶1的体积比与1.1μg/mL(混合前浓度)CD155-mFc((Acro Biosystem，货号CD5-H5254)混合，然后将混合稀释液加入96孔板中在37℃孵育1小时。在自动洗板机上用洗涤缓冲液PBST将板洗涤两次。然后将用稀释缓冲液稀释的HRP偶联山羊抗小鼠Fc抗体(Jackson Immuno Research，货号115-035-164)以50μL/孔加入板的每个孔中。之后，将ELISA板在37℃孵育1小时，然后使用洗涤缓冲液PBST在自动洗板机上洗涤板两次。最后，每孔加入50μL/孔的TMB，用1M H 2SO4终止反应。测定在450nm-620nm处的吸光度值，结果见图15。 High-binding clear polystyrene 96-well plates were coated with 0.5 μg/mL human TIGIT(M22-P141 )-Fc (SEQ ID NO: 45) in phosphate buffered saline PBS at 50 μL/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 μL of blocking buffer (PBS solution containing 1% BSA) to each well and incubate at 37° C. for 1 hour. Prepare serially diluted antibodies (6F6-H1, THC4, CT2KH and CT1KH) and negative control antibodies (Ipilimumab analog) with dilution buffer (PBS+0.5% bovine serum albumin+0.05% Tween 20) (initial concentration: 100nM (concentration before mixing), 3-fold dilution) and mixed with 1.1 μg/mL (concentration before mixing) CD155-mFc ((Acro Biosystem, Cat. Incubate the well plate at 37°C for 1 hour. Wash the plate twice with the washing buffer PBST on the automatic plate washer. Then the HRP-coupled goat anti-mouse Fc antibody (Jackson Immuno Research, Cat. No. 115) diluted with the dilution buffer -035-164) was added to each well of the plate at 50 μL/well. After that, the ELISA plate was incubated at 37 ° C for 1 hour, and then the plate was washed twice on an automatic plate washer with washing buffer PBST. Finally, each well was added 50 μL/well of TMB was used to terminate the reaction with 1M H 2 SO 4 . Measure the absorbance value at 450nm-620nm, and the results are shown in FIG. 15 .
结果显示6F6-H1、THC4、CT2KH和CT1KH可以阻断TIGIT与CD155的结合。且THC4、CT2KH和CT1KH阻断活性与6F6-H1相比无明显差异。The results showed that 6F6-H1, THC4, CT2KH and CT1KH could block the binding of TIGIT to CD155. And the blocking activity of THC4, CT2KH and CT1KH had no significant difference compared with 6F6-H1.
实施例22：抗TIGIT/CTLA-4双特异性抗体抑制人CTLA-4蛋白与人CD80蛋白结合活性Example 22: Anti-TIGIT/CTLA-4 bispecific antibody inhibits the binding activity of human CTLA-4 protein to human CD80 protein
高结合透明聚苯乙烯96孔板用在磷酸盐缓冲液PBS中的2μg/mL人CTLA-4-his(Acro Biosystem，货号CT4-H5229)以50μL/孔在4℃孵育过夜包被。然后使用洗涤缓冲液PBST(含有0.05％吐温20的PBS溶液)在自动洗板机上洗涤板一次。每孔加入200μL封闭缓冲液(含有1％BSA的PBS溶液)，室温孵育1小时。用稀释缓冲液(PBS+0.5％牛血清白蛋白+0.05％吐温20)制备逐级稀释抗体(THC4、CT2KH和CT1KH、Ipilimumab analog)和阴性对照抗体(6F6-H1)(起始浓度：100nM(混合前浓度)，3倍稀释)并以1∶1的体积比与0.02μg/mL(混合前浓度)CD80-mFc(Acro Biosystem，货号B71-H52A4)混合，然后将混合稀释液加入96孔板中并在37℃孵育1小时。在自动洗板机上用洗涤缓冲液PBST将板洗涤两次。然后将用稀释缓冲液稀释的HRP偶联山羊抗小鼠Fc抗体(Jackson Immunoresearch，Cat#115-035-164)以50μL/孔加入板的每个孔中。之后，将96孔板在37℃孵育1小时，然后使用洗涤缓冲液PBST在自动洗板机上洗涤板两次。最后，每孔加入50μL/孔的TMB，用1M H 2SO4终止反应。测定在450nm-620nm处的吸光值，结果见图16。 High-binding clear polystyrene 96-well plates were coated with 2 μg/mL human CTLA-4-his (Acro Biosystem, Cat. No. CT4-H5229) in phosphate-buffered saline PBS at 50 μL/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 μL of blocking buffer (PBS solution containing 1% BSA) to each well and incubate at room temperature for 1 hour. Prepare serially diluted antibodies (THC4, CT2KH and CT1KH, Ipilimumab analog) and negative control antibody (6F6-H1) with dilution buffer (PBS+0.5% bovine serum albumin+0.05% Tween 20) (initial concentration: 100nM (concentration before mixing), 3-fold dilution) and mixed with 0.02 μg/mL (concentration before mixing) CD80-mFc (Acro Biosystem, cat. plate and incubate at 37°C for 1 hour. Plates were washed twice with wash buffer PBST on an automatic plate washer. Then, 50 μL/well of HRP-conjugated goat anti-mouse Fc antibody (Jackson Immunoresearch, Cat# 115-035-164) diluted in dilution buffer was added to each well of the plate. Afterwards, the 96-well plate was incubated at 37°C for 1 hour, and then the plate was washed twice on an automatic plate washer using wash buffer PBST. Finally, 50 μL/well of TMB was added to each well, and the reaction was terminated with 1M H 2 SO 4 . The absorbance value at 450nm-620nm was measured, and the results are shown in Figure 16.
结果显示Ipilimumab analog、THC4、CT2KH和CT1KH可以阻断CTLA-4和CD80的结合。The results showed that Ipilimumab analog, THC4, CT2KH and CT1KH could block the binding of CTLA-4 and CD80.
实施例23：抗TIGIT/CTLA-4双特异性抗体与人TIGIT细胞结合活性Example 23: Binding activity of anti-TIGIT/CTLA-4 bispecific antibody to human TIGIT cells
通过细胞结合实验来判断抗TIGIT/CTLA-4双特异性抗体能否与稳定表达在HEK293细胞(中国科学院典型培养物保藏委员会细胞库，货号GNHu 43)上的人TIGIT蛋白结合。实验过程和方法如实施例6(1)，采用TIGIT单抗6F6-H1作为对照。Cell binding experiments were used to determine whether the anti-TIGIT/CTLA-4 bispecific antibody could bind to human TIGIT protein stably expressed on HEK293 cells (Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences, Cat. No. GNHu 43). The experimental process and method are as in Example 6 (1), using TIGIT monoclonal antibody 6F6-H1 as a control.
从图17所示的结果可知，测试的TIGIT/CTLA-4双特异性抗体THC4、CT2KH和CT1KH都能和HEK293/hTIGIT细胞结合。From the results shown in Figure 17, it can be seen that the tested TIGIT/CTLA-4 bispecific antibodies THC4, CT2KH and CT1KH can all bind to HEK293/hTIGIT cells.
序列信息：Sequence information:
Figure PCTCN2022143967-appb-000021
Figure PCTCN2022143967-appb-000021
Figure PCTCN2022143967-appb-000022
Figure PCTCN2022143967-appb-000022
Figure PCTCN2022143967-appb-000023
Figure PCTCN2022143967-appb-000023
Figure PCTCN2022143967-appb-000024
Figure PCTCN2022143967-appb-000024
Figure PCTCN2022143967-appb-000025
Figure PCTCN2022143967-appb-000025
Figure PCTCN2022143967-appb-000026
Figure PCTCN2022143967-appb-000026
Figure PCTCN2022143967-appb-000027
Figure PCTCN2022143967-appb-000027
Figure PCTCN2022143967-appb-000028
Figure PCTCN2022143967-appb-000028

Claims (35)

  1. 特异性结合TIGIT的VHH抗体，其包含A VHH antibody specifically binding to TIGIT comprising
    SEQ ID NO：1、6、9、14、16、18和21中任一项所示的VH中所含的三个互补决定区域(CDR)，The three complementarity determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21,
    优选地，所述CDR序列根据IMGT定义。Preferably, said CDR sequences are defined according to IMGT.
  2. 权利要求1的VHH抗体，其包含互补决定区域(CDR)VHH CDR1、VHH CDR2和VHH CDR3，其中The VHH antibody of claim 1, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein
    (i)VHH CDR1包含SEQ ID NO：3所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(i) 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
    (ii)VHH CDR1包含SEQ ID NO：8所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：4或23或57所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：5所示的氨基酸序列或由其组成；或(ii) 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, and VHH CDR3 comprises SEQ ID NO: or consisting of the amino acid sequence shown in 5; or
    (iii)VHH CDR1包含SEQ ID NO：11所示的氨基酸序列或由其组成，VHH CDR2包含SEQ ID NO：12所示的氨基酸序列或由其组成，VHH CDR3包含SEQ ID NO：13所示的氨基酸序列或由其组成。(iii) 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.
  3. 权利要求1的VHH抗体，其包含重链可变区或由其组成，所述重链可变区The VHH antibody of claim 1, comprising or consisting of a heavy chain variable region, said heavy chain variable region
    (i)包含与选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 90%, 91%, 92%, 93%, 94%, Amino acid sequences that are 95%, 96%, 97%, 98% or 99% identical or consist of; or
    (ii)包含选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列或由其组成；或者(ii) comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 1, 6, 9, 14, 16, 18 and 21; or
    (iii)包含与选自SEQ ID NO：1、6、9、14、16、18和21中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。(iii) 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.
  4. 特异性结合TIGIT的重链抗体，其包含权利要求1-3中任一项所述的VHH抗体。A heavy chain antibody specifically binding to TIGIT, comprising the VHH antibody of any one of claims 1-3.
  5. 权利要求4的重链抗体，其包含与抗体恒定区或Fc区连接的权利要求1-3中任一项所述的VHH抗体，优选地，所述抗体恒定区或Fc区来自人IgG1、人IgG2、人IgG3或人IgG4，任选地，所述VHH抗体与所述Fc区通过铰链区或其部分连接，优选地，所述铰链区部分的氨基酸序列为EPKSS(SEQ ID NO：43)。The heavy chain antibody of claim 4, which comprises the VHH antibody of any one of claims 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).
  6. 权利要求4的重链抗体，其包含与抗体Fc区连接的权利要求1-3中任一项所述的VHH抗体，其中所述Fc区为来自人IgG1或IgG4的Fc区，优选地，所述Fc区The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 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
    (i)包含与SEQ ID NO：40或42中所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more of the amino acid sequence shown in SEQ ID NO: 40 or 42 99% identical amino acid sequences or consisting thereof; or
    (ii)包含SEQ ID NO：40或42所示的氨基酸序列或由其组成；或者(ii) comprise or consist of the amino acid sequence shown in SEQ ID NO: 40 or 42; or
    (iii)包含与SEQ ID NO：40或42所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列。(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) 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.
  7. 权利要求5或6的重链抗体，其中所述Fc区包含改善Fc区效应子功能的突变，例如提高ADCC的突变，优选地，所述突变为如下突变组合：S239D、A330L和I332E(EU编号)，优选地，其包含与SEQ ID NO：41所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成，且包含如下突变组合：S239D、A330L和I332E(EU编号)。The heavy chain antibody of claim 5 or 6, wherein the Fc region comprises a mutation that improves the effector function of the Fc region, such as a mutation that increases ADCC, preferably, 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% or An amino acid sequence with 99% identity or consisting thereof, and comprising the following combination of mutations: S239D, A330L and I332E (EU numbering).
  8. 权利要求4的重链抗体，其The heavy chain antibody of claim 4, which
    (i)包含与选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成；或者(i) comprising at least 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist thereof; or
    (ii)包含选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列或由其组成；或者(ii) comprising or consisting of the amino acid sequence shown in any one of SEQ ID NO: 2, 7, 10, 15, 17, 19, 20 or 22; or
    (iii)包含与选自SEQ ID NO：2、7、10、15、17、19、20或22中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。(iii) 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.
  9. 权利要求权利要求1-3中任一项的VHH抗体，或权利要求4-8中任一项的重链抗体，其中所述抗体是嵌合抗体或人源化抗体。The VHH antibody of any one of claims 1-3, or the heavy chain antibody of any one of claims 4-8, wherein said antibody is a chimeric antibody or a humanized antibody.
  10. 双特异性抗体，其包含第一抗原结合区和第二抗原结合区，其中所述第一抗原结合区特异性结合TIGIT，且包含权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体。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 claims 1-3 and 9, or The heavy chain antibody of any one of claims 4-9.
  11. 权利要求10的双特异性抗体，其中第二抗原结合区特异性结合PD-1、PD-L1或PD-L2或CTLA-4，优选地，所述第二抗原结合区特异性结合PD-1，其包含来自WO2019219064A的PD1抗体或其抗原结合片段，所述抗原结合片段例如所述抗PD-1抗体的单链Fv、Fab、Fab′、(Fab)2、单域抗体、VHH或重链抗体；优选地，所述第二抗原结合区特异性结合CTLA-4，其包含来自Ipilimumab抗体或其抗原结合片段，所述抗原结合片段例如所述抗CTLA-4抗体的单链Fv、Fab、Fab′、(Fab)2、单域抗体、VHH或重链抗体。The bispecific antibody of claim 10, wherein the second antigen-binding region specifically binds to PD-1, PD-L1 or PD-L2 or CTLA-4, preferably, the second antigen-binding region specifically binds to 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 of the anti-PD-1 antibody Antibody; preferably, the second antigen-binding region specifically binds CTLA-4, which comprises an antibody from Ipilimumab or an antigen-binding fragment thereof, such as a single-chain Fv, Fab, Fab, Fab', (Fab)2, single domain antibody, VHH or heavy chain antibody.
  12. 权利要求10或11的双特异性抗体，其中VHH抗体连接到第二抗原结合区Fc片段的C末端，或连接在第二抗原结合区重链VH片段的N末端，或者可以插入第二抗原结合区的Fab片段(Fab片段重链)和Fc片段之间，即与Fab片段重链的C末端和Fc片段的N末端连接，任选地，第一和第二抗原结合区通过接头连接，优选地，接头包含(GGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。The bispecific antibody of claim 10 or 11, wherein the VHH antibody is linked to the C-terminus of the Fc fragment of the second antigen-binding region, or connected to the N-terminus of the heavy chain VH fragment of the second antigen-binding region, or can be inserted into the second antigen-binding between the Fab fragment (Fab fragment heavy chain) and the Fc fragment of the Fab fragment (Fab fragment heavy chain) and the Fc fragment, that is, the C-terminal of the Fab fragment heavy chain and the N-terminal of the Fc fragment, optionally, the first and second antigen-binding regions are connected by a linker, preferably Preferably, the linker comprises (GGS)n amino acid sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
  13. 权利要求12的双特异性抗体，其中所述双特异性抗体具有以下结构：The bispecific antibody of claim 12, wherein said bispecific antibody has the following structure:
    重链：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc-抗TIGIT VHH；或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
    从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-抗TIGIT VHH-重链恒定区Fc；或From N-terminal to C-terminal, heavy chain variable region VH-heavy chain constant region CH1-anti-TIGIT VHH-heavy chain constant region Fc of the second antigen antibody; or
    从N端至C端，抗TIGIT VHH-第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
    轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL；Light chain: from N-terminal to C-terminal, the light chain variable region of the second antigen antibody-light chain constant region CL;
    优选地，所述第二抗原选自PD-1或CTLA-4。Preferably, the second antigen is selected from PD-1 or CTLA-4.
  14. 权利要求10或11的双特异性抗体，其中所述双特异性抗体具有以下结构：The bispecific antibody of claim 10 or 11, wherein said bispecific antibody has the following structure:
    重链1：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
    重链2：1个或多个(例如2个)串联的抗TIGIT VHH-重链恒定区FcHeavy chain 2: 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
    轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL；Light chain: from N-terminal to C-terminal, the light chain variable region of the second antigen antibody-light chain constant region CL;
    优选地，所述第二抗原选自PD-1或CTLA-4，例如CTLA-4。Preferably, the second antigen is selected from PD-1 or CTLA-4, such as CTLA-4.
  15. 权利要求14的双特异性抗体，其中所述抗TIGIT-VHH通过连接肽与重链恒定区Fc的N末端连接，例如所述连接肽是来自人IgG1、2、3或4的铰链区或其部分，包括天然或突变的铰链区或其部分，例如来自人IgG1铰链区，例如所述连接肽是EPKSS(SEQ ID NO：43)。The bispecific antibody of claim 14, wherein the anti-TIGIT-VHH is connected to the N-terminus of the heavy chain constant region Fc through a linker peptide, for example, the linker peptide is from the hinge region of human IgG1, 2, 3 or 4 or its Portions, including native or mutated hinge regions or portions thereof, e.g. from human IgG1 hinge regions, e.g. the connecting peptide is EPKSS (SEQ ID NO: 43).
  16. 权利要求14或15的双特异性抗体，其中当重链2包含多个串联的抗TIGIT单域抗体VHH时，各个串联的VHH之间可以通过接头连接，优选地，接头包含(GGGGS)n氨基酸序列，n＝1，2，3，4，或5的整数，优选n＝1。The bispecific antibody according to claim 14 or 15, wherein when the heavy chain 2 comprises multiple tandem anti-TIGIT single domain antibody VHHs, each tandem VHH can be connected by a linker, preferably, the linker comprises (GGGGS)n amino acids Sequence, n=1, 2, 3, 4, or an integer of 5, preferably n=1.
  17. 权利要求10-16中任一项的双特异性抗体，其中第二抗原抗体的重链恒定区CH1来自IgG，例如IgG1、IgG2、IgG3或IgG4；优选的，所述重链恒定区CH1来自IgG1或IgG4，更优选地，所述重链恒定区CH1包含SEQ ID NO：28或31所述的氨基酸序列或由其组成。The bispecific antibody according to any one of claims 10-16, wherein 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 is from IgG1 Or IgG4, more preferably, said heavy chain constant region CH1 comprises the amino acid sequence described in SEQ ID NO: 28 or 31 or consists of it.
  18. 权利要求10-17中任一项的双特异性抗体，其中第二重链恒定区Fc如权利要求5-7中任一项所定义。The bispecific antibody according to any one of claims 10-17, wherein the second heavy chain constant region Fc is as defined in any one of claims 5-7.
  19. 权利要求14的双特异性抗体，其中重链1的Fc区与重链2的Fc区不同。The bispecific antibody of claim 14, wherein the Fc region of heavy chain 1 is different from the Fc region of heavy chain 2.
  20. 权利要求19的双特异性抗体，其中重链1的Fc区与重链2的Fc区中分别引入结(knob)突变和扣(Hole)突变。The bispecific antibody according to claim 19, wherein the Fc region of heavy chain 1 and the Fc region of heavy chain 2 are respectively introduced with a knob mutation and a buckle mutation.
  21. 权利要求21的双特异性抗体，其中The bispecific antibody of claim 21, wherein
    第一Fc区包含结突变，其The first Fc region contains a junction mutation, which
    (i)包含氨基酸序列SEQ ID NO：78或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成；或or
    (ii)包含与SEQ ID NO：78具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含S239D、A330L和I332E突变和结突变(例如S354C和T366W)；且(ii) comprising an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity to SEQ ID NO: 78 and comprising S239D, A330L and I332E mutations and knot mutations ( such as S354C and T366W); and
    第二Fc区包含扣突变，其The second Fc region contains a button mutation, which
    (i)包含氨基酸序列SEQ ID NO：77与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成；或(i) 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;
    (ii)包含与SEQ ID NO：77具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含S239D、A330L和I332E突变和扣突变。(ii) comprising an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity to SEQ ID NO: 77 and comprising S239D, A330L and I332E mutations and buckle mutations.
  22. 权利要求13的双特异性抗体，其包含The bispecific antibody of claim 13, comprising
    重链：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc-抗TIGIT VHH；且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; and
    轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
    其中所述第二抗原为PD-1；wherein the second antigen is PD-1;
    其中重链heavy chain
    (i)包含SEQ ID NO：30或33的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 30 or 33, or
    (ii)包含与所述SEQ ID NO：30、32或33所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or
    (iii)由SEQ ID NO：30或33所述的氨基酸组成；(iii) consist of amino acids described in SEQ ID NO: 30 or 33;
    和/或and / or
    轻链light chain
    (i)包含SEQ ID NO：39的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 39, or
    (ii)包含与所述SEQ ID NO：39所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 39 the amino acid sequence of
    (iii)由SEQ ID NO：39所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:39.
  23. 权利要求13的双特异性抗体，其包含The bispecific antibody of claim 13, comprising
    重链：Heavy chain:
    从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-抗TIGIT VHH-重链恒定区FcFrom N-terminal to C-terminal, the heavy chain variable region of the second antigen antibody VH-heavy chain constant region CH1-anti-TIGIT VHH-heavy chain constant region Fc
    轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
    其中所述第二抗原为PD-1；wherein the second antigen is PD-1;
    其中重链heavy chain
    (i)包含SEQ ID NO：32的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 32, or
    (ii)包含与所述SEQ ID NO：32所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 32 the amino acid sequence of
    (iii)由SEQ ID NO：32所述的氨基酸组成；(iii) consist of the amino acid described in SEQ ID NO: 32;
    和/或and / or
    轻链light chain
    (i)包含SEQ ID NO：39的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 39, or
    (ii)包含与所述SEQ ID NO：39所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 39 the amino acid sequence of
    (iii)由SEQ ID NO：39所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:39.
  24. 权利要求13的双特异性抗体，其包含The bispecific antibody of claim 13, comprising
    重链：从N端至C端，抗TIGIT VHH-第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
    轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
    其中第二抗原为CTLA-4，Wherein the second antigen is CTLA-4,
    其中重链heavy chain
    (i)包含SEQ ID NO：69的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 69, or
    (ii)包含与所述SEQ ID NO：69所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 69 the amino acid sequence of
    (iii)由SEQ ID NO：69所述的氨基酸组成；(iii) consist of the amino acid described in SEQ ID NO: 69;
    和/或and / or
    轻链light chain
    (i)包含SEQ ID NO：68的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 68, or
    (ii)包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 68 the amino acid sequence of
    (iii)由SEQ ID NO：68所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:68.
  25. 权利要求14的双特异性抗体，其包含The bispecific antibody of claim 14, comprising
    重链1：从N端至C端，第二抗原抗体的重链可变区VH-重链恒定区CH1-重链恒定区Fc；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;
    重链2：1个或多个(例如2个)串联的抗TIGIT VHH-重链恒定区FcHeavy chain 2: 1 or more (eg 2) anti-TIGIT VHH-heavy chain constant region Fc in tandem
    轻链：从N端至C端，第二抗原抗体的轻链可变区-轻链恒定区CL，Light chain: from N-terminal to C-terminal, light chain variable region of the second antigen antibody-light chain constant region CL,
    其中重链1of which heavy chain 1
    (i)包含SEQ ID NO：71的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 71, or
    (ii)包含与所述SEQ ID NO：71所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO:71 the amino acid sequence of
    (iii)由SEQ ID NO：71所述的氨基酸组成；(iii) consist of the amino acid described in SEQ ID NO: 71;
    和/或and / or
    重链2heavy chain 2
    (i)包含SEQ ID NO：72或74的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 72 or 74, or
    (ii)包含与所述SEQ ID NO：72或74所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence described in said SEQ ID NO: 72 or 74 the amino acid sequence of identity, or
    (iii)由SEQ ID NO：72或74所述的氨基酸组成；(iii) consist of amino acids described in SEQ ID NO: 72 or 74;
    和/或and / or
    轻链light chain
    (i)包含SEQ ID NO：68的氨基酸序列，或(i) comprising the amino acid sequence of SEQ ID NO: 68, or
    (ii)包含与所述SEQ ID NO：68所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或(ii) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in said SEQ ID NO: 68 the amino acid sequence of
    (iii)由SEQ ID NO：68所述的氨基酸组成。(iii) consist of the amino acid described in SEQ ID NO:68.
  26. 核酸分子，其编码权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体，或权利要求10-25中任一项的双特异性抗体中的重链和/或轻链，或由所述核酸序列组成。A nucleic acid molecule encoding the VHH antibody of any one of claims 1-3 and 9, or the heavy chain antibody of any one of claims 4-9, or the bispecific antibody of any one of claims 10-25 The heavy chain and/or light chain in, or consist of said nucleic acid sequence.
  27. 表达载体，其包含权利要求26的核酸分子，优选地，所述表达载体为pCDNA，例如pCDNA3.1。An expression vector comprising the nucleic acid molecule according to claim 26, preferably, the expression vector is pCDNA, such as pCDNA3.1.
  28. 宿主细胞，其包含权利要求26所述的核酸分子或权利要求27所述的表达载体，优选地，所述宿主细胞是原核的或真核的，例如293细胞或CHO细胞，例如293FT细胞或CHO-S细胞。A host cell comprising the nucleic acid molecule of claim 26 or the expression vector of claim 27, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293FT cells or CHO -S cells.
  29. 制备权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体，或权利要求10-25中任一项的双特异性抗体的方法，所述方法包括，在适合所述VHH抗体或重链抗体或双特异性抗体的链表达的条件下，培养权利要求包含编码VHH抗体或重链抗体的核酸、或编码双特异性抗体的各条链的核酸或包含所述核酸的表达载体的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述VHH抗体或重链抗体或双特异性抗体。A method for preparing the VHH antibody of any one of claims 1-3 and 9, or the heavy chain antibody of any one of claims 4-9, or the bispecific antibody of any one of claims 10-25, wherein The method comprises, under conditions suitable for the expression of the chains of the VHH antibody or heavy chain antibody or bispecific antibody, culturing claims comprising nucleic acid encoding a VHH antibody or heavy chain antibody, or each chain encoding a bispecific antibody A host cell of the nucleic acid or an expression vector comprising the nucleic acid, and optionally recovering the VHH antibody or heavy chain antibody or bispecific antibody from the host cell (or host cell culture medium).
  30. 免疫缀合物，其包含权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体，或权利要求10-25中任一项的双特异性抗体。An immunoconjugate comprising the VHH antibody of any one of claims 1-3 and 9, or the heavy chain antibody of any one of claims 4-9, or the bispecific antibody of any one of claims 10-25 Sexual antibodies.
  31. 药物组合物或药物或制剂，其包含权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体，或权利要求10-25中任一项的双特异性抗体，或权利要求30的免疫缀合物以及任选地药用辅料。A pharmaceutical composition or medicament or preparation comprising the VHH antibody of any one of claims 1-3 and 9, or the heavy chain antibody of any one of claims 4-9, or any one of claims 10-25 The bispecific antibody of claim 30, or the immunoconjugate of claim 30 and optionally a pharmaceutical adjuvant.
  32. 药物组合产品，其包含权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体，或权利要求10-25中任一项的双特异性抗体，或权利要求30的免疫缀合物，以及其它治疗剂。A pharmaceutical combination product comprising the VHH antibody of any one of claims 1-3 and 9, or the heavy chain antibody of any one of claims 4-9, or the bispecificity of any one of claims 10-25 Antibodies, or immunoconjugates of claim 30, and other therapeutic agents.
  33. 在受试者中预防或治疗癌症的方法，包括向受试者施用有效量的权利要求1-3和9中任一项的VHH抗体，或权利要求4-9中任一项的重链抗体，或权利要求10-25中任一项的双特异性抗体，或权利要求30的免疫缀合物，或权利要求31的药物组合物或制剂；或权利要求32的药物组合产品。A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the VHH antibody of any one of claims 1-3 and 9, or the heavy chain antibody of any one of claims 4-9 , or the bispecific antibody of any one of claims 10-25, or the immunoconjugate of claim 30, or the pharmaceutical composition or preparation of claim 31; or the pharmaceutical combination product of claim 32.
  34. 权利要求33的方法，其中所述癌症是表征为具有升高的PD-1、PD-L1或PD-L2的蛋白质水平和/或核酸水平(例如表达升高)和/或具有升高的TIGIT的的蛋白质水平和/或 核酸水平(例如表达升高)的癌症。The method of claim 33, wherein 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 and/or having elevated TIGIT Cancers at the protein level and/or nucleic acid level (e.g., elevated expression).
  35. 权利要求34的方法，其中所述方法还包括与其它疗法例如治疗方式和/或其它治疗剂组合施用。34. The method of claim 34, wherein said method further comprises administration in combination with other therapies such as treatment modalities and/or other therapeutic agents.
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