WO2025103451A1 - Trgv9结合蛋白及其医药用途 - Google Patents

Trgv9结合蛋白及其医药用途 Download PDF

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Publication number
WO2025103451A1
WO2025103451A1 PCT/CN2024/132266 CN2024132266W WO2025103451A1 WO 2025103451 A1 WO2025103451 A1 WO 2025103451A1 CN 2024132266 W CN2024132266 W CN 2024132266W WO 2025103451 A1 WO2025103451 A1 WO 2025103451A1
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linker
trgv9
seq
amino acid
gpc3
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French (fr)
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胡雅丽
施佳杰
陈思萌
张伟
廖成
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present disclosure relates to the field of biomedicine, and in particular to TRGV9 binding protein, GPC3/TRGV9 binding protein, and methods for treating cancer and pharmaceutical uses thereof.
  • ⁇ T cells are a group of T cells that naturally recognize and kill tumor cells. They have both antigen presentation and killing functions, bridging innate immunity and adaptive immunity. Unlike ⁇ TCR, the two chains that make up the TCR of ⁇ T cells are ⁇ chain and ⁇ chain, and their recognition of antigens is not restricted by MHC (Exp Mol Med. 2021Mar; 53(3): 318-327). The number of ⁇ T cells is relatively small, accounting for about 1%-5% of PBMCs. According to the difference in ⁇ chain, they can be divided into four categories: ⁇ 1, ⁇ 2, ⁇ 3, and ⁇ 5 (Front Immunol. 2022 Jun 16; 13: 915837), and ⁇ 1 and ⁇ 2 account for the largest number.
  • ⁇ 1 can pair with different ⁇ chains to form different TCRs, which are mainly distributed in skin, mucous membranes and other tissues; ⁇ 2 mainly pairs with ⁇ 9 to form the ⁇ 9 ⁇ 2 subtype, which is mainly distributed in peripheral blood, accounting for up to 95% of the total ⁇ T cells in peripheral blood (Front Immunol. 2022 Jun 16; 13:915837).
  • ⁇ T cells play an important role in anti-tumor and have multiple mechanisms for killing tumor cells, including recognizing the BTN2A/BTN3A complex activated by phosphorylated antigens on target cells through TCR, initiating killing signals and exerting killing functions; in addition, there is a killing pathway similar to NK cells, which expresses receptors such as NKG2D on the cell surface and exerts tumor killing effects by binding to corresponding ligands on tumor cells.
  • ⁇ T cells have different degrees of infiltration in various tumors. Through a large sample analysis of 25 tumor types and 5782 tumors, it was found that the infiltration of ⁇ T cells indicates a good prognosis for patients, and it is the most favorable cell population among all immune cells.
  • Glypican-3 is a potential target for the treatment of liver cancer.
  • GPC3 is a heparan sulfate proteoglycan composed of 580 amino acids, which is anchored to the cell membrane through glycosylphosphatidylinositol. GPC3 is highly expressed in a variety of tumor tissues.
  • the GPC3 positive rate in hepatocellular carcinoma is as high as 90%.
  • GPC3 expression in normal tissues is very limited, only expressed in the placenta and endometrium; and the expression level in the endometrium is much lower than that in tumor tissues, making it an ideal anti-tumor target (Sci Transl Med. 2017 Oct 4; 9(410):eaal4291).
  • the present disclosure provides an antibody targeting ⁇ TCR with a new sequence structure, and constructs a bispecific antibody with a tumor-associated antigen (e.g., GPC3) binding domain (e.g., antibody), wherein one end of the bispecific antibody binds to TAA (e.g., GPC3) and the other end binds to ⁇ TCR, which can recruit and activate ⁇ T cells to specifically kill TAA (e.g., GPC3)-positive tumor cells, which significantly improves the effectiveness of ⁇ T cell therapy.
  • the bispecific antibody disclosed in the present disclosure has good tumor killing activity, safety and drugability.
  • TCR T cell receptor
  • Glypican-3 Glypican-3
  • TCR T cell receptor
  • T cell receptor gamma variable region 9 (TRGV9) binding protein T cell receptor gamma variable region 9 (TRGV9) binding protein
  • the present disclosure provides a TRGV9 binding protein. In some embodiments, it is capable of binding to a TCR. In some embodiments, it is capable of binding to a ⁇ TCR. In some embodiments, it is capable of binding to the ⁇ 9 chain of a TCR. In some embodiments, it is capable of binding to a ⁇ 9 ⁇ 2 TCR. In other embodiments, it is capable of binding to a ⁇ 9 ⁇ 1 TCR.
  • TRGV9 binding protein comprising:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and/or CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29, for example, the immunoglobulin single variable domain comprises CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, according to the Kabat numbering system.
  • TRGV9 binding protein comprising:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and/or CDR3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; or,
  • VH and/or VL said VH comprises HCDR1, HCDR2 and/or HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13; said VL comprises LCDR1, LCDR2 and/or LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • TRGV9 binding protein comprising:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6-8, respectively; or,
  • VH and VL wherein the VH comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11-13, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 14-16, respectively.
  • the immunoglobulin single variable domain or VH or VL in the aforementioned proteins are each independently humanized, backmutated, affinity matured, T cell epitope (TCE) removed/reduced, antibody deamidation reduced and/or antibody isomerization reduced.
  • the heavy chain framework region of the human germline template used for humanization of the immunoglobulin single variable domain in the aforementioned protein is derived from IGHV3-64; the heavy chain framework region of the human germline template used in the humanization process of VH is derived from IGHV3-21, and the light chain framework region of the human germline template used in the humanization process of VL is derived from IGKV1-12.
  • TRGV9 binding protein comprising:
  • an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; or,
  • VH and VL wherein the VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-33, or a sequence that is at least 80% or at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • the TRGV9 binding protein comprises the following VH and VL:
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or having at least 80%, at least 90% identity thereto
  • VL comprises an amino acid sequence as shown in SEQ ID NO: 10, or having at least 80%, at least 90% identity thereto,
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 30, or having at least 80% or at least 90% identity thereto
  • VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80% or at least 90% identity thereto;
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 31, or having at least 80%, at least 90% identity thereto
  • VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80%, at least 90% identity thereto;
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 32, or a sequence that is at least 80% or at least 90% identical thereto
  • VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • At least 80% (sequence) identity encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity;
  • at least 90% (sequence) identity encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.
  • the aforementioned TRGV9 binding protein comprises or is an anti-TRGV9 antibody or an antigen-binding fragment thereof.
  • the anti-TRGV9 antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof.
  • the anti-TRGV9 antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody (e.g., a trispecific antibody).
  • the aforementioned TRGV9 binding protein when the aforementioned TRGV9 binding protein contains an immunoglobulin single variable domain, it can be a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof, for example, the immunoglobulin single variable domain is a single domain antibody or VHH.
  • the aforementioned TRGV9 binding protein itself is a single domain antibody or VHH.
  • the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody peptibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem di-scFv, a tandem tri-scFv.
  • the antigen-binding fragment includes but is not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv.
  • the antigen-binding fragment includes Fab, Fv, sFv, Fab', F(ab')2.
  • the aforementioned TRGV9 binding protein may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8) aforementioned immunoglobulin single variable domains.
  • the immunoglobulin single variable domains may form dimers or multimer molecules.
  • the immunoglobulin single variable domains may be homodimers or heterodimers.
  • the aforementioned TRGV9 binding protein may comprise one or more (eg, 2, 3, 4, 5, 6, 7, 8) of the aforementioned VH, VL or a combination thereof.
  • the aforementioned TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1, such as that shown in SEQ ID NO: 51 or having at least 80%, at least 90% sequence identity therewith.
  • the Fc region may be an Fc region with reduced effector function, for example, the Fc region may have a mutation, and exemplary IgG Fc regions with reduced effector function include substitutions having the following: N297A or N297Q (IgG1); L234A/L235A (IgG1); V234A/G237A (IgG2); L235A/G237A/E318A (IgG4); H268Q/V309L/A330S/A3 31S (IgG2); C220S/C226S/C229S/P238S (IgG1); C226S/C229S/E233P/L234V/L235A (IgG1); L234F/L235E/P331S (IgG1); L234F/L235E (IgG1); L234F or L235E (IgG1); L234A or L235A (IgG1); L234
  • the immunoglobulin single variable domain or VH, VL in the aforementioned TRGV9 binding protein is directly or via a linker to the Fc region.
  • the linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structures.
  • the linker is as shown in ( GmSn ) h or ( GmQn ) h or ( GGNGT ) h (SEQ ID NO: 62) or (YGNGT) h (SEQ ID NO: 63) or (EPKSS) h (SEQ ID NO: 64) or ( AmSn ) h , wherein m and n are each independently selected from integers of 1-8, and h is independently selected from integers of 1-20.
  • the linker is selected from G4S (SEQ ID NO: 65), GS, GAP, ( G4S ) 2 (SEQ ID NO: 66), ( G4S ) 3 (SEQ ID NO: 67), ( G4S ) 4 (SEQ ID NO: 68), ( G4S ) 5 (SEQ ID NO: 69), ASGS (SEQ ID NO: 70), A3S (SEQ ID NO: 71), etc.;
  • the aforementioned TRGV9 binding protein is: a protein that specifically binds to ⁇ TCR, or an anti- ⁇ TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to the ⁇ 9 chain of TCR, or an anti- ⁇ 9 chain antibody or an antigen-binding fragment thereof; a protein that specifically binds to the variable region (V ⁇ 9) of the ⁇ 9 chain of TCR, or an anti-V ⁇ 9TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to ⁇ 9 ⁇ 2 TCR, or an anti- ⁇ 9 ⁇ 2TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to ⁇ 9 ⁇ 1 TCR, or an anti- ⁇ 9 ⁇ 1 TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to TRGV9, or an anti-TRGV9 antibody or an antigen-binding fragment thereof.
  • the aforementioned TRGV9 binding protein comprises:
  • the aforementioned TRGV9 binding protein has at least one function or property selected from the following:
  • the EC50 is obtained by FACS detection, which is a commonly used affinity detection method in the art, such as described in Example 5 of the present disclosure.
  • (b) specifically binds to the ⁇ 9 chain of TCR, for example, specifically binds to ⁇ 9 ⁇ 1 TCR, ⁇ 9 ⁇ 2 TCR;
  • (b)-(e) can be obtained by conventional detection methods in the art, such as described in Example 4 of the present disclosure.
  • the K D value of the aforementioned protein binding to TRGV9 may be ⁇ 1 ⁇ 10 -7 M, such as ⁇ 1 ⁇ 10 -8 M, or ⁇ 1 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -10 M.
  • the aforementioned TRGV9 binding protein covers variants of immunoglobulin single variable domains, and the variants have one or more amino acid mutations compared to any one of SEQ ID NOs: 5, 26-29. In some embodiments, the aforementioned TRGV9 binding protein covers variants of VH and/or VL, and the variants of VH have one or more amino acid mutations compared to any one of SEQ ID NOs: 9, 30-32, and the variants of VL have one or more amino acid mutations compared to any one of SEQ ID NOs: 10, 33-35. "Multiple" covers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The amino acid mutations may be conservative replacements, substitutions or modifications, and/or deletions or additions that do not affect the function; the amino acid mutations may occur in the CDR region and/or the FR region.
  • a protein that binds to or competes for binding to the same antigenic epitope as the immunoglobulin single variable domain in the aforementioned TRGV9 binding protein of the present disclosure.
  • a protein that binds to or competes for binding with the same antigenic epitope as the VH and VL in the aforementioned TRGV9 binding protein of the present disclosure.
  • a protein is provided, the binding of which to TRGV9 is blocked by the immunoglobulin single variable domain or VH and VL in the TRGV9 binding protein of the present disclosure described above.
  • a protein or molecule which comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains disclosed above, for example, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 shown in SEQ ID NO: 6-8, or comprises the sequence shown in any one of SEQ ID NO: 5, 26-29.
  • the protein or molecule may be a conjugate or fusion protein formed with other compounds or other polypeptides, and the conjugate may, for example, comprise any detectable label.
  • a protein or molecule which comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) VH and VL disclosed above, for example, the VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11-13, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences as shown in SEQ ID NO: 14-16, respectively.
  • the protein or molecule can be a conjugate, coupling or fusion protein formed with other compounds or other polypeptides.
  • the conjugate can comprise any detectable label.
  • a protein that binds to GPC3 and TCR.
  • a protein is provided that binds to GPC3 and ⁇ TCR.
  • a protein is provided that binds to GPC3 and the ⁇ 9 chain of TCR.
  • a protein is provided that binds to GPC3 and the variable region (V ⁇ 9) of the ⁇ 9 chain of TCR.
  • a protein is provided that binds to GPC3 and ⁇ 9 ⁇ 2 TCR.
  • a protein is provided that binds to GPC3 and ⁇ 9 ⁇ 1 TCR.
  • a protein is provided that binds to GPC3 and TRGV9.
  • the "binding" is simultaneous or sequential binding.
  • the present disclosure provides binding proteins that bind to tumor-associated antigens (TAA), tumor-specific antigens, and TRGV9, which include a binding domain that binds to TAA (or tumor-specific antigen) and a binding domain that binds to TRGV9. In some embodiments, it includes a first antigen binding domain that specifically binds to TAA (or tumor-specific antigen) and a second antigen binding domain that specifically binds to TRGV9. In some embodiments, the second antigen binding domain that specifically binds to TRGV9 is the aforementioned TRGV9 binding protein of the present disclosure. In some embodiments, the second antigen binding domain that specifically binds to TRGV9 is an immunoglobulin single variable domain in the aforementioned TRGV9 binding protein of the present disclosure, and/or a combination of VH and VL.
  • TAA tumor-associated antigens
  • TRGV9 which include a binding domain that binds to TAA (or tumor-specific antigen) and a binding domain that binds to TRGV9.
  • a GPC3/TRGV9 binding protein which comprises a first antigen binding domain that specifically binds to GPC3 and a second antigen binding domain that specifically binds to TRGV9.
  • the first antigen-binding domain comprises a heavy chain variable region (VH1) and/or a light chain variable region (VL1), the VH1 comprising HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 39, and the VL1 comprising LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 40.
  • VH1 heavy chain variable region
  • VL1 light chain variable region
  • the first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprising HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 39, and the VL1 comprising LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 40, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, according to the Kabat numbering system.
  • the first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1)
  • VH1 comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 41-43
  • the VL1 comprises LCDR1, LCDR2 and LCDR3
  • the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 44-46.
  • the first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1)
  • VH1 comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NOs: 41-43, respectively
  • VL1 comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NOs: 44-46, respectively.
  • the amino acid sequence of the first antigen binding domain comprises any one or more sequences selected from SEQ ID NO: 41-46.
  • the VH1 and/or VL1 are humanized, backmutated, affinity matured, T cell epitope (TCE) removed/reduced, antibody deamidation reduced, and/or antibody isomerization reduced.
  • TCE T cell epitope
  • the VH1 comprises an amino acid sequence as shown in SEQ ID NO: 39, or having at least 80% or at least 90% sequence identity thereto, and/or VL1 comprises an amino acid sequence as shown in SEQ ID NO: 40, or having at least 80% or at least 90% sequence identity thereto.
  • amino acid sequence of VH1 is shown as SEQ ID NO: 39
  • amino acid sequence of VL1 is shown as SEQ ID NO: 40.
  • a GPC3/TRGV9 binding protein comprising any of the aforementioned first antigen binding domains that specifically bind to GPC3 and a second antigen binding domain that specifically binds to TRGV9, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and/or CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29, for example, the immunoglobulin single variable domain comprises CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29; or,
  • VH2 a heavy chain variable region
  • VL2 a light chain variable region
  • the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, e.g., according to the Kabat numbering system.
  • a GPC3/TRGV9 binding protein comprising any of the aforementioned first antigen binding domains that specifically bind to GPC3 and a second antigen binding domain that specifically binds to TRGV9, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and/or CDR3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; or,
  • VH2 and/or VL2 said VH2 comprising HCDR1, HCDR2 and/or HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13; said VL comprises LCDR1, LCDR2 and/or LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • a GPC3/TRGV9 binding protein comprising any of the aforementioned first antigen binding domains that specifically bind to GPC3 and a second antigen binding domain that specifically binds to TRGV9, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6-8, respectively; and/or,
  • VH2 and VL2 wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11-13, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 14-16, respectively.
  • the aforementioned immunoglobulin single variable domain or VH, VL is humanized, backmutated, affinity matured, T cell epitope (TCE) removed/reduced, antibody deamidation reduced and/or antibody isomerization reduced.
  • the heavy chain framework region of the human germline template used for humanization of the aforementioned immunoglobulin single variable domain is derived from IGHV3-64; the heavy chain framework region of the human germline template used in the humanization process of VH is derived from IGHV3-21, and the light chain framework region of the human germline template used in the humanization process of VL is derived from IGKV1-12.
  • a GPC3/TRGV9 binding protein comprising any of the aforementioned first antigen binding domains that specifically bind to GPC3 and a second antigen binding domain that specifically binds to TRGV9, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; or,
  • VH2 and/or VL2 wherein the VH2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-32, or a sequence that is at least 80% or at least 90% identical thereto, and the VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • the combination of VH2 and VL2 is selected from the following:
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 9, or having at least 80%, at least 90% identity thereto
  • VL2 comprises an amino acid sequence as shown in SEQ ID NO: 10, or having at least 80%, at least 90% identity thereto
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 30, or having at least 80%, at least 90% identity thereto
  • VL2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80%, at least 90% identity thereto;
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 31, or having at least 80%, at least 90% identity thereto
  • VL2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80%, at least 90% identity thereto;
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 32, or a sequence that is at least 80% or at least 90% identical thereto
  • VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • the aforementioned GPC3/TRGV9 binding protein comprises or is an anti-GPC3/TRGV9 antibody or an antigen-binding fragment thereof.
  • the anti-GPC3/TRGV9 antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof.
  • the anti-GPC3/TRGV9 antibody is a bispecific antibody, a multispecific antibody (e.g., a trispecific antibody).
  • the immunoglobulin single variable domain in the anti-GPC3/TRGV9 antibody or its antigen-binding fragment is a single domain antibody or VHH.
  • the antigen-binding fragment includes but is not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv.
  • the aforementioned GPC3/TRGV9 binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) aforementioned immunoglobulin single variable domains. In some embodiments, the aforementioned GPC3/TRGV9 binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) aforementioned combinations of VH1 and VL1. In some embodiments, the aforementioned GPC3/TRGV9 binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) aforementioned combinations of VH2 and VL2.
  • the aforementioned GPC3/TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region of human IgG1, IgG2, IgG3 or IgG4.
  • the aforementioned GPC3/TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1, such as that shown in SEQ ID NO: 51 or having at least 80%, at least 90% sequence identity therewith.
  • the Fc region may be an Fc region with reduced effector function, for example, the Fc region may have a mutation, and exemplary IgG Fc regions with reduced effector function include substitutions having the following: N297A or N297Q (IgG1); L234A/L235A (IgG1); V234A/G237A (IgG2); L235A/G237A/E318A (IgG4); H268Q/V309L/A330S/A3 31S(IgG2); C220S/C226S/C229S/P238S(IgG1); C226S/C229S/E233P/L234V/L235A(IgG1); L234F/L235E/P331S(IgG1); L234F/L235E(IgG1); L234F or L235E(IgG1); L234A or L235A(IgG1); L234
  • the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2).
  • a mutation is introduced that allows the two subunits (Fc1, Fc2) of the Fc region to pair to form a dimer, or a mutation that reduces homodimerization.
  • the first subunit and the second subunit contain knob-into-hole mutations. For example, within the CH3/CH3 interface, one, two or more amino acid residues in the CH3 domain of Fc1 are mutated to one or more amino acid residues with a larger side chain volume, thereby generating a protrusion (or knob, Knob) on the surface of the CH3 domain of Fc1.
  • one, two or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated to amino acid residues with a smaller side chain volume, thereby generating a depression (or hole, Hole) on the surface of the CH3 domain of Fc2.
  • the first subunit (Fc1) and the second subunit (Fc2) are only distinguished from the two different subunits, so the two are interchangeable.
  • the Fc1 contains one or more amino acid substitutions at positions selected from 354, 356, 358 and 366, and the Fc2 contains one or more amino acid substitutions at positions selected from 349, 356, 358, 366, 368 and 407.
  • the Fc1 contains a mutation at position 366
  • the Fc2 contains a mutation at position 366, 368 and 407, or any combination thereof.
  • the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349.
  • the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349, 366, 368 and 407.
  • the Fc1 contains one or more amino acid substitutions selected from 354C, 356E, 358M and 366W
  • the Fc2 contains one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A and 407V.
  • the Fc1 contains a 366W mutation
  • the Fc2 contains a mutation selected from 366S, 368A and 407V or any combination thereof.
  • the Fc1 contains a 354C or 356C mutation
  • the Fc2 contains a 349C mutation.
  • the Fc1 contains a 354C/366W mutation
  • the Fc2 contains a 349C/366S/368A/407V mutation.
  • the Fc1 contains a T366W mutation, and the Fc2 contains a mutation selected from T366S, L368A and Y407V or any combination thereof; the Fc1 contains a S354C or E356C mutation, and the Fc2 contains a Y349C mutation; or the Fc1 contains a S354C/T366W mutation, and the Fc2 contains a Y349C/T366S/L368A/Y407V mutation.
  • the amino acid sequence of Fc1 is shown in SEQ ID NO: 52
  • the amino acid sequence of Fc2 is shown in SEQ ID NO: 53.
  • the aforementioned GPC3/TRGV9 binding protein comprises reducing the mismatch between the light and heavy chains by mutating the amino acid size and charge of the amino acids at the interface of the heavy chain CH1 and the light chain CL.
  • Roche exchanged the domains of CH1 and CL and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187–11192(2011)), and MedImmune introduced disulfide bonds by mutating the heavy chain F126C and the light chain S121C (Mazor et al., mAbs, 7(2), pp.377–389( 2015)), Amgen further modified the CH1-CL region by electrostatic interactions (Liu et al., Journal of Biological Chemistry, 290(12), pp.7535–7562(2015)), and Lilly (Lewis et al., Nature Biotechnology, 32(2), pp.191–198(
  • the aforementioned GPC3/TRGV9 binding protein comprises Obscurin-O chain (as shown in SEQ ID NO: 47) and Titin-T chain (as shown in SEQ ID NO: 48) to prevent or reduce mismatching between different VH and VL.
  • the aforementioned GPC3/TRGV9 binding protein comprises a linker.
  • the immunoglobulin single variable domain or VH, VL in the aforementioned GPC3/TRGV9 binding protein is directly or via a linker to the Fc region.
  • the linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structures.
  • the linker is as shown in ( GmSn ) h or ( GmQn ) h or ( GGNGT ) h or (YGNGT) h or (EPKSS) h or ( AmSn ) h , wherein m and n are each independently selected from integers of 1-8, and h is independently selected from integers of 1-20.
  • the linker is selected from G4S , GS, GAP, ( G4S ) 2 , ( G4S ) 3 , ( G4S ) 4 , ( G4S ) 5 , ASGS, A3S , etc.
  • the GPC3/TRGV9 binding protein comprises a combination selected from the group consisting of:
  • the first heavy chain from N-terminus to C-terminus, is: [immunoglobulin single variable domain]-[linker 1]-[Fc1],
  • the second heavy chain from N-terminus to C-terminus, is: [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc2],
  • the first heavy chain from N-terminus to C-terminus, is: [immunoglobulin single variable domain]-[linker 1]-[Fc2],
  • the second heavy chain from N-terminus to C-terminus, is: [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc1],
  • linker 1, linker 2, linker 3 and linker 4 may be the same or different, may exist independently or not, and may be independently selected from the linkers disclosed above;
  • Linker 1 is AAAS, and Linker 2, Linker 3, and Linker 4 are absent.
  • the first heavy chain from N-terminus to C-terminus, is: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],
  • the first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 3]-[Titin-T chain],
  • the second heavy chain from N-terminus to C-terminus, is: [VH1]-[Linker 4]-[CH1]-[Linker 5]-[Fc2],
  • the second light chain from N-terminus to C-terminus, is: [VL1]-[Linker 6]-[CL];
  • the first heavy chain from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],
  • the first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 3]-[Titin-T chain],
  • the second heavy chain from N-terminus to C-terminus, is: [VH2]-[Linker 4]-[CH1]-[Linker 5]-[Fc2],
  • the second light chain from N-terminus to C-terminus, is: [VL2]-[Linker 6]-[CL]
  • linker 1, linker 2, linker 3, linker 4, linker 5 and linker 6 may be the same or different, may exist independently or not, and may be independently selected from the linkers disclosed above;
  • linker 1 and linker 3 are GGGGS, and linker 2, linker 4, linker 5, and linker 6 do not exist.
  • the first heavy chain from N-terminus to C-terminus, is: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],
  • the first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 3]-[Titin-T chain],
  • the second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 4]-[CH1]-[Linker 5]-[VH1]-[Linker 6]-[CH1]-[Linker 7]-[Fc2],
  • the second light chain from N-terminus to C-terminus, is: [VL1]-[Linker 8]-[CL];
  • the first heavy chain from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],
  • the first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 3]-[Titin-T chain],
  • the second heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 4]-[CH1]-[Linker 5]-[VH2]-[Linker 6]-[CH1]-[Linker 7]-[Fc2],
  • the second light chain from N-terminus to C-terminus, is: [VL2]-[Linker 8]-[CL];
  • linker 1, linker 2, linker 3, linker 4, linker 5, linker 6, linker 7, and linker 8 may be the same or different, may exist independently or not, and may be independently selected from the linkers disclosed above;
  • linker 1 and linker 3 are GGGGS
  • linker 5 is GGGGSGGGGS
  • linker 2 is absent
  • the molar ratio of first heavy chain:first light chain:second heavy chain:second light chain is 1:1:1:2.
  • the amino acid sequences of Obscurin-O chain and Titin-T chain are shown in SEQ ID NO: 47 and 48, respectively.
  • CL is C ⁇
  • the amino acid sequences of CH1 and C ⁇ are shown in SEQ ID NO: 49 and 50, respectively.
  • the amino acid sequences of Fc1 and Fc2 are shown in SEQ ID NO: 52 and 53, respectively.
  • a GPC3/TRGV9 binding protein is provided selected from:
  • a GPC3/TRGV9 binding protein is provided selected from the group consisting of:
  • a polypeptide combination comprising amino acid sequences as shown in SEQ ID NO: 54-56; in some embodiments, the molar ratio of the polypeptide shown in SEQ ID NO: 54: the polypeptide shown in SEQ ID NO: 55: the polypeptide shown in SEQ ID NO: 56 in the GPC3/TRGV9 binding protein is 1:1:1;
  • polypeptide combination comprising amino acid sequences as shown in SEQ ID NOs: 56-59; in some embodiments, the molar ratio of the polypeptide shown in SEQ ID NO: 56: the polypeptide shown in SEQ ID NO: 57: the polypeptide shown in SEQ ID NO: 58: the polypeptide shown in SEQ ID NO: 59 in the GPC3/TRGV9 binding protein is 1:1:1:1;
  • polypeptides comprising amino acid sequences as shown in SEQ ID NOs: 57, 58, 60, and 61; in some embodiments, the molar ratio of the polypeptide shown in SEQ ID NO: 57: the polypeptide shown in SEQ ID NO: 58: the polypeptide shown in SEQ ID NO: 60: the polypeptide shown in SEQ ID NO: 61 in the GPC3/TRGV9 binding protein is 1:1:1:2.
  • the aforementioned GPC3/TRGV9 binding protein is: a protein that specifically binds to GPC3 and ⁇ TCR, or an anti-GPC3/ ⁇ TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and the ⁇ 9 chain of TCR, or an anti-GPC3/TCR ⁇ 9 chain antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and the variable region (V ⁇ 9) of the ⁇ 9 chain of TCR, or an anti-GPC3/V ⁇ 9TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and ⁇ 9 ⁇ 2 TCR, or an anti-GPC3/ ⁇ 9 ⁇ 2 TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and ⁇ 9 ⁇ 1 TCR, or an anti-GPC3/ ⁇ 9 ⁇ 1 TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and
  • the aforementioned GPC3/TRGV9 binding protein has at least one function or property selected from the following:
  • (b) specifically binds to the ⁇ 9 chain of TCR, for example, specifically binds to ⁇ 9 ⁇ 1 TCR, specifically binds to ⁇ 9 ⁇ 2 TCR;
  • (c) does not bind to the ⁇ 8 chain of the TCR, for example, does not bind to the ⁇ 8 ⁇ 2 TCR;
  • the KD value of the aforementioned GPC3/TRGV9 binding protein binding to TRGV9 may be ⁇ 1 ⁇ 10 -7 M, such as ⁇ 1 ⁇ 10 -8 M, or ⁇ 1 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -10 M.
  • the KD value of the aforementioned GPC3/TRGV9 binding protein binding to GPC3 may be ⁇ 1 ⁇ 10 -7 M, such as ⁇ 1 ⁇ 10 -8 M, or ⁇ 1 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -10 M.
  • the immunoglobulin single variable domain in the aforementioned GPC3/TRGV9 binding protein encompasses a variant, wherein the variant has one or more amino acid mutations compared to any one of SEQ ID NO: 5, 26-29.
  • VH1 and/or VL1 in the aforementioned GPC3/TRGV9 binding protein encompasses a variant, wherein the variant of VH1 has one or more amino acid mutations compared to SEQ ID NO: 39, and the variant of VL has one or more amino acid mutations compared to SEQ ID NO: 40.
  • VH2 and/or VL2 in the aforementioned GPC3/TRGV9 binding protein encompasses a variant, wherein the variant of VH2 has one or more amino acid mutations compared to any one of 9, 30-32, and the variant of VL has one or more amino acid mutations compared to any one of SEQ ID NO: 10, 33-35.
  • “Multiple” is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the amino acid mutation may be a conservative replacement, substitution or modification, and/or a deletion or addition that does not affect the function; the amino acid mutation may occur in the CDR region and/or the FR region.
  • a protein is provided whose binding to TRGV9 and/or GPC3 is blocked by the aforementioned GPC3/TRGV9 binding protein of the present disclosure.
  • a protein or molecule which comprises any of the GPC3/TRGV9 binding proteins disclosed above.
  • the protein or molecule may be a conjugate, a coupling substance or a fusion protein formed with other compounds or other polypeptides.
  • the conjugate may comprise any detectable label.
  • the present disclosure provides polynucleotides encoding the TRGV9 binding protein, GPC3/TRGV9 binding protein of the present disclosure.
  • the nucleic acid of the present disclosure may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acid of the present disclosure is a substantially isolated nucleic acid.
  • the nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and/or can be isolated from suitable natural sources.
  • a polynucleotide which encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and/or CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29, for example, the immunoglobulin single variable domain comprises CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29; and/or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, according to the Kabat numbering system.
  • a polynucleotide which encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6-8, respectively; and/or,
  • a polynucleotide which encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises: 1) an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29 or having at least 80% or at least 90% identity thereto; and/or,
  • VH and/or VL wherein the VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-33, or a sequence that is at least 80% or at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • a polynucleotide which encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises the following VH and VL:
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 9, or having at least 80%, at least 90% identity thereto
  • VL comprises an amino acid sequence as shown in SEQ ID NO: 10, or having at least 80%, at least 90% identity thereto,
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 30, or having at least 80% or at least 90% identity thereto
  • VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80% or at least 90% identity thereto;
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 31, or having at least 80%, at least 90% identity thereto
  • VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80%, at least 90% identity thereto;
  • VH comprises an amino acid sequence as shown in SEQ ID NO: 32, or a sequence that is at least 80% or at least 90% identical thereto
  • VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • a polynucleotide which encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises:
  • a polynucleotide that encodes a GPC3/TRGV9 binding protein, wherein the GPC3/TRGV9 binding protein comprises a first antigen binding domain that specifically binds to GPC3 and a second antigen binding domain that specifically binds to TRGV9.
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the aforementioned first antigen binding domain comprises a heavy chain variable region (VH1) and/or a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 39, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 40.
  • VH1 heavy chain variable region
  • VL1 light chain variable region
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the aforementioned first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 41-43, respectively; and the VL1 comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 44-46, respectively.
  • VH1 heavy chain variable region
  • VL1 light chain variable region
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the aforementioned first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises an amino acid sequence as shown in SEQ ID NO: 39, or having at least 80%, at least 90% sequence identity thereto, and/or the VL1 comprises an amino acid sequence as shown in SEQ ID NO: 40, or having at least 80%, at least 90% sequence identity thereto.
  • VH1 heavy chain variable region
  • VL1 light chain variable region
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and/or CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29, for example, the immunoglobulin single variable domain comprises CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5 and 26-29; or,
  • VH2 a heavy chain variable region
  • VL2 a light chain variable region
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 having the amino acid sequences shown in SEQ ID NOs: 6-8, respectively; and/or,
  • VH2 and VL2 wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 11-13, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 14-16, respectively.
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the second antigen binding domain that specifically binds to TRGV9 comprises:
  • an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; or,
  • VH2 and/or VL2 wherein the VH2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-32, or a sequence that is at least 80% or at least 90% identical thereto, and the VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the second antigen binding domain that specifically binds to TRGV9 comprises the following VH2 and VL2:
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 9, or having at least 80%, at least 90% identity thereto
  • VL2 comprises an amino acid sequence as shown in SEQ ID NO: 10, or having at least 80%, at least 90% identity thereto
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 30, or having at least 80%, at least 90% identity thereto
  • VL2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80%, at least 90% identity thereto;
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 31, or having at least 80%, at least 90% identity thereto
  • VL2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 33-35, or having at least 80%, at least 90% identity thereto;
  • VH2 comprises an amino acid sequence as shown in SEQ ID NO: 32, or a sequence that is at least 80% or at least 90% identical thereto
  • VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or a sequence that is at least 80% or at least 90% identical thereto.
  • a polynucleotide which encodes a GPC3/TRGV9 binding protein, wherein the GPC3/TRGV9 binding protein is selected from:
  • the present disclosure provides recombinant host cells that express or are capable of expressing one or more TRGV9 binding proteins, GPC3/TRGV9 binding proteins of the present disclosure and/or contain polynucleotides or vectors of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).
  • Gram-negative bacterial strains e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.
  • Fungal cells include, for example, cells of species of Trichoderma, Neurospora and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica) and Hansenula.
  • Saccharomyces e.g., Saccharomyces cerevisiae
  • Schizosaccharomyces e.g., Schizosaccharomyces pombe
  • Pichia e.g., Pichia pastoris and Pichia methanolica
  • Hansenula Hansenula
  • mammalian cells examples include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.
  • the present disclosure provides a method for preparing a TRGV9 binding protein or a GPC3/TRGV9 binding protein, comprising: expressing the target protein in a host cell as described above, and isolating the target protein from the host cell.
  • a purification step may also be included, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, and then eluting the bound antibodies using a pH gradient method, detecting with SDS-PAGE, and collecting.
  • conventional methods are used for filtration and concentration. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as at -70°C, or freeze-dried.
  • the engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies.
  • the culture fluid that secretes antibodies can be purified and collected by conventional techniques.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the present disclosure provides a composition comprising the TRGV9 binding protein and/or GPC3/TRGV9 binding protein of the present disclosure.
  • a pharmaceutical composition which contains an effective amount of the TRGV9 binding protein and/or GPC3/TRGV9 binding protein for treating, alleviating or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent or adjuvant.
  • the pharmaceutical composition may contain 0.01 to 99% by weight of TRGV9 binding protein and/or GPC3/TRGV9 binding protein in a unit dosage, or the amount of TRGV9 binding protein and/or GPC3/TRGV9 binding protein in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.
  • an article or product comprising the aforementioned TRGV9 binding protein and/or GPC3/TRGV9 binding protein.
  • the article comprises a container and a label.
  • the container is, for example, a bottle, a syringe, and a test tube.
  • the container holds a pharmaceutical composition effective for treating a condition.
  • the label on or connected to the container indicates that the pharmaceutical composition is used to treat a selected condition.
  • the aforementioned disease is a cell proliferative disease or cancer.
  • the present disclosure provides the aforementioned TRGV9 binding protein, GPC3/TRGV9 binding protein, their encoding polynucleotides, and compositions (including pharmaceutical compositions) for use in methods for treating, alleviating, preventing, and diagnosing diseases or disorders.
  • a method for improving, alleviating, treating or preventing a disease comprising administering to a subject an effective amount of:
  • a method of treating a disease by combining a TRGV9 binding protein of the present disclosure with a ⁇ T cell is provided. In some embodiments, a method of treating a disease by combining a GPC3/TRGV9 binding protein of the present disclosure with a ⁇ T cell is provided. In some embodiments, the ⁇ T cell is autologous or allogeneic.
  • any of the aforementioned TRGV9 binding proteins of the present disclosure are used to treat a disease, including use in combination with ⁇ T cells.
  • ⁇ T cells are used to treat a disease, including use in combination with any of the aforementioned TRGV9 binding proteins of the present disclosure.
  • any of the aforementioned GPC3/TRGV9 binding proteins disclosed herein are used to treat a disease, including use in combination with ⁇ T cells.
  • ⁇ T cells are used to treat a disease, including use in combination with any of the aforementioned GPC3/TRGV9 binding proteins disclosed herein.
  • a method for treating a disease comprising administering a therapeutically effective amount of any TRGV9 binding protein disclosed above and ⁇ T cells to a subject in need thereof. In some embodiments, a method for treating a disease is provided, comprising administering a therapeutically effective amount of any GPC3/TRGV9 binding protein disclosed above and ⁇ T cells to a subject in need thereof.
  • the aforementioned disease is a disease or disorder caused by overexpression of GPC3.
  • the aforementioned disease is a cell proliferative disease or cancer.
  • the cancer is GPC3-positive.
  • the aforementioned disease is liver cancer.
  • the cancer is GPC3-positive liver cancer.
  • the present disclosure provides TRGV9 binding proteins, GPC3/TRGV9 binding proteins, and detection uses of polynucleotides and compositions encoding them.
  • the present disclosure also provides methods, systems or devices for in vivo or in vitro detection of GPC3 and TRGV9, which include treating a sample with the aforementioned binding proteins, polynucleotides and compositions disclosed herein.
  • a kit comprising the aforementioned TRGV9 binding protein, GPC3/TRGV9 binding protein, its encoding polynucleotide, composition, and may also include diagnostic instructions.
  • the kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent.
  • the TRGV9 binding protein, GPC3/TRGV9 binding protein, its encoding polynucleotide can be formulated as a pharmaceutical composition.
  • FIG1 is a schematic diagram of the structure of an anti-GPC3/ ⁇ TCR bispecific antibody.
  • Figures 2A to 2D show the FACS binding activity test results of the anti-GPC3/ ⁇ TCR bispecific antibody to GPC3-positive cells and human ⁇ 9 ⁇ 2 T cells.
  • Figure 2A shows the binding of the anti-GPC3/ ⁇ TCR bispecific antibody to HepG2 cells
  • Figure 2B shows the binding of the anti-GPC3/ ⁇ TCR bispecific antibody to DLD-1 cells
  • Figure 2C shows the binding of the anti-GPC3/ ⁇ TCR bispecific antibody to human ⁇ T cells
  • Figure 2D shows the binding of the anti-GPC3/ ⁇ TCR bispecific antibody to PBMCs.
  • Figures 3A and 3B show the effects of anti- ⁇ TCR antibodies on BTN2A/BTN3A-TCR native signals.
  • Figure 3A shows the effects of SDP01378 on BTN2A/BTN3A-TCR native signals;
  • Figure 3B shows the effects of SDP01315 on BTN2A/BTN3A-TCR native signals.
  • Figures 4A to 4D show the results of the killing activity of anti- ⁇ TCR antibodies against tumor cells with different GPC3 expression levels.
  • Figure 4A shows the killing activity against HepG2 cells
  • Figure 4B shows the killing activity against Huh-7 cells
  • Figure 4C shows the killing activity against MKN-45 cells
  • Figure 4D shows the killing activity against DLD-1 cells.
  • Figures 5A to 5D show the results of cytotoxicity of ⁇ T cells from different donors to HepG2 cells.
  • the ⁇ T cells used in Figures 5A to 5D were derived from the induced expansion of donors #SC12004, #SC12392, #XC11053, and #XC11061, respectively.
  • Figures 6A to 6C show the killing results of anti-GPC3/ ⁇ TCR bispecific antibodies on tumor cells with low antigen expression at different effector-target ratios.
  • Figure 6A has an effector-target ratio of 1:1
  • Figure 6B has an effector-target ratio of 10:1
  • Figure 6C has an effector-target ratio of 30:1.
  • Figures 7A to 7D show the results of increasing the proportion of PBMC to improve the killing activity of ⁇ T cells.
  • Figure 7A shows the killing of PBMC cells from donor #XC11053;
  • Figure 7B shows the killing activity of ⁇ T cells from donor #XC11053 with 30% incorporation;
  • Figure 7C shows the killing of PBMC cells from donor #SC12392;
  • Figure 7D shows the killing activity of ⁇ T cells from donor #SC12392 with 30% incorporation.
  • Figures 8A to 8D are the results of cytokine release detection, wherein Figures 8A-8B respectively show the release of IFN ⁇ and TNF ⁇ when donor #XC11053 is killed; Figures 8C-8D respectively show the release of IFN ⁇ and TNF ⁇ when donor #SC12392 is killed.
  • FIG. 9 shows the detection result of anti-GPC3/ ⁇ TCR bispecific antibody promoting the proliferation of ⁇ T cells in PBMC.
  • FIG. 10A to FIG. 10B show the anti-tumor activity of SDP01716 in the Huh-7 transplanted tumor model, wherein FIG. 10A is a graph showing the changes in tumor volume in mice, and FIG. 10B is a graph showing the changes in body weight in mice.
  • Figures 11A to 11B show the anti-tumor activity of different anti-GPC3/ ⁇ TCR bispecific antibodies in the Huh-7 transplanted tumor model, wherein Figure 11A is a graph showing the changes in mouse tumor volume, and Figure 11B is a graph showing the changes in mouse body weight.
  • TRGV9 refers to a polypeptide that can form a T cell receptor when expressed on the surface of a ⁇ T cell.
  • ⁇ T cells expressing TRGV9 are one of the earliest T cells to develop in a human fetus and are the main ⁇ T cell subsets in peripheral blood cells of healthy adults.
  • TRGV9 includes any TRGV9 variants, isoforms, and species homologs that are naturally expressed by cells (including T cells) or can be expressed on cells transfected with a gene or cDNA encoding the polypeptide.
  • TRGV9 is human TRGV9.
  • An exemplary human TRGV9 amino acid sequence is provided by GenBank Accession No. NG_001336.2.
  • GPC3 refers to Glypican 3, which is anchored to the cell membrane by phosphatidylinositol and is a marker for hepatocellular carcinoma.
  • the amino acid sequence of an exemplary human GPC3 is provided by uniprot accession number P51654.
  • TRGV9 binding protein encompasses any protein that can specifically bind to TRGV9 or any molecule comprising the protein, including but not limited to antibodies, antigen-binding fragments thereof, or fusion proteins thereof as defined in the present disclosure for TRGV9.
  • the "TRGV9 binding protein” may include at least one (e.g., 1, 2, 3, 4, 5, 6 or more) single-domain antibody or VHH that specifically binds to TRGV9 in the embodiments of the present disclosure.
  • the "TRGV9 binding protein” of the present disclosure may include at least one (e.g., 1, 2, 3, 4, 5, 6 or more) VH and VL combination that specifically binds to TRGV9 in the embodiments of the present disclosure.
  • the "TRGV9 binding protein" of the present disclosure in addition to comprising a single variable domain of an immunoglobulin or a combination of VH and VL of TRGV9, may also include a linker and/or a portion having an effector function, such as a half-life extension portion (e.g., a single variable domain of an immunoglobulin that binds to serum albumin) and/or a fusion partner (e.g., serum albumin) and/or a conjugated polymer (e.g., PEG) and/or an Fc region.
  • the “TRGV9 binding protein” encompasses the anti- ⁇ TCR antibodies or antigen-binding fragments thereof in the embodiments of the present disclosure.
  • GPC3/TRGV9 binding protein encompasses any protein that can specifically bind to GPC3 and TRGV9 or any molecule comprising the protein, including but not limited to antibodies, polypeptides, fusion proteins of antibodies and polypeptides, or conjugates thereof.
  • GPC3/TRGV9 binding protein encompasses the anti-GPC3/ ⁇ TCR bispecific antibodies in the embodiments of the present disclosure.
  • Antibodies cover various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibodies may refer to immunoglobulins, which are tetrapeptide chains formed by two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulins are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chains, ⁇ chains, ⁇ chains, ⁇ chains and ⁇ chains, respectively.
  • the same class of Ig can be divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into ⁇ chains or ⁇ chains by different constant regions.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conservative sequences.
  • the three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies disclosed herein may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic or modified forms thereof, wherein monoclonal antibodies are particularly suitable for use in a number of embodiments.
  • the antibodies disclosed herein are recombinant antibodies.
  • “recombinant” refers generally to products such as cells or nucleic acids, proteins or vectors, indicating that the cells, nucleic acids, proteins or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering natural nucleic acids or proteins, or that the cells are derived from cells so modified.
  • recombinant cells express genes that are not present in the natural (non-recombinant) cell form or express natural genes that are abnormally expressed, underexpressed or not expressed at all.
  • CDR For the determination or definition of CDR, the deterministic depiction of CDR and the identification of residues comprising the binding site of the antibody can be completed by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformational definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and ab initio approaches to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198).
  • CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between the various numbering systems is well known to those skilled in the art.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without loss of the function of the rest of the protein and/or the domain.
  • Immunoglobulin domain refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region.
  • An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.
  • an “immunoglobulin variable domain” refers to four “framework regions” referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, and “framework region 4" or “FR4", respectively, and three “complementarity determining regions” or “CDRs” of "complementarity determining region 1" or “CDR1", “complementarity determining region 2" or “CDR2", and “complementarity determining region 3" or “CDR3".
  • the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • An immunoglobulin variable domain confers specificity to an antigen by having an antigen binding site.
  • Antibody framework (FR) refers to the portion of the variable domain that serves as a scaffold for the antigen binding loops (CDRs).
  • Immunoglobulin single variable domain is generally used to refer to an immunoglobulin variable domain (which may be a heavy chain or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the required VH/VL interaction between the VH and VL domains of conventional four-chain monoclonal antibodies).
  • immunoglobulin single variable domains include nanobodies (including VHH, humanized VHH and/or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs TM ), and (single domain) antibodies that are VL domains or derived from VL domains (such as dAbs TM ).
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody” (i.e., an "antibody lacking a light chain")
  • a heavy chain antibody i.e., an "antibody lacking a light chain”
  • VHH is used to distinguish the variable domain from the heavy chain variable domain (which is referred to as “VH domain” or VH in the present disclosure) and the light chain variable domain (which is referred to as “VL domain” or VL in the present disclosure) present in conventional tetrapeptide chain structure antibodies.
  • the VHH domain specifically binds to an epitope without the need for other antigen-binding domains; this binding behavior is different from the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the VL domain recognizes the epitope together with the VH domain.
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids and then humanized, or obtained by phage display technology.
  • the total number of amino acid residues in VHH will usually be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure.
  • the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Other numbering systems or coding conventions include Chothia, IMGT, AbM.
  • Humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable region framework.
  • the strong immune response induced by chimeric antibodies due to carrying a large amount of non-human protein components can be overcome.
  • the fully human antibody variable region can be subjected to minimal reverse mutations to maintain activity.
  • humanization examples include “humanizing” a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody (also referred to as “sequence optimization" in this disclosure, in addition to humanization, “sequence optimization” may also cover other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removing potential post-translational modification sites).
  • the humanized VHH domain may contain one or more completely human framework region sequences, and in some specific embodiments, may contain human framework region sequences of IGHV3.
  • Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), that is, CDR "grafting” to other "scaffolds” (including but not limited to human scaffolds or non-immunoglobulin scaffolds).
  • Scaffolds and techniques suitable for the CDR transplantation are known in the art.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the humanized antibodies disclosed in the present invention also include humanized antibodies after further affinity maturation of CDRs by phage display.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.
  • an “affinity matured” antibody is one that has one or more changes in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such changes, such changes resulting in an improvement in the affinity of the antibody for the antigen.
  • HVRs hypervariable regions
  • an “affinity matured” TRGV9 binding protein or anti-TRGV9 antibody has one or more changes in one or more CDRs that result in an increase in affinity for the antigen compared to its parent antibody.
  • Affinity matured antibodies can be prepared, for example, by methods known in the art as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad.
  • the GPC3/TRGV9 binding proteins, TRGV9 binding proteins of the present disclosure will bind to the antigen or target protein to be bound (i.e., GPC3, TRGV9) with a dissociation constant ( KD ) of preferably 10-7 to 10-10 moles/liter (M), more preferably 10-8 to 10-10 moles/liter, even more preferably 10-9 to 10-10 or less, as measured in a Biacore or KinExA or Fortibio assay, and/or with an association constant (KA) of at least 10-7 M, preferably at least 10-8 M, more preferably at least 10-9 M, more preferably at least 10-10 M.
  • KD dissociation constant
  • KD dissociation constant
  • M dissociation constant
  • KD dissociation constant
  • KD dissociation constant
  • M dissociation constant
  • KD dissociation constant
  • KD dissociation constant
  • M preferably 10-7 to 10-10 moles/liter
  • KA association constant
  • Any KD value greater than 10-4 M is generally considered
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described in the present disclosure.
  • SPR surface plasmon resonance
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • sandwich competitive assays as described in the present disclosure.
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant ( KD ).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Other standard assays for assessing the binding ability of an antibody against a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometric analysis, as well as other assays exemplified elsewhere in this disclosure.
  • binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR), such as by using a Biacore TM system or KinExA.
  • SPR surface plasmon resonance
  • Binding affinities associated with different molecular interactions for example, comparison of the binding affinities of different antibodies for a given antigen, can be compared by comparing the KD values of the individual antibody/antigen complexes.
  • the specificity of an interaction can be assessed by determining and comparing the KD value for an interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for a non-interest interaction (e.g., a control antibody known not to bind IGF-1R or TRGV9).
  • an interaction of interest e.g., a specific interaction between an antibody and an antigen
  • a non-interest interaction e.g., a control antibody known not to bind IGF-1R or TRGV9.
  • Constant substitution refers to substitution with another amino acid residue having a property similar to the original amino acid residue.
  • lysine, arginine and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains.
  • tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when replacing an amino acid residue in a group showing similar properties as described above, it will not show a specific change in properties.
  • “Homology”, “identity” or “sequence identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same nucleotide or amino acid monomer, for example, if every position of the two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.
  • Titin-T chain refers to a peptide segment of 78-118 amino acids in the titin protein containing the titin Ig-like 152 domain or its functional variant, wherein the titin-T chain can bind to the obscurin Ig-like 1 domain to form a dimerization complex.
  • the T chain functional variant is a polypeptide that has some amino acids of the wild-type T chain mutated, but still has the ability to bind to the obscurin Ig-like 1 domain to form a dimerization complex.
  • amino acids of appropriate length can be added or shortened at the C-terminus and/or N-terminus of the Titin Ig-like 152 domain; 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues can be added or shortened; for example, five amino acids "KAGIR" in the wild-type Titin protein adjacent to the N-terminus of the Titin Ig-like 152 domain can be added to the N-terminus of the Titin Ig-like 152 domain, which still have the function of associating with the Obscurin Ig-like 1 domain to form a complex.
  • Other mutations can also be made to the amino acids of the Titin Ig-like 152 domain, for example, mutations of certain amino acids can be made to improve interchain disulfide bonds, increase the stability of the complex, etc.
  • Obscurin-O chain refers to a peptide segment of 87-117 amino acids on the Obscurin protein containing the Obscurin Ig-like 1 domain or its functional variant, wherein the Obscurin-O chain can bind to the Titin Ig-like 152 domain to form a dimerization complex.
  • the Obscurin-O chain functional variant is a polypeptide that has mutated some amino acids of the wild-type O chain but still has the ability to bind to the Titin Ig-like 152 domain to form a dimerization complex.
  • a suitable length of amino acids is added or truncated at the C-terminus and/or N-terminus of the Obscurin-O domain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are added or truncated; for example, 5 amino acids "DQPQF" adjacent to the N-terminus of the Obscurin Ig-like 1 domain in the wild-type Obscurin protein are added to the N-terminus of the Obscurin-O domain, which still has the function of binding to the Titin Ig-like 152 domain to form a dimerization complex.
  • Other mutations can also be made to some amino acids in the Obscurin Ig-like 1 domain, for example, mutations to certain amino acids are made to improve interchain disulfide bonds, or to improve antibody stability, etc.
  • Nucleic acid or “polynucleotide” are used interchangeably in this disclosure and refer to any DNA molecule or RNA molecule that is single-stranded or double-stranded and, in the case of single strands, its complementary sequence, preferably double-stranded DNA.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Host cell includes individual cells or cell cultures that can be or have been recipients of vectors for incorporation of polynucleotide inserts.
  • Host cells include progeny of a single host cell, and progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutations.
  • Host cells include cells transfected and/or transformed in vivo with the polynucleotides of the present disclosure.
  • Cell “cell line,” and “cell culture” are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny having the same function or biological activity as screened in the initially transformed cell are included.
  • Inhibit or “block” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • “Inhibit growth” (eg, involving cells) is intended to include any measurable decrease in cell growth.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, such as for treatment, pharmacokinetic, diagnostic, research, and experimental procedures.
  • Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell.
  • administering also means in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a combination composition, or by another cell.
  • it refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as any binding protein or pharmaceutical composition thereof disclosed herein, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent.
  • the amount of the therapeutic agent effective to alleviate any specific disease symptom may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of the target disease in a subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • any statistical test known in the art such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.
  • an "effective amount” includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • the subject of the present disclosure may be an animal or a human subject.
  • the “subject” and “patient” of the present disclosure refer to mammals, especially primates, and especially humans.
  • TCR ⁇ T cell receptor on the surface of ⁇ T cells is a heterodimeric membrane protein composed of ⁇ chain and ⁇ chain.
  • the amino acids 1-242 of the ⁇ 9 chain (Protein data bank, number 1HXM, chain B) of the human ⁇ TCR extracellular region protein were selected, and the 3C restriction site, Leucine zipper, and FLAG tag were added to the C-terminus in sequence;
  • the amino acids 1-229 of the ⁇ 2 chain Protein data bank, number 1HXM, chain A
  • the 3C restriction site, Leucine zipper, and His8 tag were added to the C-terminus in sequence.
  • the constant regions of the ⁇ chain and the ⁇ chain were introduced with Q180C and V168C mutations, respectively, to form interchain disulfide bonds.
  • the recombinant monkey (Macaca mulatta) ⁇ 9 ⁇ 2 TCR protein selected amino acids 1-241 of the monkey ⁇ 9 chain (sequence see published patent number: US2019144540A1 SEQ ID NO: 42), wherein the C-terminus was sequentially added with a 3C restriction site, a Leucine zipper, and a FLAG tag; the amino acids 1-229 of the monkey ⁇ 2 chain (sequence see published patent number: US20190144540A1 SEQ ID NO: 37) were selected, wherein the C-terminus was sequentially added with a 3C restriction site, a Leucine zipper, and a His8 tag; in addition, Q179C and V168C mutations were introduced into the constant regions of the ⁇ chain and ⁇ chain, respectively, to form interchain disulfide bonds.
  • Plasmids carrying target protein encoding genes were synthesized separately, human ⁇ 9 chain (SEQ ID NO: 1) and human ⁇ 2 chain (SEQ ID NO: 2) plasmids were mixed in a ratio of 1:1, monkey ⁇ 9 chain (SEQ ID NO: 3) and monkey ⁇ 2 chain (SEQ ID NO: 4) plasmids were mixed in a ratio of 1:1, and transiently transfected and expressed in Expi293 cells (purchased from Thermo) for 7 days, respectively.
  • Human ⁇ 9 ⁇ 2 TCR protein and monkey ⁇ 9 ⁇ 2 TCR protein were isolated and purified, and stored at -80°C for future use.
  • the initial immunization dose is 2E7 cells per alpaca.
  • booster immunization is performed, and the immunization dose is 2E7 cells per alpaca.
  • Each subsequent booster immunization is separated by 3 weeks. Serum samples are collected one week after each booster immunization, and the antibody titer in the alpaca serum is detected by Protein ELISA and FACS.
  • the specific detection process of Protein ELISA is as follows: dilute the recombinant human ⁇ 9 ⁇ 2 TCR protein to 2 ⁇ g/mL with 0.05M carbonate buffer (pH 9.6), 100 ⁇ L/well, and coat overnight at 4°C; block with PBST buffer containing 5% skim milk for 1 hour, wash the plate 3 times; dilute the alpaca serum in the blocking buffer by two times starting from 1:2000, incubate at 37°C for 45min, and wash the plate 5 times; add 100 ⁇ L horseradish peroxidase-labeled sheep anti-Alpaca secondary antibody (AlpVHHs, 053-404-005, diluted 1:10000 with PBS) to each well, incubate at 37°C for 45min, and wash the plate 5 times. Finally, add 100 ⁇ L TMB colorimetric solution to each well for color development, and add 50 ⁇ L stop solution after 5 minutes to terminate the reaction. Use an enzyme reader to read the absorbance value at 450nm.
  • the specific FACS detection process is as follows: collect cells and resuspend to 4E6/mL, add 50 ⁇ L of cells to gradient diluted alpaca serum, and incubate at 4°C for 1 hour. Wash twice with 1% BSA/PBS buffer and discard the supernatant, add 1:200 diluted Anti alpaca IgG iFluor647 (AlpVHHs, 053-404-009), and incubate at 4°C in the dark for 45min. Wash twice with 1% BSA/PBS buffer, resuspend in 200 ⁇ L buffer and perform FACS detection.
  • the titer of alpaca serum after immunization was greater than 128k.
  • PBMC lymphocytes
  • the phage display method was used to screen antibodies against recombinant human ⁇ 9 ⁇ 2 TCR protein
  • ELISA was used to identify antibodies that cross-bind to monkey ⁇ 9 ⁇ 2 TCR protein
  • FACS was used to determine the binding activity to in vitro expanded human ⁇ 9 ⁇ 2 T cells.
  • the positive monoclonal single domain antibodies were screened using the above ELISA and FACS detection methods, and the sequences are as follows.
  • the initial immunization dose was 1E7 per mouse.
  • booster immunization was performed with a dose of 1E7 per mouse.
  • Each subsequent booster immunization was performed at an interval of 2-3 weeks.
  • Serum samples were collected one week after each booster immunization, and the antibody activity in the mouse serum was detected by Protein ELISA and FACS.
  • the specific detection process of Protein ELISA is as follows: coat the plate with 1 ⁇ g/mL recombinant human ⁇ 9 ⁇ 2 TCR protein, incubate at 4°C overnight, block with PBST buffer containing 1% BSA for 1 hour, and wash the plate 3 times. Dilute mouse serum in a three-fold gradient starting at 1:200 in the blocking buffer, incubate at 37°C for 1 hour, wash the plate 3 times, and incubate with a 1:10000 dilution of anti-mouse IgG-Fc-HRP secondary antibody (Sigma, AP127P) for 1 hour. Wash 3 times with PBST, add 100 ⁇ L TMB colorimetric solution to each well for color development, and terminate the reaction with stop solution after 15 minutes. Use an enzyme reader to read the absorbance at 450nm.
  • the specific FACS detection process is as follows: 1E5 cells per well of the cell plate, centrifuge and discard the supernatant, add gradient dilutions of mouse serum. Incubate at 4°C for 1h. Wash twice with 1% BSA/PBS buffer and discard the supernatant, add 1:500 diluted Alexa Flour 488 goat anti-mouse secondary antibody (Jackson immuno research.115-545-071), incubate at 4°C in the dark for 30min. Wash twice with 1% BSA/PBS buffer, resuspend in 200 ⁇ L buffer and perform FACS detection. The titer of mouse serum after immunization is greater than 72900.
  • mice were killed and the spleen was taken out.
  • the spleen cells were collected by grinding and mixed with mouse myeloma cells SP2/0, and hybridoma cells were obtained by electrofusion. Positive clones with OD 450nm>0.2 were tested by FACS, and positive clones with MFI values of >5000 binding to human ⁇ 9 ⁇ 2 T cells were subcloned. After initial screening and retesting of subclones, the mouse anti- ⁇ TCR antibody was screened using the above-mentioned ELISA and FACS detection methods.
  • the sequence of SDP01315 (clone number bph-003) is as follows.
  • the single-domain antibody was prepared by cloning the nucleotide sequence encoding the antibody into the pTT5 vector and transfecting ExpiCHO cells. After 8 days, the cells were removed by centrifugation, and the cell culture medium was collected and filtered. The harvested cell culture medium was purified using a nickel affinity column (HisTrap excel, GE), and the bound antibody was eluted with 300 mM imidazole. The solution was desalted and replaced with PBS to obtain the target antibody.
  • the nucleotide sequence encoding the mouse antibody was cloned into the pTT5 vector and then transfected into ExpiCHO cells. After 8 days, the cells were removed by centrifugation, the cell culture medium was collected and filtered, and the harvested cell culture medium was purified using a Protein A affinity column (MabSelect SuRe, GE). The bound antibody was eluted with glycine, and the eluate was neutralized with 1M Tris and then desalted to obtain the target antibody.
  • a Protein A affinity column Protein A affinity column
  • the process of cell-level affinity detection of anti- ⁇ TCR single-domain antibody is as follows: the gradient diluted antibody molecules are incubated with 1 x 10 5 ⁇ 9 ⁇ 2 T cells at 4°C for 1 hour, the excess antibody is washed off, and DyLight405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) is added, incubated at 4°C for 30 minutes, and after washing off the excess antibody, it is resuspended with 200 ⁇ L 2% FBS/PBS buffer, and the fluorescence signal on the cell surface is read by Thermo Attune NxT flow cytometer. The results are shown in Table 3.
  • the process of cell-level affinity detection of mouse anti- ⁇ TCR antibody is as follows: the gradient diluted antibody molecules are incubated with 1 x 10 5 ⁇ 9 ⁇ 2 T cells at 4°C for 1 hour, the excess antibody is washed off, and the mouse Alexa Flour 647 labeled anti-human Fc antibody (Jackson, Cat#209-605-098) is added, incubated at 4°C for 30 minutes, and after washing off the excess antibody, it is resuspended with 200 ⁇ L 2% FBS/PBS buffer, and the fluorescence signal on the cell surface is read by Thermo Attune NxT flow cytometer. The results are shown in Table 3.
  • This example uses a protein-based ELISA to detect the binding region of the anti- ⁇ TCR antibody to the antigen.
  • recombinant human ⁇ 9 ⁇ 1 TCR protein (dimeric protein of SEQ ID NO: 1 and SEQ ID NO: 20), human ⁇ 8 ⁇ 2 TCR protein (dimeric protein of SEQ ID NO: 21 and SEQ ID NO: 2), human V ⁇ C ⁇ chimeric TCR protein (dimeric protein of SEQ ID NO: 22 and SEQ ID NO: 23), and human V ⁇ C ⁇ chimeric TCR protein (dimeric protein of SEQ ID NO: 24 and SEQ ID NO: 25) were prepared.
  • ELISA was used to identify the binding activity of anti- ⁇ TCR antibodies to the above antigens and determine the approximate area of antibody binding.
  • the specific detection process of Protein ELISA is as follows: the plates were coated with 1 ⁇ g/mL recombinant TCR protein, incubated at 4°C overnight, blocked with PBST buffer containing 1% BSA for 1 hour, and washed 3 times. The antibody was diluted to 30nM with blocking buffer, incubated at 37°C for 1 hour, washed 3 times, and incubated with a 1:10000 dilution of anti-mouse IgG-Fc-HRP secondary antibody (Sigma, AP127P) for 1 hour. Wash 3 times with PBST, add 100 ⁇ L TMB colorimetric solution to each well for color development, and terminate the reaction with stop solution after 15 minutes. Use an enzyme reader to read the absorbance at 450nm.
  • the ELISA results are shown in Table 4.
  • the binding absorbance values of antibodies SDP01346 and SDP01315 to different antigens are different. Both antibodies bind to the ⁇ 9 ⁇ 1 TCR protein but not to the ⁇ 8 ⁇ 2 TCR protein, indicating that the antibodies bind to the ⁇ 9 chain; these two antibodies bind to the V ⁇ C ⁇ chimeric TCR protein but not to the V ⁇ C ⁇ chimeric TCR, indicating that the antibodies bind to the TCR variable region (V region).
  • V region TCR variable region
  • variable region sequences were compared with the antibody germline database to obtain human germline templates with high homology.
  • the human germline heavy chain template used by SDP01346 was IGHV3-30; the human germline heavy chain template used by SDP01315 was IGHV3-21, and the human germline light chain template was IGKV1-12.
  • the VH, VL of the mouse antibody or the CDR of the single domain antibody was chimerized into the appropriate human GermLine framework (Bioinformation.2014;10(4):180-186;Methods Mol Biol.2019;1904:213-230), and then the back mutation was introduced.
  • the obtained humanized molecule sequence is as follows:
  • a full-length antibody was constructed by adding GGGGSHHHHHH (SEQ ID NO: 72) after the corresponding VHH.
  • a full-length antibody was constructed by adding a linker and Fc after the corresponding VHH.
  • the mouse antibody was humanized to construct a full-length antibody.
  • the following is a FACS test of the binding of humanized monoclonal antibodies to ⁇ 9 ⁇ 2 T cells.
  • Affinity detection of single domain antibody SDP01346 after humanization The gradient diluted antibody molecules were incubated with 1 x 10 5 ⁇ 9 ⁇ 2 T cells at 4°C for 1 hour, the excess antibody was washed off, and DyLight 405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) was added, incubated at 4°C for 30 minutes, and the excess antibody was washed off and resuspended in 200 ⁇ L 2% FBS/PBS buffer, and the fluorescence signal on the cell surface was read by Thermo Attune NxT flow cytometer. The results are shown in Table 6.
  • Affinity detection of humanized mouse chimeric antibody SDP01315 The gradient diluted antibody molecules were incubated with 1 x10 5 ⁇ 9 ⁇ 2 T cells at 4°C for 1 hour, the excess antibody was washed off, and mouse Alexa Flour 647 labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added, incubated at 4°C for 30 minutes, and the excess antibody was washed off and resuspended in 200 ⁇ L 2% FBS/PBS buffer, and the fluorescence signal on the cell surface was read by Thermo Attune NxT flow cytometer. The results are shown in Table 6.
  • the sequences of the heavy chain variable region and light chain variable region of the antibody G selected for GPC3 end with nanomolar affinity are as follows.
  • Antibody G binds to the C-terminal subunit and does not bind to soluble GPC3.
  • Obscurin-O and Titin-T are used as shown in SEQ ID NO: 47-48, and CH1 and C ⁇ are shown in SEQ ID NO: 49-50.
  • IgG mutations are used.
  • the full-length sequences of the bispecific antibodies are as follows, where the underlined part is the CDR sequence, the wavy underline is the Obscurin-O or Titin-T sequence, the dotted underline is CH1 or C ⁇ , the italic is the IgG1 Fc region, the italic bold is the point mutation in the Fc region, and the bold is the linker.
  • the nucleotide sequence encoding the antibody was cloned into the pTT5 vector and transfected into ExpiCHO cells. After 8 days, the cells were removed by centrifugation, the cell culture medium was collected and filtered, and the harvested cell culture medium was purified using a Protein A affinity column (MabSelect SuRe, GE). The bound antibody was eluted with glycine, and the eluate was neutralized with 1M Tris and then desalted. After testing, the target antibodies SDP01716, SDP01696 and SDP01704 were obtained.
  • Protein A biosensor chip (Cat.#29139121-AB, Cytiva) was used. Each antibody to be tested was prepared with HBS-EP+ buffer solution as a ligand to capture Protein A on the chip channel.
  • Human GPC3 (Sino Biological, Cat#10088-H08H)/cynomolgus monkey GPC3 (Acrobiosystems, Cat#GP3-C5225)/human ⁇ 9 ⁇ 2/cynomolgus monkey ⁇ 9 ⁇ 2 antigens were prepared with HBS-EP+ buffer solution as analytes, and the analytes were diluted 2 times. The diluted antigen was passed through the experimental channel and the reference channel at a flow rate of 30 ⁇ L/min, binding for 80 seconds and dissociation for 300 seconds.
  • the regeneration buffer selected was 10mM Glycine pH1.5 (GE Healthcare, BR-1003-54) and run at a flow rate of 10 ⁇ L/min for 30 seconds. Data were analyzed using Biacore 8K evaluation software.
  • Example 8 Binding activity detection of anti-GPC3/ ⁇ TCR bispecific antibody to GPC3-positive cells and human ⁇ 9 ⁇ 2 T cells
  • the binding activity of ⁇ T bispecific antibodies to HepG2 cells (purchased from ATCC) that naturally highly express human GPC3 and DLD-1 cells (purchased from ATCC) that do not express human GPC3 was detected by FACS experiments.
  • the GPC3 binding activity experiment of anti-GPC3/ ⁇ TCR bispecific antibodies on cells was evaluated by detecting the fluorescence signal of the cell surface binding antibody, and the binding strength of the antibody was evaluated according to the strength of the fluorescence signal.
  • the gradient diluted antibody molecules and control molecules were incubated at 1 x 10 5 cells at 4°C for 1 hour, the excess antibody was washed off, and the mouse Alexa Flour 647 labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added, incubated at 4°C for 30 minutes, and the excess antibody was washed off and resuspended in 200 ⁇ L 2% FBS/PBS buffer.
  • the fluorescence signal on the cell surface was read by Thermo Attune NxT flow cytometer.
  • the cells used were HepG2 cell lines.
  • the binding activity of the anti-GPC3/ ⁇ TCR bispecific antibody to human ⁇ 9 ⁇ 2 T cells and to human PBMC was detected by FACS experiment. Human ⁇ 9 ⁇ 2 T cells were induced by zoledronic acid and IL-2.
  • the binding activity experiment of the anti-GPC3/ ⁇ TCR bispecific antibody on ⁇ T cells was performed by detecting the fluorescence signal of the antibody bound to the cell surface, and the binding strength of the antibody was evaluated according to the strength of the fluorescence signal.
  • the gradient diluted antibody molecules and control molecules were incubated at 1 x 10 5 cells at 4°C for 1 hour, the excess antibody was washed off, and the mouse Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added, incubated at 4°C for 30 minutes, and the excess antibody was washed off and resuspended in 200 ⁇ L 2% FBS/PBS buffer, and the fluorescence signal on the cell surface was read by Thermo Attune NxT flow cytometer.
  • SDP01378 is a monovalent molecule at the ⁇ T end of SDP01716
  • SDP01315 is a monovalent molecule of SDP01696 and SDP01704.
  • the cell culture medium of A375-Luc is RPMI1640 containing 10% inactivated fetal bovine serum.
  • ⁇ T cells were obtained by amplification of PBMC.
  • the density of A375-Luc was adjusted, and 25 ⁇ L/well of ⁇ T cells (1 ⁇ 10 5 /well) were inoculated in a 96-well plate, and the cell amount was 1 ⁇ 10 4 /well; 25 ⁇ L/well of ⁇ T cells (1 ⁇ 10 5 /well) were mixed with A375-Luc; 25 ⁇ L/well of SDP01378 or SDP01315 (final concentration of 10 nM) and 25 ⁇ L/well of gradient dilution of BTN3A agonist antibody were added.
  • Example 10 Detection of the killing activity of ⁇ T cells against tumor cells mediated by anti-GPC3/ ⁇ TCR bispecific antibody in vitro
  • the lactate dehydrogenase (LDH) detection method was used to evaluate the antibody-mediated ⁇ T cell killing activity against target cells with different GPC3 expression levels.
  • HepG2 cells naturally highly express GPC3, and the cell culture medium is DMEM (Gibco, Cat#11995-065, the same below), containing 15% inactivated fetal bovine serum; Huh-7 expresses moderate levels of GPC3, and the cell culture medium is DMEM; MKN-45 expresses low levels of GPC3, and the cell culture medium is RPMI 1640 (Gibco, Cat#10491A-01, the same below); DLD-1 does not express GPC3, and the cell culture medium is RPMI 1640.
  • DMEM Gibco, Cat#11995-065, the same below
  • Huh-7 expresses moderate levels of GPC3, and the cell culture medium is DMEM
  • MKN-45 expresses low levels of GPC3, and the cell culture medium is RPMI 1640 (Gibco, Cat#10491A-01, the same below)
  • DLD-1 does not express GPC3, and the cell culture medium is RPMI 1640.
  • the target cells are resuspended in RPMI1640 medium containing 2% serum, and the density is adjusted to 7 ⁇ 10 4 cells/mL; then 50 ⁇ L/well are inoculated in a 96-well plate, and 50 ⁇ L of the test antibody after gradient dilution is added. 50 ⁇ L of culture medium is added to each well. ⁇ T cells are collected, resuspended in RPMI1640 containing 2% fetal bovine serum, and the cell density is adjusted. 50 ⁇ L/well was inoculated into the above experimental plate and incubated in a 37°C, 5% CO 2 incubator for 24 hours.
  • the cell culture plate was removed and centrifuged (400 g, 5 minutes) to collect the cell culture supernatant.
  • CytoTox The level of LDH was detected by Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). For specific operations, refer to the reagent instructions.
  • Anti-GPC3/ ⁇ TCR bispecific antibodies kill cells expressing different antigens
  • Example 11 Effect of anti-GPC3/ ⁇ TCR bispecific antibody on ⁇ T cell killing from different PBMC donors
  • lactate dehydrogenase (LDH) detection was used to evaluate the antibody-mediated cytotoxicity of ⁇ T cells against target cells expressing GPC3.
  • HepG2 cells were resuspended in RPMI1640 medium containing 2% serum, and the cell density was adjusted to 7 ⁇ 10 4 cells/mL; then 50 ⁇ L/well was inoculated in a 96-well plate, and 50 ⁇ L of the gradient diluted antibody to be tested was added. 50 ⁇ L of culture medium was added to each well.
  • ⁇ T cells amplified from different donors were collected, resuspended in RPMI1640 containing 2% fetal bovine serum, and the cell density was adjusted. 50 ⁇ L/well was inoculated in the above experimental plate and incubated in a 37°C, 5% CO 2 incubator for 24 hours. The cell culture plate was removed, centrifuged (400g, 5 minutes), the cell culture supernatant was collected, and CytoTox was used. The level of LDH was detected by Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780).
  • SDP01716 results are shown in an exemplary manner. As shown in Figures 5A to 5D and Table 11, SDP01716 can mediate the killing of tumor cells by ⁇ T cells from different donors, and there is no significant difference in EC 50 and maximum killing value.
  • #SC12004, #SC12392, #XC11053, #XC11061 are ⁇ T cells expanded from different donor sources (healthy allogeneic human peripheral blood), - means no killing was detected.
  • Example 12 Detection of the killing activity of anti-GPC3/ ⁇ TCR bispecific antibody against antigen low-expressing cells at different effector-target ratios
  • lactate dehydrogenase (LDH) detection was used to evaluate the antibody-mediated killing activity of ⁇ T cells against target cells with low GPC3 expression.
  • MKN-45 cells naturally low-express GPC3. After digestion, MKN-45 cells were resuspended in RPMI1640 medium containing 2% serum, and the cell density was adjusted to 7 ⁇ 10 4 cells/mL; then 50 ⁇ L/well was inoculated in a 96-well plate, and 50 ⁇ L of the antibody to be tested after gradient dilution was added. 50 ⁇ L of culture medium was added to each well. ⁇ T cells were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust the cell density.
  • SDP01716 The results of SDP01716 are shown in an exemplary manner. As shown in FIG6A , at a low efficiency-target ratio of 1:1, SDP01716 has weak killing activity against antigen-low expressing cells, with a maximum killing value of 17%; as shown in FIG6B , after increasing the efficiency-target ratio to 10:1, SDP01716 can enhance the killing activity against antigen-low expressing cells, with a maximum killing value of 64%; as shown in FIG6C , when the efficiency-target ratio is increased to 30:1, it almost reaches 100% killing. That is, the anti-GPC3/ ⁇ TCR bispecific antibody disclosed in the present invention can kill tumor target cells in an efficiency-target ratio-dependent manner.
  • Example 13 Improving the proportion of ⁇ T cells in PBMCs to enhance the killing activity of anti-GPC3/ ⁇ TCR bispecific antibodies and cytokine release detection
  • lactate dehydrogenase (LDH) detection was used to evaluate the antibody-mediated cytotoxicity of ⁇ T cells against target cells expressing GPC3.
  • HepG2 cells naturally highly express GPC3. After digestion, HepG2 cells were resuspended in RPMI1640 medium containing 2% serum, and the cell density was adjusted to 7 ⁇ 10 4 cells/mL; then 50 ⁇ L/well was inoculated in a 96-well plate, and 50 ⁇ L of the gradient diluted antibody to be tested was added. 50 ⁇ L of culture medium was added to each well. PBMC cells were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust the cell density.
  • Figures 7A to 7D show the results of SDP01716.
  • Figure 7A shows the cell killing of PBMC of donor #XC11053
  • Figure 7B shows the killing activity of ⁇ T cells spiked with 30% of PBMC of donor #XC11053
  • Figure 7C shows the cell killing of PBMC of donor #SC12392
  • Figure 7D shows the killing activity of ⁇ T cells spiked with 30% of PBMC of donor #SC12392.
  • Figures 7A and 7C show that the ability of SDP01716 to kill tumor cells in a pure PBMC system is weaker than that of the CD3 dual antibody BMK-029 (ERY974 in US20220348658) with the same target, because CD3 dual antibodies can mobilize more T cells as effector cells to kill tumors;
  • Figures 7B and 7D show that increasing the proportion of ⁇ T cells in PBMCs can enhance their killing activity.
  • the cytokine release detection results corresponding to Figures 7A to 7D are shown in Figures 8A to 8D.
  • SDP01716 induces less IFN ⁇ and TNF ⁇ release when killing tumor cells.
  • Example 14 Detection of the proliferation activity of ⁇ T cells in PBMCs promoted by anti-GPC3/ ⁇ TCR bispecific antibodies
  • flow cytometry was used to evaluate the activity of anti-GPC3/ ⁇ TCR bispecific antibodies in inducing proliferation of ⁇ T cells in PBMCs.
  • Human liver cancer Huh-7 cells (cell bank of Chinese Academy of Sciences) were subcutaneously inoculated into NSG mice (female, 6-8 weeks old, provided by Vital River) at 5 ⁇ 10 6 cells/100 ⁇ L/mouse and randomly divided into 4 groups (G1-G4), 7 mice in each group. Tumor cells were inoculated on the day of grouping, and ⁇ T cells and drugs were infused at the same time. Each group was given drugs according to the following scheme:
  • G1 medium G2: ⁇ T cells + PBS
  • Tumor volume was calculated as 1/2 ⁇ a ⁇ b 2 , where a and b represent the long and short diameters of the measured tumor, respectively;
  • T/C% (T-T0)/(C-C0) ⁇ 100;
  • TGI% 1-T/C%.
  • the results are shown in Figures 10A, 10B and Table 12.
  • the TGI of the ⁇ T cell monotherapy group (G2) was 11%, and the inhibitory effect on tumor growth was limited; SDP01716 could inhibit tumor growth in a dose-dependent manner, with the TGI reaching 58% at a dose of 3 mpk, and each dosing group had no significant effect on the weight of mice.
  • Huh-7 cells were inoculated subcutaneously into NSG mice (female, 6-8 weeks old, provided by Jicui Pharmaceutical) at 5 ⁇ 10 6 cells/100 ⁇ L/mouse and randomly divided into 6 groups, 7 mice in each group. Tumor cells were inoculated on the day of grouping, and ⁇ T cells and drugs were infused at the same time. Each group was given drugs according to the following scheme:
  • G5 ⁇ T cells + SDP01704 (5.1 mg/kg).
  • ⁇ T cells were infused and the drug was administered once a week. If ⁇ T cells and antibodies were used in combination, the drugs were administered simultaneously. Tumor volume was measured twice a week during the administration and observation period, and the measured values were recorded.
  • the results are shown in FIG. 11A , FIG. 11B and Table 13 .
  • the dual antibodies disclosed herein can effectively inhibit tumors, and no significant changes in body weight were observed in the mice in each administration group.

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Abstract

本公开涉及TRGV9结合蛋白及其医药用途。具体而言，本公开涉及TRGV9结合蛋白、GPC3/TRGV9结合蛋白及其治疗癌症的方法和制药用途。

Description

TRGV9结合蛋白及其医药用途
本申请要求2023年11月15日提交的中国专利申请CN202311518247.8的优先权。
技术领域
本公开涉及生物医药领域，具体涉及TRGV9结合蛋白、GPC3/TRGV9结合蛋白及其治疗癌症的方法和制药用途。
背景技术
γδT细胞是一群天然识别和能够杀伤肿瘤细胞的T细胞，既有抗原递呈，又有杀伤功能，桥接固有免疫和适应性免疫。与αβTCR不同，组成γδT细胞TCR的两条链分别为γ链和δ链，其对抗原的识别不受MHC限制(Exp Mol Med.2021Mar；53(3):318-327)。γδT细胞数目相对较少的，约占PBMC的1％-5％。根据δ链的不同，可分为δ1，δ2，δ3，δ5四类(Front Immunol.2022 Jun 16；13:915837)，而其中的δ1和δ2所占数目最多。δ1可与不同的γ链配对，形成不同的TCR，主要分布于皮肤，粘膜等组织；δ2主要与γ9配对，形成γ9δ2亚型，主要分布于外周血，占外周血γδT细胞总量高达95％(Front Immunol.2022 Jun 16；13:915837)。
γδT细胞在抗肿瘤中发挥着重要作用，有多种杀伤肿瘤细胞的机制，其中包括通过TCR识别靶细胞上经磷酸化抗原活化的BTN2A/BTN3A复合物，启动杀伤信号，发挥杀伤功能；此外还有与NK细胞类似的杀伤途径，其细胞表面表达NKG2D等受体，通过结合肿瘤细胞上的相应配体发挥肿瘤杀伤作用。γδT细胞在多种肿瘤中有不同程度的浸润。通过对25个肿瘤类型，5782个肿瘤的大样本分析发现，γδT细胞的浸润预示着病人的良好预后，而且是所有免疫细胞中的最有利细胞群体。对14个非脑癌实体瘤的3238份样本分析同样表明其在肿瘤中的浸润是良好预后的指征(Oncoimmunology.2017 Feb 6；6(3):e1284723)。针对γδT细胞的早期临床研究主要以体内激活和体外扩增后回输两种方式为主，尽管不同研究所显示的抗肿瘤效果比较有限，但均没有报道严重的副作用，提示了γδT细胞具有良好的安全性。
Glypican-3(GPC3)是肝癌治疗的一个潜在靶点。2020年全球肝癌新增病例90万，死亡病例83万；而在中国，2020年新增41万病例，死亡39万；全球一半左右的肝癌都发生在中国。近些年，晚期肝癌的一线或者二线治疗已经取得了长足的进展，但后线治疗仍缺乏有效治疗手段。GPC3是一种由580个氨基酸组成的硫酸乙酰肝素蛋白聚糖，通过糖基磷脂酰肌醇锚定于细胞膜。GPC3在多种肿瘤组织中高表达，例如，在肝细胞癌中GPC3阳性率高达90％。正常组织中GPC3表达则非常局限，只表达于胎盘和子宫内膜；而且子宫内膜的表达量远低于肿瘤组织，是一个理想的抗肿瘤靶点(Sci Transl Med.2017 Oct 4；9(410):eaal4291)。
本公开提供了新序列结构的靶向γδTCR的抗体，并将其与肿瘤相关抗原(例如，GPC3)结合结构域(例如，抗体)构建双特异性抗体，所述双特异性抗体一端结合TAA(例如，GPC3)，另一端结合γδTCR，能招募并激活γδT细胞特异性地杀伤TAA(例如，GPC3)阳性肿瘤细胞，这显著提高了γδT细胞治疗的有效性。本公开的双特异性抗体有良好的肿瘤杀伤活性、安全性和成药性。
发明内容
本公开提供T细胞受体(TCR)结合蛋白，磷脂酰肌醇蛋白聚糖-3(GPC3)和T细胞受体(TCR)结合蛋白，其编码核酸，其制备方法，及其用于治疗疾病的方法和用途。
T细胞受体γ可变区9(TRGV9)结合蛋白
本公开提供TRGV9结合蛋白。一些实施方案中，其能够结合TCR。一些实施方案中，其能够结合γδTCR。一些实施方案中，其能够结合TCR的γ9链。一些实施方案中，其能够结合γ9δ2 TCR。另一些实施方案中，其能够结合γ9δ1 TCR。
一些实施方案中，提供TRGV9结合蛋白，其包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR1、CDR2和/或CDR3，例如，免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR3；或，
2)重链可变区(VH)和/或轻链可变区(VL)，所述VH包含SEQ ID NO：9、30-32任一所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO：10、33-35任一所示氨基酸序列中的LCDR1、LCDR2和LCDR3，
所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如，是根据Kabat编号系统定义的。
一些实施方案中，提供TRGV9结合蛋白，其包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含CDR1、CDR2和/或CDR3，其中，CDR1包含SEQ ID NO：6所示氨基酸序列，CDR2包含SEQ ID NO：7所示氨基酸序列，CDR3包含SEQ ID NO：8所示氨基酸序列；或，
2)VH和/或VL，所述VH包含HCDR1、HCDR2和/或HCDR3，其中，HCDR1包含SEQ ID NO：11所示氨基酸序列，HCDR2包含SEQ ID NO：12所示氨基酸序列，HCDR3包含SEQ ID NO：13所示氨基酸序列；所述VL包含LCDR1、LCDR2和/或LCDR3，其中，LCDR1包含SEQ ID NO：14所示氨基酸序列，LCDR2包含SEQ ID NO：15所示氨基酸序列，LCDR3包含SEQ ID NO：16所示氨基酸序列。
一些实施方案中，提供TRGV9结合蛋白，其包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含分别如SEQ ID NO：6-8所示氨基酸序列的CDR1、CDR2和CDR3；或，
2)VH和VL，所述VH包含分别如SEQ ID NO：11-13所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：14-16所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些实施方案中，前述蛋白中的免疫球蛋白单一可变结构域或VH或VL各自独立地为人源化的、回复突变、亲合力成熟、去除/减少T细胞表位(TCE)、降低抗体脱酰胺和/或降低抗体异构化改造的。
一些实施方案中，前述蛋白中的免疫球蛋白单一可变结构域人源化所使用的人种系模板的重链框架区源自IGHV3-64；VH在人源化改造过程使用的人种系模板的重链框架区源自IGHV3-21，VL在人源化改造过程使用的人种系模板的轻链框架区源自IGKV1-12。
一些实施方案中，提供TRGV9结合蛋白，其包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含如SEQ ID NO：5、26-29任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；或，
2)VH和VL，所述VH包含如SEQ ID NO：9、30-33任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，所述VL包含如SEQ ID NO：10、33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，TRGV9结合蛋白包含如下的VH和VL：
2-1)VH包含如SEQ ID NO：9所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：10所示或与之具有至少80％、至少90％同一性的氨基酸序列，
2-2)VH包含如SEQ ID NO：30所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-3)VH包含如SEQ ID NO：31所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-4)VH包含如SEQ ID NO：32所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
本公开中，“至少80％(序列)同一性”涵盖至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性；“至少90％(序列)同一性”涵盖至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性。
一些实施方案中，前述TRGV9结合蛋白包含或为抗TRGV9抗体或其抗原结合片段。一些实施方案中，所述抗TRGV9抗体或其抗原结合片段为重组抗体或其片段。一些实施方案中，所述抗TRGV9抗体为单特异性抗体、双特异性抗体、多特异性抗体(例如，三特异性抗体)。
其中，当前述TRGV9结合蛋白中包含免疫球蛋白单一可变结构域时，其可以为骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段，例如，免疫球蛋白单一可变结构域是为单域抗体或VHH。作为一个示例，前述TRGV9结合蛋白本身即为单域抗体或VHH。一些具体实施方案中，所述抗体或其抗原结合片段为线性抗体、单链抗体、纳米抗体、肽抗体peptibody、结构域抗体和diabody、triabody和tetrabody、串联二-scFv、串联三-scFv。
其中，当前述TRGV9结合蛋白中包含VH和VL时，其可以为鼠源抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。一些具体实施方案中，所述抗原结合片段包括但不限于：Fab、Fv、sFv、Fab’、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv。一些具体实施方案中，所述抗原结合片段包括Fab、Fv、sFv、Fab’、F(ab’)2。
一些实施方案中，前述TRGV9结合蛋白中可以包含一个或多个(例如2、3、4、5、6、7、8个)前述免疫球蛋白单一可变结构域。所述免疫球蛋白单一可变结构域可以形成二聚体或多聚体分子。所述免疫球蛋白单一可变结构域可以是同二聚体或异二聚体。
一些实施方案中，前述TRGV9结合蛋白中可以包含一个或多个(例如2、3、4、5、6、7、8个)前述VH、VL或其组合。
一些实施方案中，前述TRGV9结合蛋白中还包含人免疫球蛋白Fc区；例如，所述Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区。一些实施方案中，所述Fc区是人IgG1的Fc区，例如SEQ ID NO：51所示或与之具有至少80％、至少90％序列同一性。一些具体实施方案中，所述Fc区可以是效应器功能降低的Fc区，例如，所述Fc区可以具有突变，效应器功能降低的示例性IgG Fc区包括具有以下的取代：N297A或N297Q(IgG1)；L234A/L235A(IgG1)；V234A/G237A(IgG2)；L235A/G237A/E318A(IgG4)；H268Q/V309L/A330S/A331S(IgG2)；C220S/C226S/C229S/P238S(IgG1)；C226S/C229S/E233P/L234V/L235A(IgG1)；L234F/L235E/P331S(IgG1)；L234F/L235E(IgG1)；L234F或L235E(IgG1)；L234A或L235A(IgG1)或S267E/L328F(IgG1)。例如，L234A/L235A表示为序列包含L234A和L235A。
一些实施方案中，前述TRGV9结合蛋白中免疫球蛋白单一可变结构域或VH、VL与Fc区直接或通过连接子连接。所述连接子可以是长1-20个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述连接子如(GmSn)h或(GmQn)h或(GGNGT)h(SEQ ID NO：62)或(YGNGT)h(SEQ ID NO：63)或(EPKSS)h(SEQ ID NO：64)或(AmSn)h所示，其中，m、n各自独立地选自1-8的整数，h独立地选自1-20的整数。例如，所述连接子选自G4S(SEQ ID NO：65)、GS、GAP、(G4S)2(SEQ ID NO：66)、(G4S)3(SEQ ID NO：67)、(G4S)4(SEQ ID NO：68)、(G4S)5(SEQ ID NO：69)、ASGS(SEQ ID NO：70)、A3S(SEQ ID NO：71)等；
一些实施方案中，前述TRGV9结合蛋白为：特异性结合γδTCR的蛋白，或抗γδTCR抗体或其抗原结合片段；特异性结合TCR的γ9链的蛋白，或抗TCR的γ9链的抗体或其抗原结合片段；特异性结合TCR的γ9链的可变区(Vγ9)的蛋白，或抗Vγ9TCR的抗体或其抗原结合片段；特异性结合γ9δ2 TCR的蛋白，或抗γ9δ2TCR抗体或其抗原结合片段；特异性结合γ9δ1 TCR的蛋白，或抗γ9δ1 TCR抗体或其抗原结合片段；特异性结合TRGV9的蛋白，或抗TRGV9抗体或其抗原结合片段。
一些实施方案中，前述TRGV9结合蛋白包含：
1)如SEQ ID NO：36任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列；
2)重链和轻链，其中，重链如SEQ ID NO：37任一所示或与之具有至少80％、至少90％序列同一性，轻链如SEQ ID NO：38任一所示或与之具有至少80％、至少90％序列同一性。
一些实施方案中，前述TRGV9结合蛋白具有选自以下至少一项的功能或性质：
(a)以≤10nM的EC50与γ9δ2 T细胞结合，所述EC50例如≤5nM、≤4nM、≤3nM、≤2nM、≤1nM、≤0.5nM、≤0.2nM、≤0.1nM。所述EC50是通过FACS检测获得，FACS是本领域常用的亲和力检测方法，例如本公开实施例5中所述。
(b)特异性结合TCR的γ9链，例如，特异性结合γ9δ1 TCR、γ9δ2 TCR；
(c)不结合(通过测试方法，检测不到)TCR的γ8链，例如，不结合γ8δ2 TCR；
(d)特异性结合TCR的γ9链的可变区(TRGV9)；
(e)特异性结合VγδCαβ嵌合TCR，不结合VαβCγδ嵌合TCR；
(f)结合食蟹猴γ9δ2 TCR；
其中，(b)-(e)可通过本领域常规检测方法获知，例如本公开实施例4中所述。
一些实施方案中，前述蛋白结合TRGV9的KD值可以≤1×10-7M，例如≤1×10-8M，或≤1×10-9M，或≤1×10-10M。
一些实施方案中，前述TRGV9结合蛋白涵盖免疫球蛋白单一可变结构域的变体，所述变体与SEQ ID NO：5、26-29任一相比，具有一个或多个氨基酸突变。一些实施方案中，前述TRGV9结合蛋白涵盖VH和/或VL的变体，所述VH的变体与SEQ ID NO：9、30-32任一相比，具有一个或多个氨基酸突变，所述VL的变体与SEQ ID NO：10、33-35任一相比，具有一个或多个氨基酸突变。“多个”涵盖1、2、3、4、5、6、7、8、9、10个。所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加；所述氨基酸突变可以发生在CDR区和/或FR区。
一些实施方案中，提供蛋白，其与前述本公开的TRGV9结合蛋白中的免疫球蛋白单一可变结构域结合或竞争结合相同的抗原表位。
一些实施方案中，提供蛋白，其与前述本公开的TRGV9结合蛋白中的VH和VL结合或竞争结合相同的抗原表位。
一些实施方案中，提供蛋白，其与TRGV9的结合被前述本公开的TRGV9结合蛋白中的免疫球蛋白单一可变结构域或VH和VL阻断。
一些实施方案中，提供蛋白或分子，其包含前述本公开任意一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)免疫球蛋白单一可变结构域，例如，所述免疫球蛋白单一可变结构域包含SEQ ID NO：6-8所示的CDR1、CDR2和CDR3，或包含SEQ ID NO：5、26-29任一所示序列。所述蛋白或分子可以为与其他化合物、其他多肽形成的缀合物或融合蛋白，所述缀合物例如可包含任意可检测标记。
一些实施方案中，提供蛋白或分子，其包含前述本公开任意一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)VH和VL，例如，所述VH包含SEQ ID NO：11-13所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：14-16所示氨基酸序列的LCDR1、LCDR2和LCDR3。所述蛋白或分子可以为与其他化合物、其他多肽形成的缀合物、偶联物或融合蛋白。例如，缀合物可包含任意可检测标记。
GPC3/TRGV9结合蛋白
本公开提供蛋白，其结合GPC3和TCR。一些实施方案中，提供蛋白，其结合GPC3和γδTCR。一些实施方案中，提供蛋白，其结合GPC3和TCR的γ9链。一些实施方案中，提供蛋白，其结合GPC3和TCR的γ9链的可变区(Vγ9)。一些实施方案中，提供蛋白，其结合GPC3和γ9δ2 TCR。一些实施方案中，提供蛋白，其结合GPC3和γ9δ1 TCR。一些实施方案中，提供蛋白，其结合GPC3和TRGV9。一些实施方案中，所述“结合”为同时或先后结合。
本公开提供结合肿瘤相关抗原(TAA)、肿瘤特异性抗原和TRGV9的结合蛋白，其包含结合TAA(或肿瘤特异性抗原)的结合结构域和结合TRGV9的结合结构域。一些实施方案中，其包含特异性结合TAA(或肿瘤特异性抗原)的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域。一些实施方案中，所述特异性结合TRGV9的第二抗原结合结构域为前述本公开的TRGV9结合蛋白。一些实施方案中，所述特异性结合TRGV9的第二抗原结合结构域为前述本公开的TRGV9结合蛋白中的免疫球蛋白单一可变结构域，和/或VH和VL的组合。
一些实施方案中，提供GPC3/TRGV9结合蛋白，其包含特异性结合GPC3的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域。
一些具体实施方案中，所述第一抗原结合结构域包含重链可变区(VH1)和/或轻链可变区(VL1)，所述VH1包含SEQ ID NO：39所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL1包含SEQ ID NO：40所示氨基酸序列中的LCDR1、LCDR2和LCDR3。一些具体实施方案中，所述第一抗原结合结构域包含重链可变区(VH1)和轻链可变区(VL1)，所述VH1包含SEQ ID NO：39所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL1包含SEQ ID NO：40所示氨基酸序列中的LCDR1、LCDR2和LCDR3，所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如，是根据Kabat编号系统定义的。
一些具体实施方案中，所述第一抗原结合结构域包含重链可变区(VH1)和轻链可变区(VL1)，所述VH1包含HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3分别包含如SEQ ID NO：41-43所示氨基酸序列；所述VL1包含LCDR1、LCDR2和LCDR3，所述LCDR1、LCDR2和LCDR3分别包含如SEQ ID NO：44-46所示氨基酸序列。
一些具体实施方案中，所述第一抗原结合结构域包含重链可变区(VH1)和轻链可变区(VL1)，所述VH1包含分别如SEQ ID NO：41-43所示氨基酸序列的HCDR1、HCDR2和HCDR3；所述VL1包含分别如SEQ ID NO：44-46所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，所述第一抗原结合结构域的氨基酸序列包含任意一个或多个选自SEQ ID NO：41-46的序列。
一些具体实施方案中，所述VH1和/或VL1为人源化的、回复突变、亲合力成熟、去除/减少T细胞表位(TCE)、降低抗体脱酰胺和/或降低抗体异构化改造的。
一些具体实施方案中，所述VH1包含如SEQ ID NO：39所示或与之具有至少80％、至少90％序列同一性的氨基酸序列，和/或VL1包含如SEQ ID NO：40所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些具体实施方案中，所述VH1的氨基酸序列如SEQ ID NO：39所示，所述VL1的氨基酸序列如SEQ ID NO：40所示。
一些实施方案中，提供GPC3/TRGV9结合蛋白，其包含前述任意的特异性结合GPC3的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域，其中，所述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR1、CDR2和/或CDR3，例如，免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR3；或，
2)重链可变区(VH2)和/或轻链可变区(VL2)，所述VH2包含SEQ ID NO：9、30-32任一所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL2包含SEQ ID NO：10、33-35任一所示氨基酸序列中的LCDR1、LCDR2和LCDR3，
所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如，是根据Kabat编号系统定义的。
一些实施方案中，提供GPC3/TRGV9结合蛋白，其包含前述任意的特异性结合GPC3的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域，其中，所述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含CDR1、CDR2和/或CDR3，其中，CDR1包含SEQ ID NO：6所示氨基酸序列，CDR2包含SEQ ID NO：7所示氨基酸序列，CDR3包含SEQ ID NO：8所示氨基酸序列；或，
2)VH2和/或VL2，所述VH2包含HCDR1、HCDR2和/或HCDR3，其中，HCDR1包含SEQ ID NO：11所示氨基酸序列，HCDR2包含SEQ ID NO：12所示氨基酸序列，HCDR3包含SEQ ID NO：13所示氨基酸序列；所述VL包含LCDR1、LCDR2和/或LCDR3，其中，LCDR1包含SEQ ID NO：14所示氨基酸序列，LCDR2包含SEQ ID NO：15所示氨基酸序列，LCDR3包含SEQ ID NO：16所示氨基酸序列。
一些实施方案中，提供GPC3/TRGV9结合蛋白，其包含前述任意的特异性结合GPC3的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域，其中，所述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含分别如SEQ ID NO：6-8所示氨基酸序列的CDR1、CDR2和CDR3；和/或，
2)VH2和VL2，所述VH2包含分别如SEQ ID NO：11-13所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述VL2包含分别如SEQ ID NO：14-16所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些实施方案中，前述免疫球蛋白单一可变结构域或VH、VL为人源化的、回复突变、亲合力成熟、去除/减少T细胞表位(TCE)、降低抗体脱酰胺和/或降低抗体异构化改造的。
一些实施方案中，前述免疫球蛋白单一可变结构域人源化所使用的人种系模板的重链框架区源自IGHV3-64；VH在人源化改造过程使用的人种系模板的重链框架区源自IGHV3-21，VL在人源化改造过程使用的人种系模板的轻链框架区源自IGKV1-12。
一些实施方案中，提供GPC3/TRGV9结合蛋白，其包含前述任意的特异性结合GPC3的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域，其中，所述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含如SEQ ID NO：5、26-29任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；或，
2)VH2和/或VL2，所述VH2包含如SEQ ID NO：9、30-32任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，所述VL2包含如SEQ ID NO：10、33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，VH2和VL2的组合选自如下：
2-1)VH2包含如SEQ ID NO：9所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：10所示或与之具有至少80％、至少90％同一性的氨基酸序列，
2-2)VH2包含如SEQ ID NO：30所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-3)VH2包含如SEQ ID NO：31所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-4)VH2包含如SEQ ID NO：32所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些实施方案中，前述GPC3/TRGV9结合蛋白包含或为抗GPC3/TRGV9抗体或其抗原结合片段。一些实施方案中，所述抗GPC3/TRGV9抗体或其抗原结合片段为重组抗体或其片段。一些实施方案中，所述抗GPC3/TRGV9抗体为双特异性抗体、多特异性抗体(例如，三特异性抗体)。一些实施方案中，所述抗GPC3/TRGV9抗体或其抗原结合片段中的免疫球蛋白单一可变结构域为单域抗体或VHH。一些具体实施方案中，所述抗原结合片段包括但不限于：Fab、Fv、sFv、Fab’、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv。
一些实施方案中，前述GPC3/TRGV9结合蛋白中包含一个或多个(例如2、3、4、5、6、7、8个)前述免疫球蛋白单一可变结构域。一些实施方案中，前述GPC3/TRGV9结合蛋白中包含一个或多个(例如2、3、4、5、6、7、8个)前述VH1和VL1的组合。一些实施方案中，前述GPC3/TRGV9结合蛋白中包含一个或多个(例如2、3、4、5、6、7、8个)前述VH2和VL2的组合。
一些实施方案中，前述GPC3/TRGV9结合蛋白中还包含人免疫球蛋白Fc区；例如，人IgG1、IgG2、IgG3或IgG4的Fc区。
一些实施方案中，前述GPC3/TRGV9结合蛋白中还包含人免疫球蛋白Fc区；例如，所述Fc区是人IgG1、IgG2、IgG3或IgG4的Fc区。一些实施方案中，所述Fc区是人IgG1的Fc区，例如SEQ ID NO：51所示或与之具有至少80％、至少90％序列同一性。一些具体实施方案中，所述Fc区可以是效应器功能降低的Fc区，例如，所述Fc区可以具有突变，效应器功能降低的示例性IgG Fc区包括具有以下的取代：N297A或N297Q(IgG1)；L234A/L235A(IgG1)；V234A/G237A(IgG2)；L235A/G237A/E318A(IgG4)；H268Q/V309L/A330S/A331S(IgG2)；C220S/C226S/C229S/P238S(IgG1)；C226S/C229S/E233P/L234V/L235A(IgG1)；L234F/L235E/P331S(IgG1)；L234F/L235E(IgG1)；L234F或L235E(IgG1)；L234A或L235A(IgG1)或S267E/L328F(IgG1)。
一些具体实施方案中，所述Fc区包含第一亚基(Fc1)和第二亚基(Fc2)。一些具体实施方案中，引入使Fc区的两个亚基(Fc1、Fc2)配对形成二聚体的突变，或减少同源二聚化的突变。一些实施具体方案中，所述第一亚基和所述第二亚基含有的knob-into-hole突变。例如在CH3/CH3界面内，Fc1的CH3域中的一个、二个或多个氨基酸残基突变为一个或多个具有更大侧链体积的氨基酸残基，从而在Fc1的CH3域表面产生凸起(或杵，Knob)。相应地，Fc2的CH3域中与Fc1的CH3域相互作用的一个、二个或多个氨基酸残基突变为具有更小侧链体积的氨基酸残基，从而在Fc2的CH3域表面产生凹陷(或臼，Hole)。第一亚基(Fc1)和第二亚基(Fc2)仅在于区分不同的两个亚基，因此二者是可互换的。
一些具体实施方案中，所述Fc1含有选自354、356、358和366的位点的一个或多个氨基酸取代，所述Fc2含有选自349、356、358、366、368和407的位点的一个或多个氨基酸取代。一些具体实施方案中，所述Fc1含有366位突变，Fc2含有选自366，368和407位的突变或其任意组合。一些具体实施方案中，所述Fc1含有354或356位突变，Fc2含有349位突变。一些具体实施方案中，所述Fc1含有354或356位突变，Fc2含有349，366，368和407位突变。
一些具体实施方案中，所述Fc1含有选自354C、356E、358M和366W的一个或多个氨基酸取代，所述Fc2含有选自349C、356E、358M、366S、368A和407V的一个或多个氨基酸取代。一些具体实施方案中，所述Fc1含有366W突变，Fc2含有选自366S、368A和407V的突变或其任意组合。一些具体实施方案中，所述Fc1含有354C或356C突变，Fc2含有349C突变。或一些具体实施方案中，所述Fc1含有354C/366W突变，Fc2含有349C/366S/368A/407V突变。
一些具体实施方案中，所述Fc1含有T366W突变，Fc2含有选自T366S、L368A和Y407V的突变或其任意组合；所述Fc1含有S354C或E356C突变，Fc2含有Y349C突变；或所述Fc1含有S354C/T366W突变，Fc2含有Y349C/T366S/L368A/Y407V突变。一些具体实施方案中，Fc1的氨基酸序列如SEQ ID NO：52所示，且Fc2的氨基酸序列如SEQ ID NO：53所示。
一些实施方案中，前述GPC3/TRGV9结合蛋白包含通过对重链CH1和轻链CL界面氨基酸进行氨基酸大小和电荷突变，减少了轻重链之间的错配。示例性地，Roche交换了CH1和CL的结构域，并且创造了CrossMab平台(Schaefer等，Proceedings of the National Academy of Sciences of the UnitedStates of America，108(27)，pp.11187–11192(2011))，MedImmune突变重链F126C和轻链S121C引入了二硫键(Mazor等，mAbs，7(2)，pp.377–389(2015))，Amgen在CH1-CL区进一步做了静电作用修饰(Liu等，Journal of Biological Chemistry，290(12)，pp.7535–7562(2015))，并且Lilly(Lewis等，Nature Biotechnology，32(2)，pp.191–198(2014))和Genentech(Dillon等，mAbs，9(2)，pp.213–230(2017))在可变结构域和恒定结构域均引入了突变。”药明将抗体的恒定区替换为TCR的恒定区，在CN109535257A(引用且并入全文)描述。
一些实施方案中，前述GPC3/TRGV9结合蛋白包含Obscurin-O链(如SEQ ID NO：47所示)和Titin-T链(如SEQ ID NO：48所示)，以防止或降低不同VH和VL之间的错配。
一些实施方案中，前述GPC3/TRGV9结合蛋白中包含连接子。
一些实施方案中，前述GPC3/TRGV9结合蛋白中免疫球蛋白单一可变结构域或VH、VL与Fc区直接或通过连接子连接。所述连接子可以是长1-20个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。例如，所述连接子如(GmSn)h或(GmQn)h或(GGNGT)h或(YGNGT)h或(EPKSS)h或(AmSn)h所示，其中，m、n各自独立地选自1-8的整数，h独立地选自1-20的整数。例如，所述连接子选自G4S、GS、GAP、(G4S)2、(G4S)3、(G4S)4、(G4S)5、ASGS、A3S等。
一些实施方案中，所述GPC3/TRGV9结合蛋白包含选自以下的组合：
1)第一重链、第二重链和轻链，其中，
第一重链，其从N端到C端依次为：[免疫球蛋白单一可变结构域]-[连接子1]-[Fc1]，
第二重链，其从N端到C端依次为：[VH1]-[连接子2]-[CH1]-[连接子3]-[Fc2]，
轻链，其从N端到C端依次为：[VL1]-[连接子4]-[CL]；
或
第一重链，其从N端到C端依次为：[免疫球蛋白单一可变结构域]-[连接子1]-[Fc2]，
第二重链，其从N端到C端依次为：[VH1]-[连接子2]-[CH1]-[连接子3]-[Fc1]，
轻链，其从N端到C端依次为：[VL1]-[连接子4]-[CL]；
其中，-表示肽键，所述连接子1、连接子2、连接子3和连接子4可以相同或不相同，可以独立的存在或不存在，可以独立的选自前述本公开的连接子；
一些具体实施方案中，连接子1为AAAS，连接子2、连接子3和连接子4不存在。
2)第一重链、第一轻链、第二重链和第二轻链，其中，
第一重链，其从N端到C端依次为：[VH2]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
第一轻链，其从N端到C端依次为：[VL2]-[连接子3]-[Titin-T链]，
第二重链，其从N端到C端依次为：[VH1]-[连接子4]-[CH1]-[连接子5]-[Fc2]，
第二轻链，其从N端到C端依次为：[VL1]-[连接子6]-[CL]；
或，
第一重链，其从N端到C端依次为：[VH1]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
第一轻链，其从N端到C端依次为：[VL1]-[连接子3]-[Titin-T链]，
第二重链，其从N端到C端依次为：[VH2]-[连接子4]-[CH1]-[连接子5]-[Fc2]，
第二轻链，其从N端到C端依次为：[VL2]-[连接子6]-[CL]
其中，-表示肽键，所述连接子1、连接子2、连接子3、连接子4、连接子5和连接子6可以相同或不相同，可以独立的存在或不存在，可以独立的选自前述本公开的连接子；
一些具体实施方案中，连接子1、连接子3为GGGGS，连接子2、连接子4、连接子5、连接子6不存在。
3)第一重链、第一轻链、第二重链和第二轻链，其中，
第一重链，其从N端到C端依次为：[VH2]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
第一轻链，其从N端到C端依次为：[VL2]-[连接子3]-[Titin-T链]，
第二重链，其从N端到C端依次为：[VH1]-[连接子4]-[CH1]-[连接子5]-[VH1]-[连接子6]-[CH1]-[连接子7]-[Fc2]，
第二轻链，其从N端到C端依次为：[VL1]-[连接子8]-[CL]；
或，
第一重链，其从N端到C端依次为：[VH1]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
第一轻链，其从N端到C端依次为：[VL1]-[连接子3]-[Titin-T链]，
第二重链，其从N端到C端依次为：[VH2]-[连接子4]-[CH1]-[连接子5]-[VH2]-[连接子6]-[CH1]-[连接子7]-[Fc2]，
第二轻链，其从N端到C端依次为：[VL2]-[连接子8]-[CL]；
其中，-表示肽键，所述连接子1、连接子2、连接子3、连接子4、连接子5、连接子6、连接子7、连接子8可以相同或不相同，可以独立的存在或不存在，可以独立的选自前述本公开的连接子；
一些具体实施方案中，连接子1、连接子3为GGGGS，连接子5为GGGGSGGGGS，连接子2、连接子4、连接子6、连接子7和连接子8不存在；
一些具体实施方案中，第一重链：第一轻链：第二重链：第二轻链的摩尔比为1：1：1：2。
一些具体实施方案中，Obscurin-O链和Titin-T链的氨基酸序列分别如SEQ ID NO：47、48所示。一些具体实施方案中，CL为Cκ，CH1和Cκ的氨基酸序列分别如SEQ ID NO：49、50所示。一些具体实施方案中，Fc1和Fc2的氨基酸序列分别如SEQ ID NO：52、53所示。
一些实施方案中，提供GPC3/TRGV9结合蛋白选自：
1)包含氨基酸序列如SEQ ID NO：54所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：55所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％同一性的轻链；
2)包含氨基酸序列如SEQ ID NO：57所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％同一性的第一轻链，氨基酸序列如SEQ ID NO：59所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％同一性的第二轻链；或，
3)包含氨基酸序列如SEQ ID NO：57所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％同一性的第一轻链，氨基酸序列如SEQ ID NO：60所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：61所示或与之具有至少80％、至少90％同一性的第二轻链。
一些实施方案中，提供GPC3/TRGV9结合蛋白选自：
1)包含氨基酸序列如SEQ ID NO：54-56所示的多肽组合；一些实施方案中，GPC3/TRGV9结合蛋白中SEQ ID NO：54所示多肽：SEQ ID NO：55所示多肽：SEQ ID NO：56所示多肽的摩尔比为1：1：1；
2)包含氨基酸序列如SEQ ID NO：56-59所示的多肽组合；一些实施方案中，GPC3/TRGV9结合蛋白中SEQ ID NO：56所示多肽：SEQ ID NO：57所示多肽：SEQ ID NO：58：SEQ ID NO：59所示多肽的摩尔比为1：1：1：1；
3)包含氨基酸序列如SEQ ID NO：57、58、60、61所示的多肽组合；一些实施方案中，GPC3/TRGV9结合蛋白中SEQ ID NO：57所示多肽：SEQ ID NO：58所示多肽：SEQ ID NO：60：SEQ ID NO：61所示多肽的摩尔比为1：1：1：2。
一些实施方案中，前述GPC3/TRGV9结合蛋白为：特异性结合GPC3和γδTCR的蛋白，或抗GPC3/γδTCR抗体或其抗原结合片段；特异性结合GPC3和TCR的γ9链的蛋白，或抗GPC3/TCRγ9链的抗体或其抗原结合片段；特异性结合GPC3和TCR的γ9链的可变区(Vγ9)的蛋白，或抗GPC3/Vγ9TCR的抗体或其抗原结合片段；特异性结合GPC3和γ9δ2 TCR的蛋白，或抗GPC3/γ9δ2 TCR抗体或其抗原结合片段；特异性结合GPC3和γ9δ1 TCR的蛋白，或抗GPC3/γ9δ1 TCR抗体或其抗原结合片段；特异性结合GPC3和TRGV9的蛋白，或抗GPC3/TRGV9抗体或其抗原结合片段
一些实施方案中，前述GPC3/TRGV9结合蛋白具有选自以下至少一项的功能或性质：
(a)以≤10nM的EC50与γ9δ2 T细胞结合，所述EC50例如≤5nM、≤4nM、≤3nM、≤2nM、≤1nM、≤0.5nM、≤0.2nM、≤0.1nM。所述EC50是通过FACS检测获得，FACS是本领域常用的亲和力检测方法，例如本公开实施例5中所述；
(b)特异性结合TCR的γ9链，例如，特异性结合γ9δ1 TCR，特异性结合γ9δ2TCR；
(c)不结合TCR的γ8链，例如，不结合γ8δ2 TCR；
(d)特异性结合TCR的γ9链的可变区(TRGV9)；
(e)特异性结合VγδCαβ嵌合TCR，不结合VαβCγδ嵌合TCR；
其中，(b)-(e)可通过本领域常规检测方法获知，例如本公开实施例4中所述；
(f)特异性结合GPC3蛋白或GPC3阳性细胞，不结合或几乎不结合GPC3阴性细胞(例如PBMC)，例如使用本公开实施例8检测方法；
(g)结合食蟹猴GPC3和γ9δ2蛋白；
(h)不阻断BTN2A/BTN3A-TCR天然信号，例如使用本公开实施例9检测方法；
(i)介导γδT细胞对GPC3阳性肿瘤细胞杀伤或以GPC3表达水平依赖性方式介导γδT细胞对GPC3阳性肿瘤细胞杀伤，例如使用本公开实施例10检测方法；以效靶比依赖性方式，介导γδT细胞对GPC3阳性肿瘤细胞杀伤，例如使用本公开实施例12检测方法；以低个体差异性的方式，介导γδT细胞对GPC3阳性肿瘤细胞杀伤，例如使用本公开实施例11检测方法；
(j)γδT细胞对GPC3阳性肿瘤细胞杀伤过程中，诱导更少的细胞因子(如IFNγ、TNFα)的释放，例如使用本公开实施例13检测方法；
(k)促进PBMC中γδT细胞增殖，例如使用本公开实施例14检测方法；
(l)体内抑制GPC3阳性肿瘤细胞增殖，和/或抑制GPC3阳性肿瘤生长，和/或消除GPC3阳性肿瘤；小鼠体内模型如本公开实施例15检测方法。
一些实施方案中，前述GPC3/TRGV9结合蛋白结合TRGV9的KD值可以≤1×10-7M，例如≤1×10-8M，或≤1×10-9M，或≤1×10-10M。
一些实施方案中，前述GPC3/TRGV9结合蛋白结合GPC3的KD值可以≤1×10-7M，例如≤1×10-8M，或≤1×10-9M，或≤1×10-10M。
一些实施方案中，前述GPC3/TRGV9结合蛋白中的免疫球蛋白单一可变结构域涵盖变体，所述变体与SEQ ID NO：5、26-29任一相比，具有一个或多个氨基酸突变。一些实施方案中，前述GPC3/TRGV9结合蛋白中的VH1和/或VL1涵盖变体，所述VH1的变体与SEQ ID NO：39相比，具有一个或多个氨基酸突变，所述VL的变体与SEQ ID NO：40相比，具有一个或多个氨基酸突变。一些实施方案中，前述GPC3/TRGV9结合蛋白中的VH2和/或VL2涵盖变体，所述VH2的变体与9、30-32任一相比，具有一个或多个氨基酸突变，所述VL的变体与SEQ ID NO：10、33-35任一相比，具有一个或多个氨基酸突变。“多个”例如为1、2、3、4、5、6、7、8、9、10个。所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加；所述氨基酸突变可以发生在CDR区和/或FR区。
一些实施方案中，提供蛋白，其与前述本公开GPC3/TRGV9结合蛋白结合或竞争结合相同的TRGV9和/或GPC3抗原表位。
一些实施方案中，提供蛋白，其与TRGV9和/或GPC3的结合被前述本公开GPC3/TRGV9结合蛋白阻断。
一些实施方案中，提供蛋白或分子，其包含前述本公开任意的GPC3/TRGV9结合蛋白。所述蛋白或分子可以为与其他化合物、其他多肽形成的缀合物、偶联物或融合蛋白。例如，缀合物可包含任意可检测标记。
多核苷酸和载体
本公开提供编码本公开的TRGV9结合蛋白、GPC3/TRGV9结合蛋白的多核苷酸。本公开的核酸可为RNA、DNA或cDNA。根据本公开的一些实施方案，本公开的核酸是基本上分离的核酸。
本公开的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达TRGV9结合蛋白、GPC3/TRGV9结合蛋白的载体。该表达载体通常包含至少一种本公开的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的TRGV9结合蛋白、GPC3/TRGV9结合蛋白表达有用或必需的调控元件及其他元件例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的核酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
一些实施方案中，提供多核苷酸，其编码TRGV9结合蛋白，所述TRGV9结合蛋白，其包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR1、CDR2和/或CDR3，例如，免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR3；和/或，
2)重链可变区(VH)和/或轻链可变区(VL)，所述VH包含SEQ ID NO：9、30-32任一所示氨基酸序列中的HCDR1、HCDR2和/或HCDR3，所述VL包含SEQ ID NO：10、33-35任一所示氨基酸序列中的LCDR1、LCDR2和/或LCDR3，
所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如，是根据Kabat编号系统定义的。
一些实施方案中，提供多核苷酸，其编码TRGV9结合蛋白，所述TRGV9结合蛋白，其包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含分别如SEQ ID NO：6-8所示氨基酸序列的CDR1、CDR2和CDR3；和/或，
2)VH和VL，所述VH包含分别如SEQ ID NO：11-13所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：14-16所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些实施方案中，提供多核苷酸，其编码TRGV9结合蛋白，所述TRGV9结合蛋白，其包含：1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含如SEQ ID NO：5、26-29任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；和/或，
2)VH和/或VL，所述VH包含如SEQ ID NO：9、30-33任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，所述VL包含如SEQ ID NO：10、33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，提供多核苷酸，其编码TRGV9结合蛋白，所述TRGV9结合蛋白包含如下的VH和VL：
2-1)VH包含如SEQ ID NO：9所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：10所示或与之具有至少80％、至少90％同一性的氨基酸序列，
2-2)VH包含如SEQ ID NO：30所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-3)VH包含如SEQ ID NO：31所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-4)VH包含如SEQ ID NO：32所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，提供多核苷酸，其编码TRGV9结合蛋白，所述TRGV9结合蛋白包含：
1)如SEQ ID NO：36任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列；
2)重链和轻链，其中，重链如SEQ ID NO：37任一所示或与之具有至少80％、至少90％序列同一性，轻链如SEQ ID NO：38任一所示或与之具有至少80％、至少90％序列同一性。
一些实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，所述GPC3/TRGV9结合蛋白，其包含特异性结合GPC3的第一抗原结合结构域和特异性结合TRGV9的第二抗原结合结构域。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述第一抗原结合结构域包含重链可变区(VH1)和/或轻链可变区(VL1)，所述VH1包含SEQ ID NO：39所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL1包含SEQ ID NO：40所示氨基酸序列中的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述第一抗原结合结构域所述第一抗原结合结构域包含重链可变区(VH1)和轻链可变区(VL1)，所述VH1包含分别如SEQ ID NO：41-43所示氨基酸序列的HCDR1、HCDR2和HCDR3；所述VL1包含分别如SEQ ID NO：44-46所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述第一抗原结合结构域所述第一抗原结合结构域包含重链可变区(VH1)和轻链可变区(VL1)，所述VH1包含如SEQ ID NO：39所示或与之具有至少80％、至少90％序列同一性的氨基酸序列，和/或VL1包含如SEQ ID NO：40所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR1、CDR2和/或CDR3，例如，免疫球蛋白单一可变结构域包含SEQ ID NO：5、26-29任一所示氨基酸序列中的CDR3；或，
2)重链可变区(VH2)和/或轻链可变区(VL2)，所述VH2包含SEQ ID NO：9、30-32任一所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL2包含SEQ ID NO：10、33-35任一所示氨基酸序列中的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含分别如SEQ ID NO：6-8所示氨基酸序列的CDR1、CDR2和CDR3；和/或，
2)VH2和VL2，所述VH2包含分别如SEQ ID NO：11-13所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述VL2包含分别如SEQ ID NO：14-16所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述特异性结合TRGV9的第二抗原结合结构域包含：
1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含如SEQ ID NO：5、26-29任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；或，
2)VH2和/或VL2，所述VH2包含如SEQ ID NO：9、30-32任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，所述VL2包含如SEQ ID NO：10、33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，前述特异性结合TRGV9的第二抗原结合结构域包含如下的VH2和VL2：
2-1)VH2包含如SEQ ID NO：9所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：10所示或与之具有至少80％、至少90％同一性的氨基酸序列，
2-2)VH2包含如SEQ ID NO：30所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-3)VH2包含如SEQ ID NO：31所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
2-4)VH2包含如SEQ ID NO：32所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含SEQ ID NO：33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，提供多核苷酸，其编码GPC3/TRGV9结合蛋白，所述GPC3/TRGV9结合蛋白选自：
1)包含氨基酸序列如SEQ ID NO：54所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：55所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％同一性的轻链；
2)包含氨基酸序列如SEQ ID NO：57所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％同一性的第一轻链，氨基酸序列如SEQ ID NO：59所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％同一性的第二轻链；或，
3)包含氨基酸序列如SEQ ID NO：57所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％同一性的第一轻链，氨基酸序列如SEQ ID NO：60所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：61所示或与之具有至少80％、至少90％同一性的第二轻链。
宿主细胞
本公开提供表达或能够表达一种或多种本公开的TRGV9结合蛋白、GPC3/TRGV9结合蛋白和/或含有本公开的多核苷酸或载体的重组宿主细胞。一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
制备方法
本公开提供一种用于制备TRGV9结合蛋白或GPC3/TRGV9结合蛋白的方法，包括：在如前所述的宿主细胞中表达所述目的蛋白，并自该宿主细胞中分离目的蛋白。可选地，还可以包含纯化步骤，例如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化，洗去非特异性结合的组分，再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测，收集。可选地，用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
生产和纯化抗体的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。
组合物
本公开提供组合物，包含前述本公开的TRGV9结合蛋白和/或GPC3/TRGV9结合蛋白。例如，提供药物组合物，其含有对治疗、缓解或预防疾病有效量的前述TRGV9结合蛋白和/或GPC3/TRGV9结合蛋白，和至少一种可药用的赋形剂、稀释剂或辅料。
在一些具体实施方案中，所述药物组合物单位计量中可含有0.01至99重量％的TRGV9结合蛋白和/或GPC3/TRGV9结合蛋白，或药物组合物单位剂量中含TRGV9结合蛋白和/或GPC3/TRGV9结合蛋白的量为0.1-2000mg。在一些具体实施方案中为1-1000mg。
一些实施方案中，提供制品或产品(例如药盒)，包含前述TRGV9结合蛋白和/或GPC3/TRGV9结合蛋白。可选地，制品包含容器和标签。容器例如瓶、注射器和试管。容器容纳有效于治疗病症的药物组合物。容器上或与容器相连的标签表明所述药物组合物用于治疗所选病症。
一些实施方案中，前述疾病为细胞增殖性疾病或癌症。
治疗方法和制药用途
本公开提供前述TRGV9结合蛋白、GPC3/TRGV9结合蛋白，其编码多核苷酸、组合物(包括药物组合物)用于治疗、缓解、预防、诊断疾病或病症的方法。
一些实施方案中，提供改善、缓解、治疗或预防疾病的方法，包括向受试者施用改善、缓解、治疗或预防有效量的：
1)前述本公开的TRGV9结合蛋白或其编码多核苷酸、药物组合物；
2)前述本公开的GPC3/TRGV9结合蛋白或其编码多核苷酸、药物组合物。
一些实施方案中，提供前述1)-2)项用于制备改善、缓解、治疗或预防疾病的药物的用途。
一些实施方案中，提供本公开的TRGV9结合蛋白与γδT细胞联合治疗疾病的方法。一些实施方案中，提供本公开的GPC3/TRGV9结合蛋白与γδT细胞联合治疗疾病的方法。一些实施方案中，γδT细胞是自体的或同种异体的。
一些实施方案中，本公开前述任意的TRGV9结合蛋白用于治疗疾病，包括与γδT细胞联合使用。一些实施方案中，γδT细胞用于治疗疾病，包括与本公开前述任意的TRGV9结合蛋白联合使用。
一些实施方案中，本公开前述任意的GPC3/TRGV9结合蛋白用于治疗疾病，包括与γδT细胞联合使用。一些实施方案中，γδT细胞用于治疗疾病，包括与本公开前述任意的GPC3/TRGV9结合蛋白联合使用。
一些实施方案中，提供治疗疾病的方法，包括向有需要的受试者施用治疗有效量的本公开前述任意的TRGV9结合蛋白和γδT细胞。一些实施方案中，提供治疗疾病的方法，包括向有需要的受试者施用治疗有效量的本公开前述任意的GPC3/TRGV9结合蛋白和γδT细胞。
一些实施方案中，前述疾病为与GPC3超量表达导致的疾病或病症。
一些实施方案中，前述疾病为细胞增殖性疾病或癌症。
一些具体实施方案中，为GPC3阳性癌症。
一些实施方案中，前述疾病为肝癌。
一些具体实施方案中，为GPC3阳性肝癌。
检测
本公开提供TRGV9结合蛋白、GPC3/TRGV9结合蛋白，其编码多核苷酸、组合物的检测用途。本公开还提供用于体内或体外检测GPC3、TRGV9的方法、系统或装置，其包括用本公开的前述结合蛋白、多核苷酸、组合物处理样品。
一些实施方案中，还提供试剂盒，所述试剂盒包含前述TRGV9结合蛋白、GPC3/TRGV9结合蛋白，其编码多核苷酸、组合物，还可以包含诊断使用说明。试剂盒还可以含有至少一种额外的试剂，如标记物或额外的诊断剂。对于体内使用，TRGV9结合蛋白、GPC3/TRGV9结合蛋白，其编码多核苷酸可以配制为药物组合物。
附图说明
图1为抗GPC3/γδTCR双特异性抗体结构示意图。
图2A至图2D为抗GPC3/γδTCR双特异性抗体与GPC3阳性细胞和人γ9δ2 T细胞的FACS结合活性检测结果。其中，图2A为抗GPC3/γδTCR双特异性抗体在HepG2细胞上的结合；图2B为抗GPC3/γδTCR双特异性抗体在DLD-1细胞上的结合；图2C为抗GPC3/γδTCR双特异性抗体在人γδT细胞上的结合；图2D为抗GPC3/γδTCR双特异性抗体在PBMC上的结合。
图3A至图3B为抗γδTCR抗体对BTN2A/BTN3A-TCR天然信号的影响。图3A为SDP01378对BTN2A/BTN3A-TCR天然信号的影响；图3B为SDP01315对BTN2A/BTN3A-TCR天然信号的影响。
图4A至图4D为抗γδTCR抗体对不同GPC3表达量肿瘤细胞的杀伤活性结果。其中，图4A为对HepG2细胞的杀伤活性，图4B为对Huh-7细胞的杀伤活性，图4C为对MKN-45细胞的杀伤活性，图4D为对DLD-1细胞的杀伤活性。
图5A至图5D为不同供体来源γδT细胞对HepG2杀伤活性结果。其中，图5A-图5D所采用γδT细胞分别来源于供体#SC12004，#SC12392，#XC11053，#XC11061的诱导扩增。
图6A至图6C为不同效靶比下抗GPC3/γδTCR双特异性抗体对抗原低表达肿瘤细胞的杀伤结果。其中，图6A效靶比为1:1，图6B效靶比为10:1，图6C效靶比为30:1。
图7A至图7D为提高PBMC中比例提升γδT细胞杀伤活性结果。图7A为供体#XC11053 PBMC细胞杀伤；图7B为供体#XC11053掺入30％的γδT细胞杀伤活性；图7C为供体#SC12392 PBMC细胞杀伤；图7D为供体#SC12392参入30％γδT细胞杀伤活性。
图8A至图8D为细胞因子释放检测结果。其中图8A-图8B分别为对供体#XC11053杀伤时IFNγ和TNFα的释放；图8C-8D分别为对供体#SC12392杀伤时IFNγ和TNFα的释放。
图9为抗GPC3/γδTCR双特异性抗体促进PBMC中γδT细胞增殖检测结果。
图10A至图10B为SDP01716在Huh-7移植瘤模型中的抗肿瘤活性，其中图10A为小鼠肿瘤体积变化图，图10B为小鼠体重变化图。
图11A至图11B为不同抗GPC3/γδTCR双特异性抗体在Huh-7移植瘤模型中的抗肿瘤活性，其中图11A为小鼠肿瘤体积变化图，图11B为小鼠体重变化图。
具体实施方式
定义
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本公开中的它处另有明确定义，否则本公开使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
“TRGV9”是指当在γδT细胞的表面表达时能够形成T细胞受体的多肽。表达TRGV9的γδT细胞是最早在人胎儿中发育的T细胞之一，并且是健康成人外周血细胞中的主要γδT细胞亚群。“TRGV9”包括由细胞(包括T细胞)天然表达或者能够在用编码所述多肽的基因或cDNA转染的细胞上表达的任何TRGV9变体、同种型和物种同源物。在具体的实施方案中，TRGV9是人TRGV9。示例性人TRGV9氨基酸序列由GenBank登录号NG_001336.2提供。
“GPC3”是指Glypican 3，磷脂酰肌醇蛋白聚糖3，通过磷脂酰肌醇锚定在细胞膜上，是肝细胞癌标志物。示例性人GPC3的氨基酸序列由uniprot登录号P51654提供。
“TRGV9结合蛋白”涵盖任何能够特异性结合TRGV9的蛋白或包含所述蛋白的任何分子，包括但不限于针对TRGV9的如本公开定义的抗体、其抗原结合片段或其融合蛋白。一些实施方案中，“TRGV9结合蛋白”可以包含至少一个(例如1、2、3、4、5、6或更多个)本公开实施例中特异性结合TRGV9的单域抗体或VHH。一些实施方案中，“TRGV9结合蛋白”可以包含至少一个(例如1、2、3、4、5、6或更多个)本公开实施例中特异性结合TRGV9的VH和VL组合。一些实施方案中，本公开的“TRGV9结合蛋白”除包含TRGV9的免疫球蛋白单一可变结构域或VH和VL组合外，也可包含连接子和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。一些实施方案中，“TRGV9结合蛋白”涵盖本公开实施例中的抗γδTCR抗体或其抗原结合片段。
“GPC3/TRGV9结合蛋白”涵盖任何能够特异性结合GPC3和TRGV9的蛋白或包含所述蛋白的任何分子，包括但不限于抗体、多肽、抗体和多肽的融合蛋白或其缀合物。一些实施方案中，“GPC3/TRGV9结合蛋白”涵盖本公开实施例中的抗GPC3/γδTCR双特异性抗体。
“抗体”涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的框架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
本公开的抗体可以是多克隆的、单克隆的、异种的、同种异体的、同基因的或其经过修饰的形式，其中单克隆抗体尤其适用于多个实施例中。一般来说，本公开的抗体是重组抗体。如本文所用的“重组”泛指例如细胞或核酸、蛋白质或载体等产品，表示所述细胞、核酸、蛋白质或载体已经通过引入异源核酸或蛋白质或改变天然核酸或蛋白质而加以修饰，或所述细胞来源于如此修饰的细胞。例如，重组细胞表达天然(非重组)细胞形式内不存在的基因或表达原本异常表达、低表达或完全不表达的天然基因。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；”AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的”Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal of Biological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。各种编号系统之间的对应关系是本领域技术人员熟知的。
技术人员理解，尽管在具体的实施例中、或在具体的SEQ ID NO中采用了特定的编号系统(如Kabat)，但是技术人员可以确定该序列在其他编号系统下对应的序列，这样的序列仍视为落入本公开的范围内。
多肽或蛋白的“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个功能性质，且在许多情况下可添加、移除或转移至其它蛋白而不损失蛋白的其余部分和/或结构域的功能。
“免疫球蛋白结构域”是指抗体链(例如常规四肽链结构抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”，和“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”组成。免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“抗体框架(FR)”，是指可变结构域的一部分，其用作抗原结合环(CDR)的支架。
“免疫球蛋白单一可变结构域”通常用于指可以在不与其他可变结构域相互作用的情况下(例如在没有如常规四链单克隆抗体的VH和VL结构域之间所需要的VH/VL相互作用的情况下)，形成功能性抗原结合位点的免疫球蛋白可变结构域(其可以是重链或轻链结构域，包括VH、VHH或VL结构域)。“免疫球蛋白单一可变结构域”的实例包括纳米抗体(包括VHH、人源化VHH和/或骆驼化VH，例如骆驼化人VH)、IgNAR、结构域、作为VH结构域或衍生自VH结构域的(单结构域)抗体(诸如dAbsTM)和作为VL结构域或衍生自VL结构域的(单结构域)抗体(诸如dAbsTM)。基于和/或衍生自重链可变结构域(诸如VH或VHH结构域)的免疫球蛋白单一可变结构域通常是优选的。免疫球蛋白单一可变结构域的一个具体实例为如下文定义的“VHH结构域”(或简称为“VHH”)。
“VHH”亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段、VHH抗体、纳米抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.:”Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用“VHH”以将所述可变结构域与存在于常规四肽链结构抗体中的重链可变结构域(其在本公开中称为“VH结构域”或VH)以及轻链可变结构域(其在本公开中称为“VL结构域”或VL)进行区分。VHH结构域特异性结合表位，而无需其他抗原结合结构域；这种结合行为与常规四肽链结构抗体中的VH或VL结构域不同，在该情况下VL结构域与VH结构域一起识别表位。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”、以及“结构域”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。VHH包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的。VHH中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本公开所述的目的。获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。其它的编号系统或编码规则包括Chothia、IMGT、AbM。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。“人源化”的例子包括可将源自骆驼科的VHH结构域通过以人常规四肽链结构抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而“人源化”(本公开中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其它修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列，且在一些具体实施方案中，可含IGHV3的人框架区序列。人源化方法例如蛋白表面氨基酸人源化(resurfacing)及抗体人源化通用框架移植法(CDR grafting to a universal framework)，即将CDR“移植”于其它“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。如人重链和轻链可变区基因的种系DNA序列可以在VBase人种系序列数据库，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲合力成熟后的人源化抗体。此外，为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“亲和力成熟的”抗体指与不拥有此类改变的亲本抗体相比，在一个或多个高变区(HVR)中具有一处或多处改变的抗体，此类改变导致该抗体对抗原的亲和力改善。例如，“亲合力成熟”的TRGV9结合蛋白或抗TRGV9抗体，在一个或多个CDR中具有一个或多个变化，所述变化导致对抗原的亲合力相比于其亲本抗体有所增加。亲合力成熟的抗体可通过例如由以下所述的本领域中已知的方法来制备：Marks等人，1992，Biotechnology 10：779-783或Barbas等人，1994，Proc.Nat.Acad.Sci，USA 91：3809-3813.；Shier等人，1995，Gene 169：147-155；Yelton等人，1995，Immunol.155：1994-2004；Jackson等人，1995，J.Immunol.154(7)：3310-9；及Hawkins等人，1992，J.MoI.Biol.226(3)：889896；KS Johnson及RE Hawkins，”Affinity maturation of antibodies using phage display”，Oxford University Press 1996。
通常，本公开的GPC3/TRGV9结合蛋白、TRGV9结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10-7至10-10摩尔/升(M)、更优选10-8至10-10摩尔/升、甚至更优选10-9至10-10或更低的解离常数(KD)，和/或以至少10-7M、优选至少10-8M、更优选至少10-9M，更优选至少10-10M的缔合常数(KA)结合所要结合的抗原或靶蛋白(即，GPC3、TRGV9)。任何大于10-4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本公开所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定)。
“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。KD通过方程KD＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且甚至可通过例如Caceci等人(1984，Byte 9：340-362)中所述的那些方法对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定KD。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开其它地方例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用BiacoreTM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的KD值来比较与不同分子相互作用相关的结合亲和力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的KD值与非目的相互作用(例如已知不结合IGF-1R或TRGV9的对照抗体)的KD值进行评价。
“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
“同源性”、“同一性”或“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“Titin-T链”或“T链”是指Titin蛋白中一段长度为78-118个氨基酸的包含Titin Ig-样152结构域的肽段或其功能变体，所述Titin-T链能够与Obscurin Ig-样1结构域彼此结合形成二聚化复合物。所述T链功能变体是对野生型T链的部分氨基酸进行突变，但依然具有与Obscurin Ig-样1结构域彼此结合形成二聚化复合物的多肽。例如，在Titin Ig-样152结构域C端和/或N端增加或截短合适长度的氨基酸；可增加或截短1、2、3、4、5、6、7、8、9或10个氨基酸残基；例如在Titin Ig-样152结构域N端增加5个野生型Titin蛋白中紧邻Titin Ig-样152结构域N端的“KAGIR”这5个氨基酸，其仍具有与Obscurin Ig-样1结构域彼此缔合形成复合物的功能。还可对Titin Ig-样152结构域氨基酸进行其它突变，例如，对某些氨基酸突变以便改善链间二硫键，提高复合物稳定性等。
“Obscurin-O链”或“O链”是指Obscurin蛋白上一段长度为87-117个氨基酸的包含Obscurin Ig-样1结构域的肽段或其功能变体，所述Obscurin-O链能够与Titin Ig-样152结构域相互结合形成二聚化复合物。所述的Obscurin-O链功能变体是对野生型O链的部分氨基酸进行突变，但依然具有与Titin Ig-样152结构域相互结合形成二聚化复合物的多肽。例如，在Obscurin-O结构域C端和/或N端增加或截短合适长度的氨基酸，例如增加或截短1、2、3、4、5、6、7、8、9或10个氨基酸；例如在Obscurin-O结构域N端增加5个野生型Obscurin蛋白中紧邻Obscurin Ig-样1结构域N端的“DQPQF”这5个氨基酸，其仍具有与Titin Ig-样152结构域相互结合形成二聚化复合物的功能。还可对Obscurin Ig-样1结构域部分氨基酸进行其它突变，例如，对某些氨基酸突变以便改善链间二硫键，或提高抗体稳定性等。
“核酸”或“多核苷酸”在本公开中可互换使用，指的是单链或双链的任何DNA分子或RNA分子以及在单链的情况下，它的互补序列的分子，优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
“宿主细胞”包括各个细胞或细胞培养物，其可为或已是用于掺入多核苷酸插入片段的载体的受体。宿主细胞包括单个宿主细胞的子代，并且由于天然、偶然或有意的突变，子代可不一定与原始亲本细胞完全相同(在形态学或基因组DNA互补体中)。宿主细胞包括用本公开的多核苷酸在体内转染和/或转化的细胞。“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触，例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开的任一种结合蛋白或其药物组合物作为治疗剂，所述受试者已经患有、疑似患有、倾向于患有一种或多种增殖性疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床能测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。本公开的受试者可以是动物或人类受试者。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。“和/或”应视为特定揭示两种指定特征或组分中的每一者具有或不具有另一者。因此，诸如本公开中“A和/或B”的词组中所用的术语“和/或”包括“A及B”、“A或B”、“A”(单独)及“B”(单独)。除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。在本公开中Fc区所包含的突变的上下文中，“/”表示“和”，例如，“354C/366W”表示“354C和366W”，即，Fc中包含354C和366W突变；本公开Fc区的突变的氨基酸位置均是根据EU编号系统编号的。
本公开的“受试者”、“患者”意指哺乳动物，尤其灵长类动物，尤其是人。
实施例
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗原的设计与制备
γδT细胞表面的TCR(γδT cell receptor)是由γ链和δ链复合形成异质二聚体膜蛋白。选取人γδTCR胞外区蛋白γ9链(Protein data bank，编号1HXM，chain B)的1-242位氨基酸，其中C端依次添加3C酶切位点、Leucine zipper、FLAG标签；选取人γδTCR胞外区蛋白δ2链(Protein data bank，编号1HXM，chain A)的1-229位氨基酸，其中C端依次添加3C酶切位点、Leucine zipper、His8标签。此外，γ链和δ链的恒定区分别引入Q180C和V168C突变，形成链间二硫键。
重组猴(Macaca mulatta)γ9δ2 TCR蛋白选取猴γ9链(序列见公开专利号：US2019144540A1 SEQ ID NO：42)的1-241位氨基酸，其中C端依次添加3C酶切位点、Leucine zipper、FLAG标签；选取猴δ2链(序列见公开专利号：US20190144540A1 SEQ ID NO：37)的1-229位氨基酸，其中C端依次添加3C酶切位点、Leucine zipper、His8标签；此外，γ链和δ链的恒定区分别引入Q179C和V168C突变，形成链间二硫键。
>重组人γ9链
>重组人δ2链
>重组猴γ9链
>重组猴δ2链
分别合成携带目的蛋白编码基因的质粒，将人γ9链(SEQ ID NO：1)和人δ2链(SEQ ID NO：2)质粒按1：1的比例进行混合，将猴γ9链(SEQ ID NO：3)和猴δ2链(SEQ ID NO：4)质粒按1：1的比例进行混合，分别于Expi293细胞(购自Thermo)中瞬时转染表达7天，分离、纯化，获得人γ9δ2 TCR蛋白和猴γ9δ2TCR蛋白，-80℃储存备用。
实施例2.抗γδTCR抗体的筛选
1.抗γδTCR单域抗体筛选
使用体外扩增的人γ9δ2 T细胞作为免疫原免疫成年健康羊驼(Alpaca)，采用噬菌体展示的方法使用实施例1中的重组人γ9δ2 TCR蛋白筛选特异性抗体。
具体地，初次免疫剂量为每只羊驼2E7细胞数。初次免疫3周后，加强免疫，免疫剂量为每只羊驼2E7细胞数。以后每次加强免疫间隔3周。每次加强免疫一周后采集血清样品，采用Protein ELISA和FACS检测羊驼血清中抗体效价。
Protein ELISA具体检测过程如下：用0.05M碳酸盐buffer(pH 9.6)将重组人γ9δ2 TCR蛋白稀释至2μg/mL，100μL/孔，4℃包被过夜；用含5％脱脂牛奶的PBST缓冲液封闭1小时，洗板3次；在封闭缓冲液中以1：2000开始，两倍梯度稀释羊驼血清，37℃孵育45min，洗板5次；每孔加入100μL辣根过氧化物酶标记的羊抗Alpaca二抗(AlpVHHs,053-404-005,用PBS按1:10000稀释)，37℃孵育45min，洗板5次。最后每孔加入100μL TMB显色液显色，5分钟后加入50μL终止液终止反应。使用酶标仪读取450nm处的吸光值。
FACS具体检测过程如下：收集细胞重悬至4E6/mL，50μL细胞加入梯度稀释的羊驼血清，4℃孵育1小时。用1％BSA/PBS缓冲液洗涤两遍后弃上清，加入1：200稀释的Anti alpaca IgG iFluor647(AlpVHHs,053-404-009)，4℃避光孵育45min。用1％BSA/PBS缓冲液洗涤两遍，200μL缓冲液重悬后FACS检测。
经检测，免疫后羊驼血清效价大于128k。
免疫三次后每次免疫后一周采50mL外周血分离淋巴细胞(PBMC)，用RNAiso Plus试剂提取PBMC总RNA进行噬菌体文库建立，获得滴度为3.08×E13 cfu/mL的噬菌体文库。
采用噬菌体展示的方法对针对重组人γ9δ2 TCR蛋白的抗体进行淘选，用ELISA鉴定出交叉结合于猴γ9δ2 TCR蛋白的抗体，用FACS确定对体外扩增的人γ9δ2 T细胞的结合活性。使用上述ELISA和FACS检测方法筛选出阳性单克隆单域抗体，序列如下。
>SDP01346可变区(克隆号ac-025)
表1.抗人γδTCR单域抗体的CDR序列(Kabat编号规则)
2.鼠源抗γδTCR抗体筛选
使用体外扩增的人γ9δ2 T细胞作为免疫原，免疫6-8周龄的Balb/c和SJL小鼠，采用杂交瘤融合的方法使用实施例1中的重组人γ9δ2 TCR蛋白筛选特异性抗体。
具体地，初次免疫剂量为每只小鼠1E7。初次免疫2周后，加强免疫，免疫剂量为每只小鼠1E7。以后每次加强免疫间隔2-3周。每次加强免疫一周后采集血清样品，用Protein ELISA和FACS检测小鼠血清中抗体活性。
Protein ELISA具体检测过程如下：用1μg/mL重组人γ9δ2 TCR蛋白包板，4℃过夜，用含1％BSA的PBST缓冲液封闭1小时，洗板3次。在封闭缓冲液中以1：200开始，三倍梯度稀释小鼠血清，37℃孵育1小时，洗板3次，与1：10000稀释的抗小鼠IgG-Fc-HRP的二抗(Sigma,AP127P)孵育1小时。PBST洗涤3次，每孔加入100μL TMB显色液显色，15min后用终止液终止反应。使用酶标仪读取450nm处的吸光值。
FACS具体检测过程如下：细胞铺板每孔1E5细胞，离心弃上清加入梯度稀释的小鼠血清。4℃孵育1h。用1％BSA/PBS缓冲液洗涤两遍后弃上清，加入1：500稀释的Alexa Flour 488羊抗鼠二抗(Jackson immuno research.115-545-071)，4℃避光孵育30min。用1％BSA/PBS缓冲液洗涤两遍，200μL缓冲液重悬后FACS检测。经免疫后小鼠血清效价大于72900。
最后一次免疫腹腔注射50μg重组人γ9δ2 TCR蛋白，4天后处死小鼠后取脾脏，碾磨收集脾细胞与小鼠骨髓瘤细胞SP2/0混合，电融合获得杂交瘤细胞。将OD 450nm>0.2的阳性克隆加测FACS，将与人γ9δ2 T细胞结合MFI值>5000的阳性克隆进行亚克隆。经过亚克隆初筛与复测，使用上述ELISA和FACS检测方法筛选出鼠源抗γδTCR抗体，SDP01315(克隆号bph-003)序列如下。
>SDP01315(克隆号bph-003)VH
>SDP01315(克隆号bph-003)VL
表2.鼠源抗γδTCR抗体的CDR序列(Kabat编号规则)
实施例3.抗γδTCR嵌合抗体的构建和功能效果验证
1.嵌合抗体的表达
单域抗体将编码抗体的核苷酸序列克隆到pTT5载体上后转染ExpiCHO细胞，8天后离心去除细胞收集并过滤细胞培养液，将收获的细胞培养液使用镍亲和柱(HisTrap excel，GE)纯化，使用300mM咪唑洗脱结合的抗体，脱盐换液至PBS，获得目的抗体。
>SDP01346全长
鼠源抗体将编码抗体的核苷酸序列克隆到pTT5载体上后转染ExpiCHO细胞，8天后离心去除细胞收集并过滤细胞培养液，将收获的细胞培养液使用Protein A亲和柱(MabSelect SuRe，GE)纯化，使用甘氨酸洗脱结合的抗体，1M Tris中和洗脱液后脱盐，获得目的抗体。
>SDP01315重链全长
>SDP01315轻链全长
2.嵌合抗体与γ9δ2 T细胞的结合
抗γδTCR单域抗体细胞水平亲和力检测过程如下：将梯度稀释的抗体分子于1 x 105个γ9δ2 T细胞，4℃孵育1小时，洗掉多余的抗体，加入DyLight405-AffiniPure Goat Anti-Alpaca IgG,VHH domain抗体(Jackson，Cat#128-475-232)，在4℃孵育30分钟，洗掉多余的抗体后，用200μL 2％FBS/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号。结果如表3所示。
鼠源抗γδTCR抗体细胞水平亲和力检测过程如下：将梯度稀释的抗体分子于1 x 105个γ9δ2 T细胞，4℃孵育1小时，洗掉多余的抗体，加入鼠源Alexa Flour 647标记的抗人Fc抗体(Jackson，Cat#209-605-098)，在4℃孵育30分钟，洗掉多余的抗体后，用200μL 2％FBS/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号。结果如表3所示。
表3.抗γδTCR嵌合抗体的结合能力
结果显示，SDP01346、SDP01315在γ9δ2 T细胞上具有良好的结合活性。
实施例4.抗γδTCR抗体与抗原结合区域的鉴定
本实施例采用基于蛋白的ELISA检测抗γδTCR抗体与抗原的结合区域。采用实施例1类似的方法，制备重组人γ9δ1 TCR蛋白(SEQ ID NO：1和SEQ ID NO：20的二聚体蛋白)、人γ8δ2 TCR蛋白(SEQ ID NO：21和SEQ ID NO：2的二聚体蛋白)、人VγδCαβ嵌合TCR蛋白(SEQ ID NO：22和SEQ ID NO：23的二聚体蛋白)以及人VαβCγδ嵌合TCR蛋白(SEQ ID NO：24和SEQ ID NO：25的二聚体蛋白)。
采用ELISA分别鉴定抗γδTCR抗体与上述抗原的结合活性，判断抗体结合的大致区域。Protein ELISA具体检测过程如下：用1μg/mL重组TCR蛋白分别包板，4℃过夜，用含1％BSA的PBST缓冲液封闭1小时，洗板3次。用封闭缓冲液将抗体稀释至30nM，37℃孵育1小时，洗板3次，与1：10000稀释的抗小鼠IgG-Fc-HRP的二抗(Sigma,AP127P)孵育1小时。PBST洗涤3次，每孔加入100μL TMB显色液显色，15min后用终止液终止反应。使用酶标仪读取450nm处的吸光值。
>重组人δ1链
>重组人γ8链
>重组人Vγ9Cβ链

>重组人Vδ2Cα链
>重组人Vβ7Cγ链
>重组人Vα13Cδ链
ELISA结果如表4所示，抗体SDP01346和SDP01315对不同抗原的结合吸光值是不同的，这两个抗体均是结合γ9δ1 TCR蛋白而不结合γ8δ2 TCR蛋白，说明抗体结合的是γ9链；这两个抗体结合VγδCαβ嵌合TCR蛋白而不结合VαβCγδ嵌合TCR，说明抗体结合的是TCR可变区(V区)，综上可以明确抗体SDP01346和SDP01315结合的是TRGV9。
表4.嵌合抗体与抗原结合区域的鉴定结果
实施例5.抗γδTCR抗体的人源化改造
将可变区序列与抗体种系数据库比较，获得同源性高的人种系模板。其中，SDP01346所使用的人种系重链模板为IGHV3-30；SDP01315使用的人种系重链模板为IGHV3-21，人种系轻链模板为IGKV1-12。通过同源建模预测单抗的结构后，将鼠源抗体的VH、VL或单域抗体的CDR嵌合到合适的人GermLine框架上(Bioinformation.2014；10(4):180-186；Methods Mol Biol.2019；1904:213-230)，随后引入回复突变。
获得的人源化分子序列如下：
>SDP01346 VH1
>SDP01346 VH2
>SDP01346 VH3
>SDP01346 VH4
>SDP01315 VH1
>SDP01315 VH2
>SDP01315 VH3
>SDP01315 VL1
>SDP01315 VL2
>SDP01315 VL3
单域抗体人源化后构建全长抗体，在对应VHH后加入GGGGSHHHHHH(SEQ ID NO：72)。或单域抗体人源化后构建全长抗体，在对应VHH后加入接头和Fc。
鼠源抗体人源化后构建全长抗体，轻链恒定区使用的是Cκ，重链恒定区使用的是带L234F，L235E突变的IgG1。
表5.抗体名称与抗体结构的对应关系。
以下示例性给出SDP01378、SDP05576和SDP01369的全长序列。
>SDP01378全长
>SDP05576

>SDP01369重链全长
>SDP01369轻链全长
以下进行人源化单抗与γ9δ2 T细胞结合的FACS检测。
单域抗体SDP01346人源化后亲和力检测：将梯度稀释的抗体分子于1 x 105个γ9δ2 T细胞，4℃孵育1小时，洗掉多余的抗体，加入DyLight 405-AffiniPure Goat Anti-Alpaca IgG,VHH domain抗体(Jackson，Cat#128-475-232)，在4℃孵育30分钟，洗掉多余的抗体后200μL 2％FBS/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号。结果如表6所示。
鼠源嵌合抗体SDP01315人源化后亲和力检测：将梯度稀释的抗体分子于1 x105个γ9δ2 T细胞，4℃孵育1小时，洗掉多余的抗体，加入鼠源Alexa Flour 647标记的抗人Fc抗体(Jackson，Cat#209-605-098)，在4℃孵育30分钟，洗掉多余的抗体后200μL 2％FBS/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号。结果如表6所示。
表6.SDP01346和SDP01315人源化分子亲和力检测

结果说明，单域抗体人源化分子SDP01378亲和力最强，SDP01346人源化分子SDP01365-SDP01373亲和力相当，选择回复突变数目居中的SDP01369。
实施例6.抗GPC3/γδTCR双特异性抗体的设计和制备
GPC3端选择的具有纳摩尔级亲和力的抗体G，重链可变区和轻链可变区的序列如下。抗体G结合的是C-末端亚基，不结合可溶性GPC3。
>GPC3 VH
>GPC3 VL
表7.抗体G的VH和VL(Kabat编号规则)
我们设计了多种GPC3/γδTCR双抗，最终选择了与SDP01346H4(即，SDP01378)组成1+1非对称结构，采用KIH(knob into hole)避免重链错配；与SDP01315H2L2组成2+1非对称结构，采用KIH避免重链错配，HOT避免轻链错配。结构示意图见图1。
为防止、降低重链和轻链错配，使用的Obscurin-O和Titin-T见SEQ ID NO：47-48，CH1和Cκ见SEQ ID NO：49-50。为防止、降低两条重链之间错配，使用了IgG的突变。双抗全长序列如下，其中，下划线部分为CDR序列，波浪下划线的为Obscurin-O或Titin-T序列，点下划线为CH1或Cκ，斜体为IgG1 Fc区，斜体加粗为Fc区的点突变，粗体为连接子。
>Obscurin-O
>Titin-T
>CH1
>Cκ
>IgG1 Fc(WT)
>IgG1 Fc1(突变为S354C/T366W/L234F/L235E)
>IgG1 Fc2(突变为Y349C/T366S/L368A/Y407V/L234F/L235E)
>SDP01716 H1

>SDP01716 H2
>SDP01716 L
>SDP01696 H1
>SDP01696 L1
>SDP01696 H2

>SDP01696 L2
同SDP01716 L(SEQ ID NO：56)。
>SDP01704 H1
同SDP01696 H1(SEQ ID NO：57)。
>SDP01704 L1
同SDP01696 L1(SEQ ID NO：58)。
>SDP01704 H2
>SDP01704 L2
将编码抗体的核苷酸序列克隆到pTT5载体上后转染ExpiCHO细胞，8天后离心去除细胞收集并过滤细胞培养液，将收获的细胞培养液使用Protein A亲和柱(MabSelect SuRe，GE)纯化，使用甘氨酸洗脱结合的抗体，1M Tris中和洗脱液后脱盐，经检测，获得目的抗体SDP01716、SDP01696和SDP01704。
实施例7.抗GPC3/γδTCR双特异性抗体与人和猴抗原结合亲和力检测
采用Protein A生物传感芯片(Cat.#29139121-AB,Cytiva)。用HBS-EP+缓冲溶液分别配制各待测抗体作为配体与芯片通道上的Protein A进行捕获，人GPC3(Sino Biological,Cat#10088-H08H)/食蟹猴GPC3(Acrobiosystems,Cat#GP3-C5225)/人γ9δ2/食蟹猴γ9δ2抗原作为分析物用HBS-EP+缓冲溶液进行配制，分析物进行2倍梯度稀释。将稀释好的抗原在30μL/min的流速下流过实验通道和参比通道，结合80秒，解离300秒。再生缓冲液选择10mM Glycine pH1.5(GE Healthcare，BR-1003-54)在10μL/min的流速下运行30秒。数据用Biacore 8K evaluation software软件进行分析。
结果如表8和表9所示，三个抗体对人、食蟹猴GPC3蛋白的抗原结合亲和力均在2nM左右，与抗体G的亲和力相近；三个抗体与食蟹猴γ9δ2 TCR蛋白的结合KD均为50nM左右。
表8.抗体与人/食蟹猴GPC3蛋白的结合动力学参数
表9.抗体与人/食蟹猴γ9δ2蛋白的结合动力学参数
实施例8.抗GPC3/γδTCR双特异性抗体与GPC3阳性细胞和人γ9δ2 T细胞的结合活性检测
用FACS实验检测γδT双抗与天然高表达人GPC3的HepG2细胞(购自ATCC)和不表达人GPC3的DLD-1细胞(购自ATCC)的结合活性。抗GPC3/γδTCR双特异性抗体在细胞上的GPC3结合活性实验通过检测细胞表面结合抗体的荧光信号，根据荧光信号的强弱来评价抗体的结合强度。具体地，将梯度稀释的抗体分子和对照分子于1 x 105个细胞，4℃孵育1小时，洗掉多余的抗体，加入鼠源Alexa Flour 647标记的抗人Fc抗体(Jackson，Cat#209-605-098)，在4℃孵育30分钟，洗掉多余的抗体后200μL 2％FBS/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号，所使用的细胞为HepG2细胞系。
用FACS实验检测抗GPC3/γδTCR双特异性抗体与人γ9δ2 T细胞的结合活性以及与人PBMC结合活性。人γ9δ2 T细胞采用唑来膦酸和IL-2进行诱导获得。抗GPC3/γδTCR双特异性抗体在γδT细胞上的结合活性实验通过检测细胞表面结合抗体的荧光信号，根据荧光信号的强弱来评价抗体的结合强度。具体地，将梯度稀释的抗体分子和对照分子于1 x 105个细胞，4℃孵育1小时，洗掉多余的抗体，加入鼠源Alexa Flour 647标记的抗人Fc抗体(Jackson，Cat#209-605-098)，在4℃孵育30分钟，洗掉多余的抗体后200μL 2％FBS/PBS缓冲液重悬，通过Thermo Attune NxT流式细胞仪读取细胞表面的荧光信号。
结果如图2A-图2D所示，其中，如图2A和图2B，SDP01716和SDP01696的GPC3端亲和力相当，EC50分别为4.853nM和5.166nM。SDP01704显示出二价结合特征，EC50为2.466nM；所测抗体在GPC3阴性细胞上均没有非特异性结合。如图2C和图2D，SDP01716对γ9δ2 T细胞的亲和力为0.9064nM，SDP01696为2.676nM，SDP01704为4.289nM；所测抗体在PBMC上均没有非特异性结合。
实施例9.抗GPC3/γδTCR双特异性抗体对BTN2A/BTN3A-TCR天然信号的影响
在天然状态下，细胞内的磷酸化抗原与BTN3A结合后，改变了BTN3A的分子张力；2个BTN3A分子和2个BTN2A分子结合形成异源四聚体，进而与TCR相互作用，激活TCR，诱导γδT细胞的活化、增殖、杀伤等功能。由于抗γδTCR抗体同样结合TCR，因此，本实施例评估本公开的抗体对BTN2A/BTN3A-TCR天然信号影响。SDP01378是SDP01716的γδT端单价分子，SDP01315是SDP01696和SDP01704的单价分子。
A375-Luc细胞培养基为RPMI1640，含有10％灭活胎牛血清。γδT细胞由PBMC扩增获得。调整A375-Luc密度，以25μL/孔每孔接种于96孔板，细胞量为1×104/孔；取25μL/孔γδT细胞(1×105/孔)与A375-Luc混合；再加入25μL/孔SDP01378或者SDP01315(终浓度为10nM)，以及25μL/孔梯度稀释的BTN3A激动性抗体。在37℃、5％CO2培养箱中孵育24小时。24小时后加入100μL/孔Bright-GloTM Luciferase Assay System(Promega，E2650)试剂进行检测。具体操作参考试剂说明书。
结果如图3A和图3B所示，SDP01378和SDP01315都不阻断BTN2A/BTN3A-TCR天然信号。因此，可以认为，本公开的抗GPC3/γδTCR双特异性抗体不阻断BTN2A/BTN3A-TCR天然信号。
实施例10.抗GPC3/γδTCR双特异性抗体体外介导γδT细胞对肿瘤细胞杀伤活性检测
本实施例使用乳酸脱氢酶(LDH)检测法评估抗体介导γδT细胞对不同GPC3表达量靶细胞的杀伤活性。
HepG2细胞天然高表达GPC3，细胞培养基为DMEM(Gibco，Cat#11995-065，下同)，含有15％灭活胎牛血清；Huh-7表达中等水平GPC3，细胞培养基为DMEM；MKN-45表达低水平GPC3，细胞培养基为RPMI 1640(Gibco，Cat#10491A-01，下同)；DLD-1不表达GPC3，细胞培养基为RPMI 1640。将靶细胞消化后用2％血清的RPMI1640培养基重悬，密度调整为7×104细胞/mL；随后以50μL/孔接种于96孔板，并加入50μL梯度稀释后的待测抗体。每孔补50μL培养基。收集γδT细胞，重悬于含有2％胎牛血清的RPMI1640中，调整细胞密度。以50μL/孔接种于上述实验板，在37℃、5％CO2培养箱中孵育24小时。取出细胞培养板，离心(400g，5分钟)收集细胞培养上清，采用CytoToxNon-Radioactive Cytotoxicity Assay试剂盒(Promega，G1780)检测LDH的水平。具体操作参考试剂说明书。
结果如图4A至图4D、表10显示，随着抗原表达量的降低，本公开的双抗对肿瘤细胞的杀伤活性减弱；抗体γδT端亲和力强弱与对肿瘤细胞杀伤活性成正相关，且GPC3端二价分子强于单价分子。
表10.抗GPC3/γδTCR双特异性抗体对不同抗原表达细胞杀伤
注：-为没有检测到杀伤活性。
实施例11.抗GPC3/γδTCR双特异性抗体对不同PBMC供体来源γδT细胞杀伤的影响
本实施例使用乳酸脱氢酶(LDH)检测法评估抗体介导γδT细胞对表达GPC3的靶细胞的杀伤活性。
将HepG2细胞消化后用2％血清的RPMI1640培养基重悬，细胞密度调整为7×104个/mL；随后以50μL/孔接种于96孔板，并加入50μL梯度稀释后的待测抗体。每孔补50μL培养基。收集不同供体扩增的γδT细胞，重悬于含有2％胎牛血清的RPMI1640中，调整细胞密度。以50μL/孔接种于上述实验板，在37℃、5％CO2培养箱中孵育24小时。取出细胞培养板，离心(400g，5分钟)收集细胞培养上清，采用CytoToxNon-Radioactive Cytotoxicity Assay试剂盒(Promega，G1780)检测LDH的水平。
示例性展示了SDP01716的结果。结果如图5A至图5D、表11所示，SDP01716能介导不同供体来源的γδT细胞杀伤肿瘤细胞，EC50及最大杀伤值没有较大差异。
表11.抗体介导不同供体来源的γδT细胞对HepG2杀伤活性
注：#SC12004、#SC12392、#XC11053、#XC11061为不同供体来源(健康同种异体的人类外周血)扩增的γδT细胞，-为未检测到杀伤。
实施例12.抗GPC3/γδTCR双特异性抗体对抗原低表达细胞不同效靶比下的杀伤活性检测
本实施例使用乳酸脱氢酶(LDH)检测法评估抗体介导γδT细胞对低表达GPC3靶细胞的杀伤活性。
MKN-45细胞天然低表达GPC3。将MKN-45细胞消化后用2％血清的RPMI1640培养基重悬，细胞密度调整为7×104个/mL；随后以50μL/孔接种于96孔板，并加入50μL梯度稀释后的待测抗体。每孔补50μL培养基。收集γδT细胞，重悬于含有2％胎牛血清的RPMI1640中，调整细胞密度。以50μL/孔接种于上述实验板，最终效靶比(γδT细胞：MKN-45细胞)为1:1；10:1；30:1。在37℃、5％CO2培养箱中孵育24小时。取出细胞培养板，离心(400g，5分钟)收集细胞培养上清，采用CytoToxNon-Radioactive Cytotoxicity Assay试剂盒(Promega，G1780)检测LDH的水平。具体操作参考试剂说明书。
示例性展示了SDP01716的结果。如图6A所示，1:1低效靶比时，SDP01716对抗原低表达细胞杀伤活性弱，最大杀伤值为17％；如图6B所示，提升效靶比到10:1后，SDP01716能够增强对抗原低表达细胞的杀伤活性，最大杀伤达到64％；如图6C所示，当效靶比提升到30:1时，几乎达到100％杀伤。即，本公开的抗GPC3/γδTCR双特异性抗体能够效靶比依赖性的杀伤肿瘤靶细胞。
实施例13.提高PBMC中γδT细胞比例增强抗GPC3/γδTCR双特异性抗体的杀伤活性及细胞因子释放检测
本实施例使用乳酸脱氢酶(LDH)检测法评估抗体介导γδT细胞对表达GPC3靶细胞的杀伤活性。
HepG2细胞天然高表达GPC3，将HepG2细胞消化后用2％血清的RPMI1640培养基重悬，细胞密度调整为7×104个/mL；随后以50μL/孔接种于96孔板，并加入50μL梯度稀释后的待测抗体。每孔补50μL培养基。收集PBMC细胞，重悬于含有2％胎牛血清的RPMI1640中，调整细胞密度。以50μL/孔接种于上述实验板；或者在PBMC中加入30％的γδT细胞，以50μL/孔接种于上述实验板，在37℃、5％CO2培养箱中孵育24小时。取出细胞培养板，离心(400g，5分钟)收集细胞培养上清，采用CytoToxNon-Radioactive Cytotoxicity Assay试剂盒(Promega，G1780)检测LDH的水平。同时采用试剂盒检测IFNγ(Cisbio，S62HIFNGPEG)，TNFα(Cisbio，62HTNFAPEH)检测相应细胞因子分泌水平具体操作参考试剂说明书。
示例性展示了SDP01716的结果。结果如图7A至7D所示，其中，图7A为供体#XC11053的PBMC细胞杀伤；图7B为供体#XC11053的PBMC掺入30％的γδT细胞杀伤活性；图7C为供体#SC12392的PBMC细胞杀伤；图7D为供体#SC12392的PBMC掺入30％的γδT细胞杀伤活性。
图7A和图7C说明，SDP01716在纯PBMC体系中肿瘤细胞的杀伤能力弱于同靶点CD3双抗BMK-029(为US20220348658中的ERY974)，因为CD3双抗可以动用更多的T细胞作为效应细胞进行杀伤肿瘤；图7B和图7D说明，在PBMC中提高γδT细胞的比例可提升其杀伤活性。与此同时，与图7A至图7D对应的细胞因子释放检测结果如图8A至图8D所示，SDP01716在杀伤肿瘤细胞时诱导更少的IFNγ和TNFα释放。
实施例14.抗GPC3/γδTCR双特异性抗体促进PBMC中γδT细胞增殖活性检测
本实施例使用流式细胞检测法评估抗GPC3/γδTCR双特异性抗体诱导PBMC中γδT细胞增殖的活性。
取PBMC 800μL(1×106细胞)加入24孔板中；HepG2细胞PBS洗两次后调整细胞密度5×105细胞/mL，取1mL加入对应孔中；待测抗体用培养基配置成相应浓度后取200μL加入孔中。37℃、5％CO2培养箱中孵育，每3天进行板换液。6至7天后细胞用PBS洗两遍，CD3抗体(BD,Cat#564713)，Vδ2 TCR抗体(BD,Cat#555739)，Vγ9TCR抗体(BD,Cat#555732)染色，鉴定γδT细胞占比变化。
结果如图9所示，三个供体#XC11211、#XC11061、#XC11251来源的PBMC细胞中，经6-7天的刺激，SDP01716、SDP01696和SDP01704均能显著提升γδT细胞占比。
实施例15.小鼠的人肝癌Huh-7移植瘤模型验证
1.将人肝癌Huh-7细胞(中科院细胞库)以5×106个/100μL/只接种于NSG小鼠(雌性，6-8周龄，由维通利华提供)皮下并随机分组，每组7只，共4组(G1-G4)，在分组当天接种肿瘤细胞，同时输注γδT细胞和药物，各组分别按照如下方案给药：
G1：介质G2：γδT细胞+PBS
G3：γδT细胞+SDP01716(3mg/kg)
G4：γδT细胞+SDP01716(0.3mg/kg)
之后每周输注γδT细胞和给药1次。给药和观察期间每周测量2次肿瘤体积，并记录测量值。计算公式如下(下同)：
计算肿瘤体积(tumor volume,TV)＝1/2×a×b2，其中a，b分别代表测量肿瘤的长径和短径；
相对肿瘤增值率T/C％＝(T-T0)/(C-C0)×100；
抑瘤率TGI％＝1-T/C％。
结果见图10A、图10B和表12所示，γδT细胞单用组(G2)的TGI为11％，抑制肿瘤生长作用有限；SDP01716能剂量依赖性抑制肿瘤生长，3mpk剂量下TGI达到58％，且各给药组均未对小鼠体重产生显著影响。
表12.SDP01716在Huh-7移植瘤模型中的抗肿瘤活性
注：*代表P<0.05；**代表P<0.01。
2.将Huh-7细胞以5×106个/100μL/只接种于NSG小鼠(雌性，6-8周龄，由集萃药康提供)皮下并随机分组，每组7只，共6组，在分组当天接种肿瘤细胞，同时输注γδT细胞和药物，各组分别按照如下方案给药：
G1：介质
G2：γδT细胞
G3：γδT细胞+SDP01716(3mg/kg)
G4：γδT细胞+SDP01696(3.9mg/kg)
G5：γδT细胞+SDP01704(5.1mg/kg)。
之后每周输注γδT细胞和给药1次，如是γδT细胞和抗体联用，则是同时给药。给药和观察期间每周测量2次肿瘤体积，并记录测量值。
结果见图11A、图11B和表13所示，本公开的双抗均能够有效抑制肿瘤，各给药组小鼠均未出现体重的明显改变。
表13.双抗在Huh-7移植瘤模型中的抗肿瘤活性
注：**代表P<0.01。

Claims (27)

  1. GPC3/TRGV9结合蛋白，其包含：
    特异性结合GPC3的第一抗原结合结构域；和
    特异性结合TRGV9的第二抗原结合结构域，其中，
    其中：
    所述第一抗原结合结构域包含重链可变区(VH1)和轻链可变区(VL1)，
    所述VH1包含SEQ ID NO：39所示氨基酸序列中的HCDR1、HCDR2和HCDR3，
    所述VL1包含SEQ ID NO：40所示氨基酸序列中的LCDR1、LCDR2和LCDR3，
    所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
    优选地，所述CDR是根据Kabat编号系统定义的；
    优选地，所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：41-43所示，所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：44-46所示。
  2. 如权利要求1所述的GPC3/TRGV9结合蛋白，所述第二抗原结合结构域包含免疫球蛋白单一可变结构域，或包含重链可变区(VH2)和轻链可变区(VL2)，其中，
    所述免疫球蛋白单一可变结构域包含SEQ ID NO：29、26-28、5任一所示氨基酸序列中的CDR1、CDR2和CDR3，
    所述VH2包含SEQ ID NO：31、9、30、32任一所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL2包含SEQ ID NO：34、10、33、35任一所示氨基酸序列中的LCDR1、LCDR2和LCDR3，
    所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
    优选地，所述CDR是根据Kabat编号系统定义的；
    优选地，所述免疫球蛋白单一可变结构域包含分别如SEQ ID NO：6-8所示氨基酸序列的CDR1、CDR2和CDR3，
    所述VH2的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO：11-13所示，所述VL2的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO：14-16所示。
  3. 如权利要求1或2所述的GPC3/TRGV9结合蛋白，其中，
    所述免疫球蛋白单一可变结构域、所述重链可变区和/或所述轻链可变区为人源化、回复突变、亲合力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的；
    优选地，所述免疫球蛋白单一可变结构域在人源化改造过程使用的人种系模板的框架区源自IGHV3-64；
    优选地，所述VH2在人源化改造过程使用的人种系模板的框架区源自IGHV3-21，和/或所述VL2在人源化改造过程使用的人种系模板的框架区源自IGKV1-12。
  4. 如权利要求1至3任一项所述的GPC3/TRGV9结合蛋白，其中，
    所述第一抗原结合结构域中的VH1包含如SEQ ID NO：39所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL1包含如SEQ ID NO：40所示或与之具有至少80％、至少90％同一性的氨基酸序列。
  5. 如权利要求1至4任一项所述的GPC3/TRGV9结合蛋白，其中，
    所述免疫球蛋白单一可变结构域包含如SEQ ID NO：29、26-28、5任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
    所述第二抗原结合结构域中的VH2包含如SEQ ID NO：9、30-32任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，VL2包含如SEQ ID NO：10、33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列。
  6. 如权利要求1至5任一项所述的GPC3/TRGV9结合蛋白，其还包含免疫球蛋白的Fc区；
    优选地，所述Fc区是人IgG1、人IgG2、人IgG3或人IgG4的Fc区；
    更优选地，所述Fc区是人IgG1的Fc区；
    最优选地，所述Fc区是包含L234F和/或L235E突变的IgG1的Fc区。
  7. 如权利要求6所述的GPC3/TRGV9结合蛋白，其中，所述Fc区包含第一亚基和第二亚基；
    优选地，所述Fc区的第一亚基和第二亚基包含knob-into-hole突变；
    优选地，所述Fc区的第一亚基含有366位突变，第二亚基含有选自366、368或407位的突变或其任意组合；所述Fc区的第一亚基含有354或356位突变，第二亚基含有349位突变；或
    所述Fc区的第一亚基含有354或356位突变，第二亚基含有349、366、368或407位突变或其任意组合；
    更优选地，所述Fc区的第一亚基含有366W突变，第二亚基含有选自366S、368A和407V的突变或其任意组合；所述Fc区的第一亚基含有354C或356C突变，第二亚基含有349C突变；或所述Fc区的第一亚基含有354C/366W突变，第二亚基含有349C/366S/368A/407V突变；
    突变位点的编码规则为Eu编码。
  8. 如权利要求1至7任一项所述GPC3/TRGV9结合蛋白，其还含有连接子；
    优选地，所述连接子如(GmSn)h或(GmQn)h或(GGNGT)h或(YGNGT)h或(EPKSS)h或(AmSn)h所示，其中，m、n各自独立地选自1-8的整数，h独立地选自1-20的整数；
    更优选地，所述连接子为A3S或G4S。
  9. 如权利要求1至8任一项所述GPC3/TRGV9结合蛋白，其包含选自(1)-(3)任一组的多肽链：
    (1)第一重链、第二重链和轻链，其中，
    第一重链，其从N端到C端依次为：[免疫球蛋白单一可变结构域]-[连接子1]-[Fc1]，
    第二重链，其从N端到C端依次为：[VH1]-[连接子2]-[CH1]-[连接子3]-[Fc2]，
    轻链，其从N端到C端依次为：[VL1]-[连接子4]-[CL]；
    其中，所述连接子1、连接子2、连接子3和连接子4可以相同或不相同，可以独立的存在或不存在，可以独立的选自权利要求8所限定的连接子；
    优选地，连接子1为AAAS，连接子2、连接子3和连接子4不存在；
    (2)第一重链、第一轻链、第二重链和第二轻链，其中，
    第一重链，其从N端到C端依次为：[VH2]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
    第一轻链，其从N端到C端依次为：[VL2]-[连接子3]-[Titin-T链]，
    第二重链，其从N端到C端依次为：[VH1]-[连接子4]-[CH1]-[连接子5]-[Fc2]，
    第二轻链，其从N端到C端依次为：[VL1]-[连接子6]-[CL]；
    或，
    第一重链，其从N端到C端依次为：[VH1]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
    第一轻链，其从N端到C端依次为：[VL1]-[连接子3]-[Titin-T链]，
    第二重链，其从N端到C端依次为：[VH2]-[连接子4]-[CH1]-[连接子5]-[Fc2]，
    第二轻链，其从N端到C端依次为：[VL2]-[连接子6]-[CL]；
    其中，所述连接子1、连接子2、连接子3、连接子4、连接子5和连接子6可以相同或不相同，可以独立的存在或不存在，可以独立的选自权利要求8限定的连接子；
    优选地，连接子1、连接子3为G4S，连接子2、连接子4、连接子5、连接子6不存在；
    (3)第一重链、第一轻链、第二重链和第二轻链，其中，
    第一重链，其从N端到C端依次为：[VH2]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
    第一轻链，其从N端到C端依次为：[VL2]-[连接子3]-[Titin-T链]，
    第二重链，其从N端到C端依次为：[VH1]-[连接子4]-[CH1]-[连接子5]-[VH1]-[连接子6]-[CH1]-[连接子7]-[Fc2]，
    第二轻链，其从N端到C端依次为：[VL1]-[连接子8]-[CL]；
    或，
    第一重链，其从N端到C端依次为：[VH1]-[连接子1]-[Obscurin-O链]-[连接子2]-[Fc1]，
    第一轻链，其从N端到C端依次为：[VL1]-[连接子3]-[Titin-T链]，
    第二重链，其从N端到C端依次为：[VH2]-[连接子4]-[CH1]-[连接子5]-[VH2]-[连接子6]-[CH1]-[连接子7]-[Fc2]，
    第二轻链，其从N端到C端依次为：[VL2]-[连接子8]-[CL]；
    其中，所述连接子1、连接子2、连接子3、连接子4、连接子5、连接子6、连接子7、连接子8、连接子9、连接子10可以相同或不相同，可以独立的存在或不存在，可以独立的选自权利要求8所述的连接子；
    优选地，连接子1、连接子3为G4S，连接子5为(G4S)2，连接子2、连接子4、连接子6、连接子7和连接子8不存在；
    以上(1)-(3)中，-表示肽键，
    优选地，所述Obscurin-O链和Titin-T链的氨基酸序列分别如SEQ ID NO：47、48所示；
    优选地，所述CL为Cκ，所述CH1和Cκ的氨基酸序列分别如SEQ ID NO：49、50所示；
    优选地，所述Fc1和Fc2的氨基酸序列分别如SEQ ID NO：52、53所示。
  10. 如权利要求1至9任一项所述的GPC3/TRGV9结合蛋白，其包含选自(1)-(3)任一组的多肽链：
    (1)氨基酸序列如SEQ ID NO：54所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：55所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％同一性的轻链；
    (2)氨基酸序列如SEQ ID NO：57所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％同一性的第一轻链，氨基酸序列如SEQ ID NO：59所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：56所示或与之具有至少80％、至少90％同一性的第二轻链；或，
    (3)氨基酸序列如SEQ ID NO：57所示或与之具有至少80％、至少90％同一性的第一重链，氨基酸序列如SEQ ID NO：58所示或与之具有至少80％、至少90％同一性的第一轻链，氨基酸序列如SEQ ID NO：60所示或与之具有至少80％、至少90％同一性的第二重链，氨基酸序列如SEQ ID NO：61所示或与之具有至少80％、至少90％同一性的第二轻链；
    优选地，所述GPC3/TRGV9结合蛋白包含选自(1)-(3)任一组的多肽链：
    (1)氨基酸序列如SEQ ID NO：54-56所示的多肽链；
    (2)氨基酸序列如SEQ ID NO：56-59所示的多肽链；
    (3)氨基酸序列如SEQ ID NO：57、58、60、61所示的多肽链；
    更优选地，所述GPC3/TRGV9结合蛋白包含选自(1)-(3)任一组的多肽链：
    (1)摩尔比为1：1：1的氨基酸序列如SEQ ID NO：54-56所示的多肽链；
    (2)摩尔比为1：1：1：1的氨基酸序列如SEQ ID NO：56-59所示的多肽链；
    (3)摩尔比为1：1：1：2的氨基酸序列如SEQ ID NO：57、58、60、61所示的多肽链。
  11. 如权利要求1至10任一项所述的GPC3/TRGV9结合蛋白，其为抗体或其抗原结合片段，优选为抗GPC3/TRGV9抗体或其抗原结合片段。
  12. TRGV9结合蛋白，其包含：
    (1)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含SEQ ID NO：29、26-28、5任一所示氨基酸序列中的CDR1、CDR2和CDR3，优选地，包含SEQ ID NO：6-8所示氨基酸序列中的CDR1、CDR2和CDR3；或，
    (2)重链可变区(VH)和/或轻链可变区(VL)，所述VH包含SEQ ID NO：9、30-32任一所示氨基酸序列中的HCDR1、HCDR2和HCDR3，所述VL包含SEQ ID NO：10、33-35任一所示氨基酸序列中的LCDR1、LCDR2和LCDR3；
    所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的，例如，是根据Kabat编号系统定义的。
  13. 如权利要求12所述的TRGV9结合蛋白，所述免疫球蛋白单一可变结构域、VH和/或VL为人源化、回复突变、亲合力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的；
    优选地，所述免疫球蛋白单一可变结构域在人源化改造过程使用的人种系模板的框架区源自IGHV3-64；
    优选地，所述VH在人源化改造过程使用的人种系模板的框架区源自IGHV3-21，和/或所述VL在人源化改造过程使用的人种系模板的框架区源自IGKV1-12。
  14. 如权利要求12或13所述的TRGV9结合蛋白，其中，
    (1)免疫球蛋白单一可变结构域包含如SEQ ID NO：29、26-28、5任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；或，
    (2)VH和VL，所述VH包含如SEQ ID NO：9、30-32任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，所述VL包含如SEQ ID NO：10、33-35任一所示或与之具有至少80％、至少90％同一性的氨基酸序列；
    优选地，所述TRGV9结合蛋白是抗TRGV9抗体或其抗原结合片段；
    更优选地，当所述TRGV9结合蛋白包含免疫球蛋白单一可变结构域时，其是抗TRGV9单域抗体或VHH。
  15. 如权利要求12至14任一项所述的TRGV9结合蛋白，其还包含免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1、人IgG2、人IgG3或人IgG4的Fc区，所述Fc区更优选为人IgG1的Fc区；
    优选地，所述Fc区是包含L234F和/或L235E突变的IgG1的Fc区。
  16. 如权利要求12至15任一项所述的TRGV9结合蛋白，其进一步包含肿瘤相关抗原(TAA)结合结构域、或肿瘤特异性抗原结合结构域，优选为特异性结合GPC3的抗原结合结构域。
  17. 如权利要求1至11任一项所述的GPC3/TRGV9结合蛋白或权利要求12至16任一项所述的TRGV9结合蛋白，其具有选自(a)-(d)任一项或多项的性质或功能：
    (a)特异性结合TCR的γ9链，优选地，特异性结合γ9δ1TCR、γ9δ2TCR；
    (b)检测不到和TCR的γ8链结合，优选地，检测不到和γ8δ2TCR结合；
    (c)特异性结合TCR的γ9链的可变区(TRGV9)；
    (d)特异性结合VγδCαβ嵌合TCR，检测不到和VαβCγδ嵌合TCR结合。
  18. 多核苷酸，其编码权利要求1至11任一项所述的GPC3/TRGV9结合蛋白或权利要求12至16任一项所述的TRGV9结合蛋白，或其组合；
    优选地，所述多核苷酸为DNA或RNA。
  19. 载体，其包含权利要求18所述的多核苷酸，
    优选地，所述载体表达权利要求18所述的多核苷酸。
  20. 宿主细胞，其含有或表达权利要求18所述的多核苷酸或权利要求19所述的载体。
  21. 制备权利要求1至11任一项所述的GPC3/TRGV9结合蛋白或权利要求12至16任一项所述的TRGV9结合蛋白的方法，包括：
    在权利要求20所述的宿主细胞中表达权利要求18所述的多核苷酸或权利要求19所述的载体，以及从所述宿主细胞中分离表达的GPC3/TRGV9结合蛋白或TRGV9结合蛋白；
    可选地，进一步包含纯化所述GPC3/TRGV9结合蛋白或TRGV9结合蛋白的步骤。
  22. 药物组合物，其包含权利要求1至11任一项所述的GPC3/TRGV9结合蛋白、权利要求12至16任一项所述的TRGV9结合蛋白、权利要求18所述的多核苷酸和/或权利要求19所述的载体；
    优选地，所述药物组合物进一步包含T细胞，所述T细胞优选为γδT细胞；
    优选地，所述药物组合物进一步包含一种或多种可药用的赋形剂、稀释剂或辅料。
  23. 组合或者药盒，其包含：
    (1)权利要求1至11任一项所述的GPC3/TRGV9结合蛋白和T细胞，或
    (2)权利要求12至16任一项所述的TRGV9结合蛋白和T细胞；
    所述T细胞优选为γδT细胞。
  24. 权利要求1至11任一项所述的GPC3/TRGV9结合蛋白、权利要求12至16任一项所述的TRGV9结合蛋白、权利要求18所述的多核苷酸、权利要求19所述的载体、权利要求23的组合或者药盒在制备治疗疾病的药物中的用途；
    所述疾病优选为癌症，
    更优选为肝癌或GPC3阳性癌症，最优选为GPC3阳性肝癌。
  25. 权利要求1至11任一项所述的GPC3/TRGV9结合蛋白、权利要求12至16任一项所述的TRGV9结合蛋白、权利要求18所述的多核苷酸、权利要求19所述的载体在制备治疗疾病的药物中的用途，其中：
    所述GPC3/TRGV9结合蛋白、所述TRGV9结合蛋白、所述多核苷酸、或所述载体与T细胞联用；
    所述T细胞优选为γδT细胞；
    所述疾病优选为癌症，更优选为肝癌或GPC3阳性癌症，最优选为GPC3阳性肝癌。
  26. T细胞用于制备治疗疾病的药物中的用途，其中：
    所述T细胞与权利要求1至11任一项所述的GPC3/TRGV9结合蛋白、权利要求12至16任一项所述的TRGV9结合蛋白、权利要求18所述的多核苷酸、或权利要求19所述的载体联用；
    所述T细胞优选为γδT细胞；
    所述疾病优选为癌症，更优选为肝癌或GPC3阳性癌症，最优选为GPC3阳性肝癌。
  27. 治疗或缓解疾病的方法，包括向有需要的受试者施用治疗或缓解有效量的权利要求1至11任一项所述的GPC3/TRGV9结合蛋白、权利要求12至16任一项所述的TRGV9结合蛋白、权利要求18所述的多核苷酸、权利要求19所述的载体、权利要求22的药物组合物或权利要求23的组合或者药盒；
    所述疾病优选为癌症，更优选为肝癌或GPC3阳性癌症，最优选为GPC3阳性肝癌。
PCT/CN2024/132266 2023-11-15 2024-11-15 Trgv9结合蛋白及其医药用途 Pending WO2025103451A1 (zh)

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