TRGV9 Binding Protein and Its Medicinal Uses

Abstract

The present disclosure relates to TRGV9 binding proteins and the pharmaceutical uses thereof. Specifically, the present disclosure relates to TRGV9 binding proteins, GPC3 / TRGV9 binding proteins, methods for treating cancer and pharmaceutical uses thereof.

Description

Technology Field

[0001] This application claims priority to Chinese patent application CN202311518247.8 filed on November 15, 2023.

[0002] Technology field

[0003] The present disclosure relates to the biomedical field, specifically to a TRGV9 binding protein, a GPC3 / TRGV9 binding protein, a method for treating cancer, and pharmaceutical uses thereof. Background Technology

[0004] γδ T cells are a population of T cells capable of naturally recognizing and killing tumor cells; possessing both antigen-presenting and apoptotic functions, they bridge innate and adaptive immunity. Unlike αβ TCRs, the two chains constituting the TCR of γδ T cells are the γ chain and the δ chain, respectively, and their antigen recognition is not restricted by MHC (Exp Mol Med. 2021 Mar;53(3):318-327). The number of γδ T cells is relatively small, accounting for approximately 1% to 5% of PBMCs. Based on the differences in the δ chain, they can be classified into four categories: δ1, δ2, δ3, and δ5 (Front Immunol. 2022 Jun 16;13:915837), with δ1 and δ2 accounting for the largest numbers. δ1 can pair with different γ chains to form different TCRs and is mainly distributed in tissues such as the skin and mucous membranes; δ2 mainly pairs with γ9 to form the γ9δ2 subtype, is mainly distributed in peripheral blood, and accounts for up to 95% of the total γδ T cells in peripheral blood (Front Immunol. 2022 Jun 16;13:915837).

[0005] γδ T cells play a crucial role in anti-tumor activity and possess various mechanisms for inducing tumor cell death. These include recognizing the BTN2A / BTN3A complex, activated by phosphorylated antigens on target cells via the TCR, initiating death signals, and exercising death functions; additionally, they exhibit death pathways similar to those of NK cells, exerting tumor-killing effects by expressing receptors such as NKG2D on their cell surfaces and binding to corresponding ligands on tumor cells. γδ T cells infiltrate to varying degrees in different tumors. A large-scale sample analysis of 5,782 tumors across 25 tumor types revealed that γδ T cell infiltration indicates a favorable patient prognosis and is the most advantageous cell population among all immune cells. Similarly, an analysis of 3,238 samples from 14 non-brain cancer solid tumors showed that their infiltration into the tumor was an indicator of a favorable prognosis (Oncoimmunology. 2017 Feb 6;6(3):e1284723). Early clinical studies on γδ T cells mainly consisted of two methods: in vivo activation and in vitro amplification followed by re-injection. Although the antitumor effects observed in various studies were relatively limited, no serious side effects were reported, suggesting that γδ T cells have good safety.

[0006] Glypican-3 (GPC3) is a potential target for the treatment of liver cancer. In 2020, there were 900,000 new cases and 830,000 deaths from liver cancer worldwide; in contrast, China recorded 410,000 new cases and 390,000 deaths in 2020, accounting for approximately half of all liver cancer cases globally. While first- or second-line treatments for advanced liver cancer have made significant progress in recent years, effective therapeutic options remain lacking for subsequent treatments. GPC3 is a heparan sulfate proteoglycan composed of 580 amino acids, anchored to the cell membrane via glycosylphosphatidylinositol. GPC3 is highly expressed in various tumor tissues; for example, the positivity rate in hepatocellular carcinoma reaches up to 90%. In normal tissues, GPC3 expression is highly localized, appearing only in the placenta and endometrium; In addition, expression levels in the endometrium are much lower than in tumor tissue, making it an ideal antitumor target (Sci Transl Med. 2017 Oct 4;9(410):eaal4291).

[0007] The present disclosure provides an antibody targeting a γδTCR of a novel 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 a TAA (e.g., GPC3) and the other end binds to a γδTCR, thereby recruiting and activating γδ T cells to specifically kill TAA (e.g., GPC3)-positive tumor cells, which significantly enhances the efficacy of γδ T cell therapy. The bispecific antibody of the present disclosure possesses good tumor-killing activity, safety, and pharmacological potential.

[0008] The present disclosure provides a T cell receptor (TCR) binding protein, phosphatidylinositol proteoglycan-3 (GPC3) and a T cell receptor (TCR) binding protein, the coding nucleic acid thereof, a method for preparing the same, and a method and use thereof for treating diseases.

[0009] T cell receptor γ-variable region 9 (TRGV9) binding protein

[0010] The present disclosure provides a TRGV9 binding protein. In some embodiments, it may bind to a TCR. In some embodiments, it may bind to a γδTCR. In some embodiments, it may bind to the γ9 chain of a TCR. In some embodiments, it may bind to a γ9δ2 TCR. In other embodiments, it may bind to a γ9δ1 TCR.

[0011] In some embodiments, a TRGV9 binding protein is provided, which:

[0012] 1) An immunoglobulin monovariable domain comprising CDR1, CDR2, and / or CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29, e.g., an immunoglobulin monovariable domain comprising CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29; or,

[0013] 2) comprising a heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 9, 30–32, and / or a light chain variable region (VL) including LCDR1, LCDR2, and LCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 10, 33–35, and

[0014] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, according to the Kabat numbering system.

[0015] In some embodiments, a TRGV9 binding protein is provided, which:

[0016] 1) an immunoglobulin monovariable domain comprising CDR1, CDR2 and / or CDR3, wherein CDR1 comprises the amino acid sequence presented in SEQ ID NO: 6, CDR2 comprises the amino acid sequence presented in SEQ ID NO: 7, and CDR3 comprises the amino acid sequence presented in SEQ ID NO: 8; or,

[0017] 2) VH comprising HCDR1, HCDR2 and / or HCDR3, wherein HCDR1 comprises the amino acid sequence presented in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence presented in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence presented in SEQ ID NO: 13; and / or VL comprising LCDR1, LCDR2 and / or LCDR3, wherein LCDR1 comprises the amino acid sequence presented in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence presented in SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence presented in SEQ ID NO: 16.

[0018] In some embodiments, a TRGV9 binding protein is provided, which:

[0019] 1) an immunoglobulin monovariable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as presented in SEQ ID NO: 6–8, respectively; or,

[0020] 2) VH comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as presented in SEQ ID NO: 11~13, respectively, and VL comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as presented in SEQ ID NO: 14~16, respectively.

[0021] In some embodiments, the immunoglobulin single variable domain or VH or VL of the aforementioned proteins is each independently modified by humanization, reverse mutation, affinity maturation, removal / reduction of T cell epitopes (TCEs), reduction of antibody deamidation and / or reduction of antibody isomerization.

[0022] In some embodiments, the heavy chain framework region of the human germline template used in the 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 modification process for VH is derived from IGHV3-21, and the light chain framework region of the human germline template used in the humanization modification process for VL is derived from IGKV1-12.

[0023] In some embodiments, a TRGV9 binding protein is provided, which:

[0024] 1) An immunoglobulin monovariable domain comprising an amino acid sequence that is the same as or has at least 80% or at least 90% identity with any one of SEQ ID NOs: 5, 26–29; or,

[0025] 2) Includes VH having an amino acid sequence that is the same as or has at least 80% and at least 90% identity with any one of SEQ ID NO: 9, 30~33, and VL having an amino acid sequence that is the same as or has at least 80% and at least 90% identity with any one of SEQ ID NO: 10, 33~35.

[0026] In some specific embodiments, the TRGV9 binding protein is the following VH and VL:

[0027] 2-1) VH comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 9, VL comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 10,

[0028] 2-2) VH comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 30, and VL comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0029] 2-3) VH comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 31, and VL comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0030] 2-4) Includes VH containing an amino acid sequence that is the same as or has at least 80% to 90% identity with SEQ ID NO: 32, and VL containing an amino acid sequence that is the same as or has at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35.

[0031] In the present disclosure, "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% (order) 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% (order) identity.

[0032] In some embodiments, the aforementioned TRGV9 binding protein comprises or is equivalent to an anti-TRGV9 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-TRGV9 antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof. In some embodiments, the anti-TRGV9 antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody (e.g., a trispecific antibody).

[0033] Here, where the aforementioned TRGV9 binding protein includes an immunoglobulin single variable domain, it may 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. As an example, the aforementioned TRGV9 binding protein itself is a single-domain antibody or VHH. In some specific embodiments, the antibody or its antigen-binding fragment is a linear antibody, a short-chain antibody, a nanobody, a peptibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem D-scFv, or a tandem tri-scFv.

[0034] Here, where VH and VL are included in the aforementioned TRGV9 binding protein, it may be a mouse-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antigen-binding fragment includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibody, short-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, diabody, triabody, and tetrabody, serial D-scFv, and serial tri-scFv. In some specific embodiments, the antigen-binding fragment includes Fab, Fv, sFv, Fab', and F(ab')2.

[0035] In some embodiments, the aforementioned TRGV9 binding protein may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin monovariable domains. The immunoglobulin monovariable domains may form dimers or polymer molecules. The immunoglobulin monovariable domains may be homodimers or heterodimers.

[0036] In some embodiments, the aforementioned TRGV9 binding protein may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned VH, VL, or combinations thereof.

[0037] In some embodiments, the aforementioned TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, said Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, said Fc region is the Fc region of human IgG1, for example, as presented in SEQ ID NO: 51 or having at least 80% to 90% sequence identity with it. In some specific embodiments, said Fc region may be an Fc region with reduced effector function, for example, said Fc region may have mutations, and exemplary IgG Fc regions with reduced effector function have the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); Includes H268Q / V309L / A330S / A331S(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) or S267E / L328F(IgG1). For example, L234A / L235A indicates that the sequence includes L234A and L235A.

[0038] In some embodiments, the immunoglobulin single variable domain or the VH, VL, and Fc regions of the aforementioned TRGV9 binding protein are connected directly or via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structures. For example, the linker is (G m S n ) h or (G m Q n ) h or (GGNGT) h(SEQ ID NO: 62) or (YGNGT) h (SEQ ID NO: 63) or (EPKSS) h (SEQ ID NO: 64) or (A m S n ) h As presented in [ ], where m and n are each independently selected from integers 1 to 8, and h is independently selected from integers 1 to 20. For example, 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.;

[0039] In some embodiments, the above-mentioned TRGV9 binding protein is: a protein that specifically binds to γδTCR, or an anti-γδTCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to the γ9 chain of TCR, or an antibody against the γ9 chain of TCR or an antigen-binding fragment thereof; a protein that specifically binds to the variable region (Vγ9) of the γ9 chain of TCR, or an antibody against Vγ9 TCR or an antigen-binding fragment thereof; a protein that specifically binds to γ9δ2 TCR, or an anti-γ9δ2 TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to γ9δ1 TCR, or an anti-γ9δ1 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.

[0040] In some embodiments, the aforementioned TRGV9 binding protein is:

[0041] 1) An amino acid sequence as presented in SEQ ID NO: 36 or having at least 80% to 90% sequence identity with respect to it;

[0042] 2) It includes a heavy chain that is as presented in SEQ ID NO: 37 or has at least 80% to 90% sequence identity therewith, and a light chain that is as presented in any one of SEQ ID NO: 38 or has at least 80% to 90% sequence identity therewith.

[0043] In some embodiments, the above-described TRGV9 binding protein has a function or property selected from at least one of the following:

[0044] (a) EC of ≤10 nM 50 It binds to γ9δ2 T cells, and the above EC 50 is, for example, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. The above EC 50 It is obtained through FACS detection, and FACS is an affinity detection method commonly used in the art, as described, for example, in Example 5 of the present disclosure.

[0045] (b) specifically binds to the γ9 chain of the TCR, for example, specifically binds to γ9δ1 TCR, γ9δ2 TCR;

[0046] (c) does not bind to the γ8 chain of the TCR (not detected by test methods), e.g., does not bind to the γ8δ2 TCR;

[0047] (d) specifically binds to the variable region (TRGV9) of the γ9 chain of the TCR;

[0048] (e) specifically binds to VγδCαβ chimeric TCRs and does not bind to VαβCγδ chimeric TCRs;

[0049] (f) Synomolgus binds to the γ9δ2 TCR of monkeys;

[0050] Here, (b) to (e) can be determined through general detection methods in the art, for example, as described in Example 4 of the present disclosure.

[0051] In some embodiments, the aforementioned protein binds to TRGV9 K D The value is ≤1×10 -7 It can be M, for example ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 It is M.

[0052] In some embodiments, the aforementioned TRGV9 binding protein comprises variants of the immunoglobulin single variable domain, said variants having one or more amino acid mutations compared to any one of SEQ ID NO: 5, 26–29. In some embodiments, the aforementioned TRGV9 binding protein comprises variants of VH and / or VL, said variants of VH having one or more amino acid mutations compared to any one of SEQ ID NO: 9, 30–32, and said variants of VL having one or more amino acid mutations compared to any one of SEQ ID NO: 10, 33–35. “Plural” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. said amino acid mutations may be conservative substitutions, substitutions or modifications, and / or deletions or additions that do not affect function; said amino acid mutations may occur in the CDR region and / or FR region.

[0053] In some embodiments, the present disclosure provides a protein that binds to an immunoglobulin single variable domain of the TRGV9 binding protein of the present disclosure described above or competitively binds to the same antigen epitope.

[0054] In some embodiments, the present disclosure provides a protein that binds to VH and VL among the TRGV9 binding proteins of the present disclosure described above, or competitively binds to the same antigen epitope.

[0055] In some embodiments, the present invention provides a protein in which binding with TRGV9 is blocked by an immunoglobulin single variable domain or VH and VL of the TRGV9 binding protein of the present disclosure described above.

[0056] In some embodiments, the present invention provides a protein or molecule comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of any of the above-described immunoglobulin monovariable domains, for example, said immunoglobulin monovariable domains include CDR1, CDR2, and CDR3 presented in SEQ ID NO: 6–8, or include a sequence presented in any one of SEQ ID NO: 5, 26–29. The protein or molecule may be a conjugate or fusion protein formed with another compound or another polypeptide, said conjugate may include, for example, any detectable marker.

[0057] In some embodiments, a protein or molecule is provided comprising any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the above-described VH and VL of the present disclosure, for example, the VH comprises HCDR1, HCDR2, and HCDR3 as presented in SEQ ID NO: 11–13, and the VL comprises LCDR1, LCDR2, and LCDR3 of amino acid sequences as presented in SEQ ID NO: 14–16, respectively. The protein or molecule may be a conjugate, conjugate, or fusion protein formed with another compound or another polypeptide. For example, the conjugate may comprise any detectable marker.

[0058] GPC3 / TRGV9 binding protein

[0059] The present disclosure provides a protein that binds to GPC3 and TCR. In some embodiments, a protein that binds to GPC3 and γδTCR. In some embodiments, a protein that binds to the γ9 chain of GPC3 and TCR. In some embodiments, a protein that binds to the variable region (Vγ9) of the γ9 chain of GPC3 and TCR. In some embodiments, a protein that binds to GPC3 and γ9δ2 TCR. In some embodiments, a protein that binds to GPC3 and γ9δ1 TCR. In some embodiments, a protein that binds to GPC3 and TRGV9. In some embodiments, the "binding" is simultaneous or sequential binding.

[0060] The present disclosure provides a binding protein that binds to a tumor-associated antigen (TAA), a tumor-specific antigen, and TRGV9, comprising a binding domain that binds to the TAA (or tumor-specific antigen) and a binding domain that binds to TRGV9. In some embodiments, it comprises a first antigen-binding domain that specifically binds to the 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 TRGV9 binding protein of the present disclosure described above. In some embodiments, the second antigen-binding domain that specifically binds to TRGV9 is an immunoglobulin single variable domain of the TRGV9 binding protein of the present disclosure described above, and / or a combination of VH and VL.

[0061] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising a first antigen-binding domain that specifically binds to GPC3 and a second antigen-binding domain that specifically binds to TRGV9.

[0062] In some specific embodiments, the first antigen binding domain comprises a heavy chain variable region (VH1) and / or a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence presented in SEQ ID NO: 39, and VL1 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence presented in SEQ ID NO: 40. In some specific embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 among the amino acid sequences presented in SEQ ID NO: 39, and VL1 comprises LCDR1, LCDR2, and LCDR3 among the amino acid sequences presented in SEQ ID NO: 40, and CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, defined according to the Kabat numbering system.

[0063] In some specific embodiments, the first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 each comprise an amino acid sequence as presented in SEQ ID NO: 41 to 43; and VL1 comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 each comprise an amino acid sequence as presented in SEQ ID NO: 44 to 46.

[0064] In some specific embodiments, the first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as presented in SEQ ID NO: 41–43, respectively; and VL1 comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as presented in SEQ ID NO: 44–46, respectively.

[0065] In some specific embodiments, the amino acid sequence of the first antigen-binding domain comprises any one or more sequences selected from SEQ ID NO: 41 to 46.

[0066] In some specific embodiments, the VH1 and / or VL1 are modified by humanization, reverse mutation, affinity maturation, removal / reduction of T cell epitopes (TCEs), reduction of antibody deamidation and / or reduction of antibody isomerization.

[0067] In some specific embodiments, the VH1 comprises an amino acid sequence that is the same as or has at least 80% to 90% sequence identity with SEQ ID NO: 39, and / or the VL1 comprises an amino acid sequence that is the same as or has at least 80% to 90% sequence identity with SEQ ID NO: 40.

[0068] In some specific embodiments, the amino acid sequence of VH1 is as presented in SEQ ID NO: 39, and the amino acid sequence of VL1 is as presented in SEQ ID NO: 40.

[0069] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising a first antigen-binding domain specifically binding to any of the aforementioned GPC3 and a second antigen-binding domain specifically binding to TRGV9, wherein the second antigen-binding domain specifically binding to TRGV9 is:

[0070] 1) An immunoglobulin monovariable domain comprising CDR1, CDR2, and / or CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29, e.g., an immunoglobulin monovariable domain comprising CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29; or,

[0071] 2) comprising a heavy chain variable region (VH2) including HCDR1, HCDR2, and HCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 9, 30–32, and / or a light chain variable region (VL2) including LCDR1, LCDR2, and LCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 10, 33–35, and

[0072] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, according to the Kabat numbering system.

[0073] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising a first antigen-binding domain specifically binding to any of the aforementioned GPC3 and a second antigen-binding domain specifically binding to TRGV9, wherein the second antigen-binding domain specifically binding to TRGV9 is:

[0074] 1) an immunoglobulin monovariable domain comprising CDR1, CDR2 and / or CDR3, wherein CDR1 comprises the amino acid sequence presented in SEQ ID NO: 6, CDR2 comprises the amino acid sequence presented in SEQ ID NO: 7, and CDR3 comprises the amino acid sequence presented in SEQ ID NO: 8; or,

[0075] 2) VH2 comprising HCDR1, HCDR2 and / or HCDR3, wherein HCDR1 comprises the amino acid sequence presented in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence presented in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence presented in SEQ ID NO: 13; and / or VL comprising LCDR1, LCDR2 and / or LCDR3, wherein LCDR1 comprises the amino acid sequence presented in SEQ ID NO: 14, LCDR2 comprises the amino acid sequence presented in SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence presented in SEQ ID NO: 16.

[0076] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising a first antigen-binding domain specifically binding to any of the aforementioned GPC3 and a second antigen-binding domain specifically binding to TRGV9, wherein the second antigen-binding domain specifically binding to TRGV9 is:

[0077] 1) an immunoglobulin monovariable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as presented in SEQ ID NO: 6–8, respectively; and / or,

[0078] 2) VH2 comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as presented in SEQ ID NO: 11~13, respectively, and VL2 comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as presented in SEQ ID NO: 14~16, respectively.

[0079] In some embodiments, the aforementioned immunoglobulin single variable domain or VH, VL is modified by humanization, reverse mutation, affinity maturation, removal / reduction of T cell epitopes (TCE), reduction of antibody deamidation and / or reduction of antibody isomerization.

[0080] In some embodiments, the heavy chain framework region of the human germline template used in the 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 modification process for VH is derived from IGHV3-21, and the light chain framework region of the human germline template used in the humanization modification process for VL is derived from IGKV1-12.

[0081] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising a first antigen-binding domain specifically binding to any of the aforementioned GPC3 and a second antigen-binding domain specifically binding to TRGV9, wherein the second antigen-binding domain specifically binding to TRGV9 is:

[0082] 1) An immunoglobulin monovariable domain comprising an amino acid sequence that is the same as or has at least 80% or at least 90% identity with any one of SEQ ID NOs: 5, 26–29; or,

[0083] 2) VH2 comprising an amino acid sequence identical to or having at least 80% and at least 90% identity with any one of SEQ ID NO: 9, 30~32, and / or VL2 comprising an amino acid sequence identical to or having at least 80% and at least 90% identity with any one of SEQ ID NO: 10, 33~35.

[0084] In some specific embodiments, the combination of VH2 and VL2 is:

[0085] 2-1) VH2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 9, VL2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 10,

[0086] 2-2) VH2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 30, and VL2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0087] 2-3) VH2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 31, and VL2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0088] 2-4) Selected from VH2 containing an amino acid sequence that is the same as or has at least 80% to 90% identity with SEQ ID NO: 32, and VL2 containing an amino acid sequence that is the same as or has at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35.

[0089] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises or is equivalent to an anti-GPC3 / TRGV9 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-GPC3 / TRGV9 antibody or its antigen-binding fragment is a recombinant antibody or a fragment thereof. In some embodiments, the anti-GPC3 / TRGV9 antibody is a bispecific antibody or a multispecific antibody (e.g., a trispecific antibody). In some embodiments, the immunoglobulin monovariable domain in the anti-GPC3 / TRGV9 antibody or its antigen-binding fragment is a monodomain antibody or VHH. In some specific embodiments, the antigen-binding fragment comprises, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibody, short-chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody, domain antibody, diabody, triabody and tetrabody, serial D-scFv, serial Tri-scFv.

[0090] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin monovariable domains. In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) combinations of the aforementioned 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) combinations of the aforementioned VH2 and VL2.

[0091] In some embodiments, 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.

[0092] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, said Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, said Fc region is the Fc region of human IgG1, for example, as presented in SEQ ID NO: 51 or having at least 80% to 90% sequence identity with it. In some specific embodiments, said Fc region may be an Fc region with reduced effector function, for example, said Fc region may have mutations, and exemplary IgG Fc regions with reduced effector function have the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); Includes having H268Q / V309L / A330S / A331S(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) or S267E / L328F(IgG1).

[0093] In some specific embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2). In some specific embodiments, a mutation is introduced that causes two subunits (Fc1, Fc2) of the Fc region to pair up to form a dimer, or a mutation that reduces homomerization. In some specific embodiments, the first subunit and the second subunit contain a knob-into-hole mutation. For example, within a CH3 / CH3 interface, one, two, or more amino acid residues of the CH3 domain of Fc1 are mutated into one or more amino acid residues having a larger side chain volume to create a protrusion (or knob) on the surface of the CH3 domain of Fc1. Correspondingly, one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated into amino acid residues having a smaller side chain volume, thereby creating an indentation (or hole) on the surface of the CH3 domain of Fc2. Since the first subunit (Fc1) and the second subunit (Fc2) are merely for distinguishing two different subunits, the two may be interchangeable.

[0094] In some specific embodiments, Fc1 contains one or more amino acid substitutions selected from sites 354, 356, 358, and 366, and Fc2 contains one or more amino acid substitutions selected from sites 349, 356, 358, 366, 368, and 407. In some specific embodiments, Fc1 contains a mutation at position 366, and Fc2 contains a mutation selected from positions 366, 368, and 407, or any combination thereof. In some specific embodiments, Fc1 contains a mutation at position 354 or 356, and Fc2 contains a mutation at position 349. In some specific embodiments, Fc1 contains a mutation at position 354 or 356, and Fc2 contains mutations at positions 349, 366, 368, and 407.

[0095] In some specific embodiments, Fc1 contains one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and Fc2 contains one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some specific embodiments, Fc1 contains a 366W mutation, and Fc2 contains a mutation selected from 366S, 368A, and 407V, or any combination thereof. In some specific embodiments, Fc1 contains a 354C or 356C mutation, and Fc2 contains a 349C mutation. Or in some specific embodiments, Fc1 contains a 354C / 366W mutation, and Fc2 contains a 349C / 366S / 368A / 407V mutation.

[0096] In some specific embodiments, Fc1 contains a T366W mutation and Fc2 contains a mutation selected from T366S, L368A, and Y407V or any combination thereof; Fc1 contains an S354C or E356C mutation and Fc2 contains a Y349C mutation; or Fc1 contains an S354C / T366W mutation and Fc2 contains a Y349C / T366S / L368A / Y407V mutation. In some specific embodiments, the amino acid sequence of Fc1 is as presented in SEQ ID NO: 52 and the amino acid sequence of Fc2 is as presented in SEQ ID NO: 53.

[0097] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein includes mutating the size and charge of amino acids at the interface between the heavy chain CH1 and the light chain CL to reduce the mismatch between the light chain and the heavy chain. For example, Roche created the CrossMab platform by exchanging CH1 and CL domains (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187-11192(2011)), MedImmune introduced disulfide bonds to the mutant heavy chain F126C and light chain S121C (Mazor et al., mAbs, 7(2), pp.377-389(2015)), Amgen further modified the CH1-CL region through electrostatic action (Liu et al., Journal of Biological Chemistry, 290(12), pp.7535-7562(2015)), Lilly (Lewis et al., Nature Biotechnology, 32(2), pp.191-198(2014)) and Genentech (Dillon et al., mAbs, 9(2), pp.213-230 (2017)) introduced mutations into both the variable domain and the constant domain. WuXi Biologics replaced the constant domain of the antibody with the constant domain of the TCR as described in CN109535257A (included by citation in the full text).

[0098] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises an Obscurin-O chain (as presented in SEQ ID NO: 47) and a Titin-T chain (as presented in SEQ ID NO: 48) to prevent or reduce mismatch between different VH and VL.

[0099] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein includes a linker.

[0100] In some embodiments, the immunoglobulin single variable domain or the VH, VL, and Fc regions of the aforementioned GPC3 / TRGV9 binding protein are connected directly or via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and lacking secondary or higher structures. For example, the linker is (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h or (A m S n ) h As presented in [the example], where m and n are each independently selected from integers 1 to 8, and h is independently selected from integers 1 to 20. For example, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, A3S, etc.

[0101] In some embodiments, the GPC3 / TRGV9 binding protein comprises a combination selected from the following:

[0102] 1) First heavy chain, second heavy chain and light chain, where,

[0103] In the first heavy chain, from the N-terminus to the C-terminus, it is sequentially [immunoglobulin monovariable domain]-[linker 1]-[Fc1], and

[0104] In the second heavy chain, from stage N to stage C, sequentially [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc2], and

[0105] In the light chain, from the N-stage to the C-stage, it is sequentially [VL1]-[linker 4]-[CL];

[0106] or

[0107] In the first heavy chain, from the N-terminus to the C-terminus, it is sequentially [immunoglobulin monovariable domain]-[linker 1]-[Fc2], and

[0108] In the second heavy chain, from stage N to stage C, sequentially [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc1], and

[0109] In the light chain, from the N-stage to the C-stage, it is sequentially [VL1]-[linker 4]-[CL];

[0110] Here, - indicates a peptide bond, and the linkers 1, 2, 3, and 4 may be the same or different, may exist independently or may not exist, and may be independently selected from the linkers of the present disclosure described above;

[0111] In some specific embodiments, linker 1 is AAAS, and linker 2, linker 3, and linker 4 do not exist.

[0112] 2) First heavy chain, first light chain, second heavy chain and second light chain, where,

[0113] In the first heavy chain, from the N stage to the C stage, sequentially [VH2]-[linker 1]-[Obscurin-O chain]-[linker 2]-[Fc1], and

[0114] In the first light chain, from the N-stage to the C-stage, it is sequentially [VL2]-[linker 3]-[Titin-T chain], and

[0115] In the second heavy chain, from stage N to stage C, sequentially [VH1]-[Linker 4]-[CH1]-[Linker 5]-[Fc2], and

[0116] In the second light chain, sequentially from N to C is [VL1]-[linker 6]-[CL];

[0117] or,

[0118] In the first heavy chain, from the N stage to the C stage, sequentially [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], and

[0119] In the first light chain, from the N-stage to the C-stage, it is sequentially [VL1]-[linker 3]-[Titin-T chain], and

[0120] In the second heavy chain, from the N stage to the C stage, sequentially [VH2]-[Linker 4]-[CH1]-[Linker 5]-[Fc2], and

[0121] In the second light chain, from the N stage to the C stage, sequentially [VL2]-[linker 6]-[CL];

[0122] Here, - indicates a peptide bond, and the linkers 1, 2, 3, 4, 5, and 6 may be the same or different, may exist independently or may not exist, and may be independently selected from the linkers of the present disclosure described above;

[0123] In some specific embodiments, linker 1 and linker 3 are GGGGS, and linker 2, linker 4, linker 5, and linker 6 do not exist.

[0124] 3) First heavy chain, first light chain, second heavy chain and second light chain, where,

[0125] In the first heavy chain, from the N stage to the C stage, sequentially [VH2]-[linker 1]-[Obscurin-O chain]-[linker 2]-[Fc1], and

[0126] In the first light chain, from the N-stage to the C-stage, it is sequentially [VL2]-[linker 3]-[Titin-T chain], and

[0127] In the second heavy chain, from stage N to stage C, sequentially [VH1]-[Linker 4]-[CH1]-[Linker 5]-[VH1]-[Linker 6]-[CH1]-[Linker 7]-[Fc2], and

[0128] In the second light chain, sequentially from N to C is [VL1]-[linker 8]-[CL];

[0129] or,

[0130] In the first heavy chain, from the N stage to the C stage, sequentially [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], and

[0131] In the first light chain, from the N-stage to the C-stage, it is sequentially [VL1]-[linker 3]-[Titin-T chain], and

[0132] In the second heavy chain, from stage N to stage C, sequentially [VH2]-[Linker 4]-[CH1]-[Linker 5]-[VH2]-[Linker 6]-[CH1]-[Linker 7]-[Fc2], and

[0133] In the second light chain, from the N stage to the C stage, sequentially [VL2]-[linker 8]-[CL];

[0134] Here, - indicates a peptide bond, and the linkers 1, 2, 3, 4, 5, 6, 7, and 8 may be the same or different, may exist independently or may not exist, and may be independently selected from the linkers of the present disclosure described above;

[0135] In some specific embodiments, Linker 1 and Linker 3 are GGGGS, Linker 5 is GGGGSGGGGS, and Linker 2, Linker 4, Linker 6, Linker 7, and Linker 8 do not exist;

[0136] In some specific embodiments, the molar ratio of the first heavy chain:first light chain:second heavy chain:second light chain is 1:1:1:2.

[0137] In some specific embodiments, the amino acid sequences of the Obscurin-O chain and the Titin-T chain are as presented in SEQ ID NO: 47, 48, respectively. In some specific embodiments, CL is Cκ, and the amino acid sequences of CH1 and Cκ are as presented in SEQ ID NO: 49, 50, respectively. In some specific embodiments, the amino acid sequences of Fc1 and Fc2 are as presented in SEQ ID NO: 52, 53, respectively.

[0138] In some embodiments, the provided GPC3 / TRGV9 binding protein is:

[0139] 1) comprising a first heavy chain having an amino acid sequence identical to that presented in SEQ ID NO: 54 or having at least 80% to 90% identity therewith, a second heavy chain having an amino acid sequence identical to that presented in SEQ ID NO: 55 or having at least 80% to 90% identity therewith, and a light chain having an amino acid sequence identical to that presented in SEQ ID NO: 56 or having at least 80% to 90% identity therewith;

[0140] 2) comprising a first heavy chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 57, a first light chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 58, a second heavy chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 59, and a second light chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 56; or,

[0141] 3) The amino acid sequence is selected from a first heavy chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 57, a first light chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 58, a second heavy chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 60, and a second light chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 61.

[0142] In some embodiments, the provided GPC3 / TRGV9 binding protein is:

[0143] 1) The amino acid sequence comprises polypeptide combinations as presented in SEQ ID NO: 54–56; in some embodiments, the molar ratio of the polypeptide presented in SEQ ID NO: 54: polypeptide presented in SEQ ID NO: 55: polypeptide presented in SEQ ID NO: 56 in the GPC3 / TRGV9 binding protein is 1:1:1;

[0144] 2) The amino acid sequence comprises polypeptide combinations as presented in SEQ ID NO: 56–59; in some embodiments, the molar ratio of the polypeptide presented in SEQ ID NO: 56: polypeptide presented in SEQ ID NO: 57: polypeptide presented in SEQ ID NO: 58: polypeptide presented in SEQ ID NO: 59 in the GPC3 / TRGV9 binding protein is 1:1:1:1;

[0145] 3) The amino acid sequence comprises polypeptide combinations as presented in SEQ ID NO: 57, 58, 60, and 61; in some embodiments, the molar ratio of polypeptide presented in SEQ ID NO: 57: polypeptide presented in SEQ ID NO: 58: polypeptide presented in SEQ ID NO: 60: polypeptide presented in SEQ ID NO: 61 among the GPC3 / TRGV9 binding proteins is selected from 1:1:1:2.

[0146] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein is: a protein that specifically binds to GPC3 and γδTCR, or an anti-GPC3 / γδTCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to the γ9 chain of GPC3 and TCR, or an anti-GPC3 / TCR antibody against the γ9 chain or an antigen-binding fragment thereof; a protein that specifically binds to the variable region (Vγ9) of the γ9 chain of GPC3 and TCR, or an anti-GPC3 / Vγ9 TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and γ9δ2 TCR, or an anti-GPC3 / γ9δ2 TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and γ9δ1 TCR, or an anti-GPC3 / γ9δ1 TCR antibody or an antigen-binding fragment thereof; It is a protein that specifically binds to GPC3 and TRGV9, or an anti-GPC3 / TRGV9 antibody or its antigen-binding fragment.

[0147] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein has a function or property selected from at least one of the following:

[0148] (a) EC of ≤10 nM 50 It binds to γ9δ2 T cells, and the above EC 50 is, for example, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. The above EC 50 It is obtained through FACS detection, and FACS is an affinity detection method commonly used in the art, as described, for example, in Example 5 of the present disclosure;

[0149] (b) specifically binds to the γ9 chain of the TCR, e.g., specifically binds to the γ9δ1 TCR and specifically binds to the γ9δ2 TCR;

[0150] (c) does not bind to the γ8 chain of the TCR, e.g., does not bind to the γ8δ2 TCR;

[0151] (d) specifically binds to the variable region (TRGV9) of the γ9 chain of the TCR;

[0152] (e) specifically binds to VγδCαβ chimeric TCRs and does not bind to VαβCγδ chimeric TCRs;

[0153] Here, (b) to (e) can be determined through general detection methods in the art, for example as described in Example 4 of the present disclosure;

[0154] (f) specifically binding to GPC3 protein or GPC3-positive cells, and not binding to or hardly binding to GPC3-negative cells (e.g., PBMCs), using, for example, the detection method of Example 8 of the present disclosure;

[0155] (g) Synomolgus binds to monkey GPC3 and γ9δ2 proteins;

[0156] (h) does not block the natural BTN2A / BTN3A-TCR signal, and, for example, uses the detection method of Example 9 of the present disclosure;

[0157] (i) mediating the death of γδ T cells in GPC3-positive tumor cells or mediating the death of γδ T cells in GPC3-positive tumor cells in a manner dependent on the GPC3 expression level, for example, using the detection method of Example 10 of the present disclosure; mediating the death of γδ T cells in GPC3-positive tumor cells in a manner dependent on the effector-target-ratio, for example, using the detection method of Example 12 of the present disclosure; mediating the death of γδ T cells in GPC3-positive tumor cells in a manner of low individual variability, for example, using the detection method of Example 11 of the present disclosure;

[0158] (j) Inducing the release of fewer cytokines (e.g., IFNγ, TNFα) during the process of γδ T cell death against GPC3-positive tumor cells, using, for example, the detection method of Example 13 of the present disclosure;

[0159] (k) Promotes γδ T cell proliferation in PBMCs, using, for example, the detection method of Example 14 of the present disclosure;

[0160] (l) inhibiting the proliferation of GPC3-positive tumor cells in vivo, and / or inhibiting the growth of GPC3-positive tumors, and / or eliminating GPC3-positive tumors; the mouse in vivo model is the same as the detection method of Example 15 of the present disclosure.

[0161] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein binds to TRGV9 K D The value is ≤1×10 -7 It can be M, for example ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 It is M.

[0162] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein binds to K of GPC3. D The value is ≤1×10 -7 It can be M, for example ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 It is M.

[0163] In some embodiments, the immunoglobulin single variable domain of the aforementioned GPC3 / TRGV9 binding protein comprises variants, said variants having one or more amino acid mutations compared to any one of SEQ ID NO: 5, 26–29. In some embodiments, VH1 and / or VL1 of the aforementioned GPC3 / TRGV9 binding protein comprise variants, said variants of VH1 having one or more amino acid mutations compared to SEQ ID NO: 39, and said variants of VL having one or more amino acid mutations compared to SEQ ID NO: 40. In some embodiments, VH2 and / or VL2 of the aforementioned GPC3 / TRGV9 binding protein comprise variants, said variants of VH2 having one or more amino acid mutations compared to any one of 9, 30–32, and said variants of VL having one or more amino acid mutations compared to any one of SEQ ID NO: 10, 33–35. "Plural" is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The amino acid mutations may be conservative substitutions, replacements, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0164] In some embodiments, the present invention provides a protein that binds to the GPC3 / TRGV9 binding protein of the aforementioned disclosure or competitively binds to the same TRGV9 and / or GPC3 antigen epitope.

[0165] In some embodiments, a protein is provided in which binding with TRGV9 and / or GPC3 is blocked by the GPC3 / TRGV9 binding protein of the present disclosure described above.

[0166] In some embodiments, a protein or molecule comprising any of the GPC3 / TRGV9 binding proteins of the foregoing disclosure is provided. The protein or molecule may be a conjugate formed with another compound or another polypeptide, a conjugate, or a fusion protein. For example, the conjugate may include any detectable marker.

[0167] Polynucleotides and Vectors

[0168] The present disclosure provides a polynucleotide encoding the TRGV9 binding protein of the present disclosure and the GPC3 / TRGV9 binding protein. 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 substantially isolated nucleic acid.

[0169] The nucleic acid of the present disclosure may also be in the form of a vector, may be present within a vector and / or be part of a vector, such vector being, for example, a plasmid, cosmid, YAC, or viral vector. The vector may be, in particular, an expression vector, that is, a vector that provides expression of TRGV9 binding protein, GPC3 / TRGV9 binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression regulatory elements (e.g., promoter, amplifier, terminator, etc.). It is common knowledge of those skilled in the art to select said elements and their sequences for expression in a particular host. Regulatory elements and other elements useful or necessary for the expression of the TRGV9 binding protein, GPC3 / TRGV9 binding protein of the present disclosure are, for example, promoters, amplifiers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0170] The nucleic acids of the present disclosure may be prepared or obtained by a known method (e.g., automated DNA synthesis and / or recombinant DNA technology) based on information regarding the amino acid sequence of the polypeptide of the present disclosure and / or isolated from a suitable natural source.

[0171] In some embodiments, a polynucleotide encoding a TRGV9 binding protein is provided, said TRGV9 binding protein is:

[0172] 1) An immunoglobulin monovariable domain comprising CDR1, CDR2, and / or CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29, e.g., an immunoglobulin monovariable domain comprising CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29; and / or,

[0173] 2) comprising a heavy chain variable region (VH) containing HCDR1, HCDR2 and / or HCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 9, 30–32, and / or a light chain variable region (VL) containing LCDR1, LCDR2 and / or LCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 10, 33–35, and

[0174] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, for example, according to the Kabat numbering system.

[0175] In some embodiments, a polynucleotide encoding a TRGV9 binding protein is provided, said TRGV9 binding protein is:

[0176] 1) an immunoglobulin monovariable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as presented in SEQ ID NO: 6–8, respectively; and / or,

[0177] 2) VH comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as presented in SEQ ID NO: 11~13, respectively, and VL comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as presented in SEQ ID NO: 14~16, respectively.

[0178] In some embodiments, a polynucleotide encoding a TRGV9 binding protein is provided, said TRGV9 binding protein comprising: 1) an immunoglobulin monovariable domain comprising an amino acid sequence that is as presented in any one of SEQ ID NOs: 5, 26–29 or has at least 80% to 90% identity therewith; and / or,

[0179] 2) VH comprising an amino acid sequence identical to or having at least 80% and at least 90% identity with any one of SEQ ID NO: 9, 30~33, and / or VL comprising an amino acid sequence identical to or having at least 80% and at least 90% identity with any one of SEQ ID NO: 10, 33~35.

[0180] In some specific embodiments, a polynucleotide encoding a TRGV9 binding protein is provided, said TRGV9 binding protein having the following VH and VL:

[0181] 2-1) VH comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 9, VL comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 10,

[0182] 2-2) VH comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 30, and VL comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0183] 2-3) VH comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 31, and VL comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0184] 2-4) Includes VH containing an amino acid sequence that is the same as or has at least 80% to 90% identity with SEQ ID NO: 32, and VL containing an amino acid sequence that is the same as or has at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35.

[0185] In some specific embodiments, a polynucleotide encoding a TRGV9 binding protein is provided, said TRGV9 binding protein is:

[0186] 1) An amino acid sequence as presented in SEQ ID NO: 36 or having at least 80% to 90% sequence identity with respect to it;

[0187] 2) It includes a heavy chain that is as presented in SEQ ID NO: 37 or has at least 80% to 90% sequence identity therewith, and a light chain that is as presented in any one of SEQ ID NO: 38 or has at least 80% to 90% sequence identity therewith.

[0188] In some embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, said 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.

[0189] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein the first antigen-binding domain described above comprises a heavy chain variable region (VH1) and / or a light chain variable region (VL1), said VH1 comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence presented in SEQ ID NO: 39, and said VL1 comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence presented in SEQ ID NO: 40.

[0190] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein the first antigen-binding domain described above comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as presented in SEQ ID NO: 41–43, respectively; and VL1 comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as presented in SEQ ID NO: 44–46, respectively.

[0191] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein the aforementioned first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), said VH1 comprises an amino acid sequence that is as presented in SEQ ID NO: 39 or has at least 80% to 90% sequence identity therewith, and / or VL1 comprises an amino acid sequence that is as presented in SEQ ID NO: 40 or has at least 80% to 90% sequence identity therewith.

[0192] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein a second antigen-binding domain that specifically binds to the aforementioned TRGV9 is:

[0193] 1) An immunoglobulin monovariable domain comprising CDR1, CDR2, and / or CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29, e.g., an immunoglobulin monovariable domain comprising CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 5, 26–29; or,

[0194] 2) It includes a heavy chain variable region (VH2) comprising HCDR1, HCDR2, and HCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 9, 30~32, and / or a light chain variable region (VL2) comprising LCDR1, LCDR2, and LCDR3 in the amino acid sequence presented in any one of SEQ ID NO: 10, 33~35.

[0195] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein a second antigen-binding domain that specifically binds to the aforementioned TRGV9 is:

[0196] 1) an immunoglobulin monovariable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as presented in SEQ ID NO: 6–8, respectively; and / or,

[0197] 2) VH2 comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as presented in SEQ ID NO: 11~13, respectively, and VL2 comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as presented in SEQ ID NO: 14~16, respectively.

[0198] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein a second antigen-binding domain that specifically binds to the aforementioned TRGV9 is:

[0199] 1) An immunoglobulin monovariable domain comprising an amino acid sequence that is the same as or has at least 80% or at least 90% identity with any one of SEQ ID NOs: 5, 26–29; or,

[0200] 2) VH2 comprising an amino acid sequence identical to or having at least 80% and at least 90% identity with any one of SEQ ID NO: 9, 30~32, and / or VL2 comprising an amino acid sequence identical to or having at least 80% and at least 90% identity with any one of SEQ ID NO: 10, 33~35.

[0201] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, wherein a second antigen-binding domain that specifically binds to the aforementioned TRGV9 is the following VH2 and VL2:

[0202] 2-1) VH2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 9, VL2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 10,

[0203] 2-2) VH2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 30, and VL2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0204] 2-3) VH2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 31, and VL2 comprising an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35;

[0205] 2-4) Includes VH2 having an amino acid sequence that is the same as or has at least 80% to 90% identity with SEQ ID NO: 32, and VL2 having an amino acid sequence that is the same as or has at least 80% to 90% identity with any one of SEQ ID NO: 33 to 35.

[0206] In some specific embodiments, a polynucleotide encoding a GPC3 / TRGV9 binding protein is provided, said GPC3 / TRGV9 binding protein is:

[0207] 1) comprising a first heavy chain having an amino acid sequence identical to that presented in SEQ ID NO: 54 or having at least 80% to 90% identity therewith, a second heavy chain having an amino acid sequence identical to that presented in SEQ ID NO: 55 or having at least 80% to 90% identity therewith, and a light chain having an amino acid sequence identical to that presented in SEQ ID NO: 56 or having at least 80% to 90% identity therewith;

[0208] 2) comprising a first heavy chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 57, a first light chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 58, a second heavy chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 59, and a second light chain having an amino acid sequence identical to or having at least 80% to 90% identity with SEQ ID NO: 56; or,

[0209] 3) The amino acid sequence is selected from a first heavy chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 57, a first light chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 58, a second heavy chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 60, and a second light chain having at least 80% and at least 90% identity as presented in SEQ ID NO: 61.

[0210] host cell

[0211] The present disclosure provides a recombinant host cell that expresses or can express one or more of the TRGV9 binding proteins, GPC3 / TRGV9 binding proteins of the present disclosure and / or contains a polynucleotide or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0212] Bacterial cells are, for example, Gram-negative bacterial strains (e.g., E. coli ( Escherichia coli ) strain, Proteus( Proteus ) strains and Pseudomonas ( Pseudomonas) strains) and Gram-positive bacterial strains (e.g., Bacillus species ( Bacillus ) strain, Streptomyces( Streptomyces ) strain, Staphylococcus (Staphylococcus ) strains and Lactococcus( Lactococcus It includes cells of the strain.

[0213] Fungal cells are, for example, Trichoderma ( Trichoderma ), Neurospora( Neurospora ) and Aspergillus( Aspergillus ) containing cells of the species; or Saccharomyces ( Saccharomyces )(e.g., Saccharomyces cerevisiae( Saccharomyces cerevisiae )), Schizocaromyces( Schizosaccharomyces )(e.g., Schizocaromyces pombe( Schizosaccharomyces pombe )), Peachia( Pichia )(e.g., Piccia Pastoris( Pichia Pastoris ) and Peachia methaneolica( Pichia methanolica )) and Hansenula( Hansenula It includes cells of the species.

[0214] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0215] However, the present disclosure may use amphibian cells, insect cells, plant cells, and any other cells in the art for expressing heterologous proteins.

[0216] Manufacturing method

[0217] The present disclosure provides a method for preparing a TRGV9 binding protein or a GPC3 / TRGV9 binding protein, comprising the steps of expressing said target protein in a host cell as previously described and isolating the target protein from said host cell. Optionally, the method may also include a purification step of, for example, purifying with an A or G Sepharose FF column containing a modified buffer, washing non-specifically bound components, eluting the bound antibody by a pH gradient, and detecting and collecting by SDS-PAGE. Optionally, filtration and concentration are performed by conventional methods. Soluble mixtures and polymers may also be removed by conventional methods such as molecular sieves or ion exchange. The obtained product must be immediately frozen, for example, at -70°C, or freeze-dried.

[0218] Methods for generating and purifying antibodies in the prior art are well known and can be found, for example, Cold Spring’s antibody testing technical guidelines (Chapters 5–8 and Chapter 15).

[0219] The processed antibody or antigen-binding fragment of the present disclosure can be prepared and purified by conventional methods. For example, the cDNA sequence encoding the heavy and light chains can be cloned into an expression vector and recombined. The recombined immunoglobulin expression vector can stably transfect CHO cells. The mammalian expression system results in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. A stable clone is obtained by expressing an antibody that specifically binds to the humanized antigen. The positive clone is extended in serum-free medium in a bioreactor to produce the antibody. The culture medium from which the antibody has been secreted can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods such as molecular sieves and ion exchange.

[0220] composition

[0221] The present disclosure provides a composition comprising the TRGV9 binding protein and GPC3 / TRGV9 binding protein of the present disclosure as described above. For example, a pharmaceutical composition containing an effective amount of the aforementioned TRGV9 binding protein and / or GPC3 / TRGV9 binding protein for the treatment, alleviation, or prevention of disease, and at least one pharmaceutically acceptable excipient, diluent, or adjuvant.

[0222] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99 weight% of TRGV9 binding protein and / or GPC3 / TRGV9 binding protein per unit dose, or the amount of TRGV9 binding protein and / or GPC3 / TRGV9 binding protein per unit dose of the pharmaceutical composition is 0.1 to 2000 mg. In some specific embodiments, it is 1 to 1000 mg.

[0223] In some embodiments, a manufactured product or product (e.g., a kit) comprising the aforementioned TRGV9 binding protein and / or GPC3 / TRGV9 binding protein is provided. Optionally, the manufactured product comprises a container and a label. The container is, for example, a flask, a syringe, and a test tube. The container contains a pharmaceutical composition effective for treating a disease. The container or a label attached to the container indicates that the pharmaceutical composition is used to treat a selected disease.

[0224] In some embodiments, the aforementioned disease is a proliferative disease or cancer.

[0225] Treatment methods and pharmaceutical uses

[0226] The present disclosure provides a method for treating, alleviating, preventing, or diagnosing a disease or pathology of the aforementioned TRGV9 binding protein, GPC3 / TRGV9 binding protein, its coding polynucleotide, and a composition (including a pharmaceutical composition).

[0227] In some embodiments, a method for improving, alleviating, treating, or preventing a disease is provided, comprising the step of administering an effective amount of the following to a subject:

[0228] 1) The TRGV9 binding protein of the present disclosure or its coding polynucleotide, pharmaceutical composition;

[0229] 2) The GPC3 / TRGV9 binding protein of the present disclosure or its coding polynucleotide, pharmaceutical composition.

[0230] In some embodiments, the uses of 1) to 2) described above are provided in the manufacture of a drug for improving, alleviating, treating, or preventing a disease.

[0231] In some embodiments, a method for treating a disease is provided by using the TRGV9 binding protein of the present disclosure in combination with γδ T cells. In some embodiments, a method for treating a disease is provided by using the GPC3 / TRGV9 binding protein of the present disclosure in combination with γδ T cells. In some embodiments, the γδ T cells are autologous or allogeneic.

[0232] In some embodiments, any of the TRGV9 binding proteins described above of the present disclosure are for the treatment of a disease and include combination with γδ T cells. In some embodiments, γδ T cells are for the treatment of a disease and include combination with any of the TRGV9 binding proteins described above of the present disclosure.

[0233] In some embodiments, any of the aforementioned GPC3 / TRGV9 binding proteins of the present disclosure are for the treatment of a disease and include combination with γδ T cells. In some embodiments, γδ T cells are for the treatment of a disease and include combination with any of the aforementioned GPC3 / TRGV9 binding proteins of the present disclosure.

[0234] In some embodiments, a method for treating a disease is provided, comprising the step of administering a therapeutically effective amount of any of the TRGV9 binding proteins and γδ T cells described above of the present disclosure to a subject who requires it. In some embodiments, a method for treating a disease is provided, comprising the step of administering a therapeutically effective amount of any of the GPC3 / TRGV9 binding proteins and γδ T cells described above of the present disclosure to a subject who requires it.

[0235] In some embodiments, the aforementioned disease is a disease or pathology caused by the overexpression of GPC3.

[0236] In some embodiments, the aforementioned disease is a proliferative disease or cancer.

[0237] In some specific embodiments, it is a GPC3-positive cancer.

[0238] In some embodiments, the aforementioned disease is liver cancer.

[0239] In some specific embodiments, it is GPC3-positive liver cancer.

[0240] Detection

[0241] The present disclosure provides a TRGV9 binding protein, a GPC3 / TRGV9 binding protein, a polynucleotide coding for the same, and a composition for detection. The present disclosure also provides a method, system, or apparatus for detecting GPC3, TRGV9 in vivo or in vitro, comprising treating a sample with the aforementioned binding protein, polynucleotide, or composition of the present disclosure.

[0242] In some embodiments, a kit is also provided that includes the aforementioned TRGV9 binding protein, GPC3 / TRGV9 binding protein, and its coding polynucleotide, and may include a diagnostic instruction manual. The kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the TRGV9 binding protein, GPC3 / TRGV9 binding protein, and its coding polynucleotide may be formulated into a pharmaceutical composition. Brief explanation of the drawing

[0243] Figure 1 is a schematic diagram of the structure of an anti-GPC3 / γδTCR bispecific antibody. Figures 2A to 2D show the results of detecting the FACS binding activity of the anti-GPC3 / γδTCR bispecific antibody with GPC3-positive cells and human γ9δ2 T cells. Here, Figure 2A shows the binding of the anti-GPC3 / γδTCR bispecific antibody in HepG2 cells; Figure 2B shows the binding of the anti-GPC3 / γδTCR bispecific antibody in DLD-1 cells; Figure 2C shows the binding of the anti-GPC3 / γδTCR bispecific antibody in human γδ T cells; and Figure 2D shows the binding of the anti-GPC3 / γδTCR bispecific antibody in PBMCs. Figures 3A and 3B show the effect of anti-γδTCR antibodies on the natural BTN2A / BTN3A-TCR signal. Figure 3A shows the effect of SDP01378 on the natural BTN2A / BTN3A-TCR signal; Figure 3B shows the effect of SDP01315 on the natural BTN2A / BTN3A-TCR signal. Figures 4A to 4D show the results of the apoptotic activity of anti-γδTCR antibodies against tumor cells with different GPC3 expression levels. Here, Figure 4A shows the apoptotic activity against HepG2 cells, Figure 4B shows the apoptotic activity against Huh-7 cells, Figure 4C shows the apoptotic activity against MKN-45 cells, and Figure 4D shows the apoptotic activity against DLD-1 cells. Figures 5A to 5D show the results of the apoptotic activity of different donor-derived γδ T cells against HepG2. Here, the γδ T cells used in Figures 5A to 5D are derived from the induction amplification of donors #SC12004, #SC12392, #XC11053, and #XC11061, respectively. Figures 6A to 6C show the results of killing antigen-low expression tumor cells with anti-GPC3 / γδTCR bispecific antibodies under different effector-target ratios. Here, the effector-target ratio in Figure 6A is 1:1, the effector-target ratio in Figure 6B is 10:1, and the effector-target ratio in Figure 6C is 30:1. Figures 7A to 7D show the results of apoptosis activity after increasing the ratio of γδ T cells in PBMCs. Figure 7A shows the apoptosis of donor #XC11053 PBMC cells; Figure 7B shows the apoptosis activity after incorporating 30% γδ T cells into the PBMC cells of donor #XC11053; Figure 7C shows the apoptosis of donor #SC12392 PBMC cells; and Figure 7D shows the apoptosis activity after incorporating 30% γδ T cells into the PBMC cells of donor #SC12392. Figures 8A to 8D are the results of cytokine release detection. Here, Figures 8A to 8B show the release of IFNγ and TNFα upon death of donor #XC11053, respectively; Figures 8C to 8D show the release of IFNγ and TNFα upon death of donor #SC12392, respectively. Figure 9 shows the detection results of the anti-GPC3 / γδTCR bispecific antibody promoting γδ T cell proliferation in PBMCs. Figures 10A and 10B show the antitumor activity of SDP01716 in a Huh-7 transplanted tumor model, where Figure 10A is a graph of mouse tumor volume change and Figure 10B is a graph of mouse body weight change. Figures 11A and 11B show the antitumor activity of different anti-GPC3 / γδTCR bispecific antibodies in a Huh-7 transplanted tumor model, where Figure 11A is a change in mouse tumor volume and Figure 11B is a change in mouse body weight. Specific details for implementing the invention

[0244] definition

[0245] To facilitate a better understanding of the present disclosure, specific technical and scientific terms are defined below. Unless otherwise explicitly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meaning commonly understood by those skilled in the art to which the present disclosure pertains.

[0246] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0247] "TRGV9" refers to a polypeptide capable of forming a T cell receptor when expressed on the surface of γδ T cells. γδ T cells expressing TRGV9 are one of the first T cells to develop in human fetuses and are the major γδ T cell subgroup in healthy adult peripheral blood cells. "TRGV9" includes any TRGV9 variants, isotypes, and species homologues that may be naturally expressed by cells (including T cells) or expressed in cells transfected with a gene or cDNA encoding said polypeptide. In a specific embodiment, TRGV9 is human TRGV9. An exemplary human TRGV9 amino acid sequence is provided by GenBank registry number NG_001336.2.

[0248] "GPC3" refers to Glypican 3, phosphatidylinositol proteoglycan-3, which is anchored to the cell membrane via phosphatidylinositol and is a marker for hepatocellular carcinoma. An exemplary amino acid sequence of human GPC3 is provided by uniprot registry number P51654.

[0249] "TRGV9 binding protein" encompasses any protein capable of specifically binding to TRGV9, or any molecule comprising said protein, including but not limited to antibodies, antigen-binding fragments thereof, or conjugates and fusion proteins as defined in the present disclosure for TRGV9. In some embodiments, "TRGV9 binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6 or more) single-domain antibodies or VHHs of TRGV9 that specifically bind to TRGV9 in the embodiments of the present disclosure. In some embodiments, "TRGV9 binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6 or more) VH and VL combination of TRGV9 that specifically bind to TRGV9 in the embodiments of the present disclosure. In some embodiments, the “TRGV9 binding protein” of the present disclosure, in addition to comprising the immunoglobulin monovariable domain of TRGV9 or a combination of VH and VL, may also comprise a portion having linker and / or effector functions, for example, a half-life extension portion (e.g., an immunoglobulin monovariable domain 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. In some embodiments, the “TRGV9 binding protein” comprises an anti-γδTCR antibody or an antigen-binding fragment thereof according to an embodiment of the present disclosure.

[0250] "GPC3 / TRGV9 binding protein" encompasses any protein capable of specifically binding to GPC3 and TRGV9, or any molecule comprising said protein, including but not limited to antibodies, polypeptides, fusion proteins of antibodies and polypeptides, or conjugates thereof. In some embodiments, "GPC3 / TRGV9 binding protein" encompasses bispecific antibodies against GPC3 / γδTCR according to embodiments of the present disclosure.

[0251] The term "antibody" encompasses various antibody structures that exhibit desired antigen-binding activity, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions). Antibodies may refer to immunoglobulins, which are tetrapeptide chain structures formed by connecting two heavy chains and two light chains via interchain disulfide bonds. Because the amino acid composition and sequence of the constant regions of immunoglobulin heavy chains differ, their antigenicity also differs. Therefore, immunoglobulins can be classified into five types, or referred to as isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being the μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig can be classified into different subclasses based on differences in the amino acid composition of its hinge region and the number and location of heavy chain disulfide bonds, just as IgG is classified into IgG1, IgG2, IgG3, and IgG4. The light chain is classified as either a κ chain or a λ chain depending on the difference in the invariant region. In all five types of Ig, each type may contain either a κ chain or a λ chain. Approximately 110 amino acid sequences adjacent to the N-terminus of the antibody heavy and light chains constitute a highly variable region (V region); the remaining amino acid sequences adjacent to the C-terminus constitute a relatively stable and invariant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody and are also referred to as complementarity determining regions (CDR). The light chain variable region (VL) and heavy chain variable region (VH) of each strand are composed of three CDR regions and four FR regions, arranged sequentially from the amino group to the carboxyl group in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The three CDR regions of the light chain point to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain point to HCDR1, HCDR2, and HCDR3.

[0252] The antibodies of the present disclosure may be polyclonal, monoclonal, heterogeneous, homologous, homologous, or modified forms thereof, wherein monoclonal antibodies are particularly suitable in various embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" broadly refers to a product such as a cell or nucleic acid, protein, or vector, which indicates that said cell, nucleic acid, protein, or vector has been modified through the introduction of a heterologous nucleic acid or protein or a modification of a natural nucleic acid or protein, or that said cell is derived from a cell thus modified. For example, a recombinant cell expresses a gene that is absent in the natural (non-recombinant) cell form, or expresses a natural gene that was originally abnormally expressed, underexpressed, or not fully expressed.

[0253] Regarding the determination or definition of a CDR, the definitive description of the CDR and the identification of residues containing the antibody's binding site can be accomplished through the structural fractionation of the antibody and / or the antibody-ligand complex. This can be implemented by any various techniques known to those skilled in the art, such as X-ray crystallography. The CDR can be identified using various analytical methods, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, Contact definitions, and structural definitions. The Kabat numbering system is a standard for numbering antibody residues 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 location of specific structural ring 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 to model antibody structures (see, e.g., Martin et al., 1989, ProcNatl 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 a knowledge database and the first-principles method (ab initio method) to model the tertiary structure of antibodies from basic sequences (Samudrala et al., 1999, "Ab" in PROTEINS, Structure, Function and Genetics Suppl.(See the content described in "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in , 3:194-198). Contack definitions are based on the analysis of available complex crystal structures (e.g., see MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In structural definitions, the position of the CDR can be identified as a residue that contributes enthalpy to antigen binding (e.g., see Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Additionally, other CDR boundary definitions may not strictly follow one of the methods described above, but may still overlap with at least some of the Kabat CDRs and may be shortened or extended based on predictions or experimental results that a specific residue or group of residues does not have a significant effect on antigen binding. As used in this disclosure, CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. Correspondences between various numbering systems are well known to those skilled in the art.

[0254] Those skilled in the art understand that even if a specific numbering system (e.g., Kabat) is adopted in a specific embodiment or in a specific SEQ ID NO, those skilled in the art may determine a sequence corresponding to that sequence under a different numbering system, and such sequence is still considered to be within the scope of this disclosure.

[0255] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional characteristic of the protein, and in many cases, it can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain.

[0256] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a general tetrapeptide chain structure antibody or a chain of a heavy chain antibody) or substantially a polypeptide composed of such globular regions. The immunoglobulin domain is characterized by maintaining the immunoglobulin folding characteristics of the antibody molecule.

[0257] The "immunoglobulin variable domain" substantially refers to a domain composed of four "framework regions"—"framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4"—and three "complementary determination regions" or "CDRs"—"complementary determination region 1" or "CDR1", "complementary determination region 2" or "CDR2", and "complementary determination region 3" or "CDR3". The general structure or sequence of the immunoglobulin variable domain may be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 as follows. The immunoglobulin variable domain has an antigen binding site that confers specificity to an antigen.

[0258] "Antibody Framework (FR)" refers to a portion of the variable domain used as a stent of the antigen-binding ring (CDR).

[0259] "Immunoglobulin monovariable domain" is typically used to refer to an immunoglobulin variable domain capable of forming a functional antigen-binding site in the absence of interaction with other variable domains (e.g., in the case of necessary VH / VL interactions between VH and VL domains in the absence of a typical quadruplet-chain monoclonal antibody) (which may be a heavy or light chain domain containing a VH, VHH, or VL domain). Examples of "immunoglobulin monovariable domains" include nanobodies (VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNARs, domains, antibodies derived as or from a VH domain (single domain) (e.g., dAbs TM ) and antibodies (single domain) as or derived from the VL domain (e.g., dAbs TM Includes ). Immunoglobulin monovariable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are typically preferred. One specific example of an immunoglobulin monovariable domain is the "VHH domain" (or abbreviated as "VHH") as defined below.

[0260] "VHH" is a heavy chain single-domain antibody, VHH, V HIt is a variable domain of an antigen-binding immunoglobulin referred to as a "heavy chain antibody" (i.e., "antibody lacking a light chain"), also called the H domain, VHH antibody fragment, VHH antibody, or nanobody (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH" is used to distinguish the variable domain from the heavy chain variable domain (referred to as the "VH domain" or VH in this disclosure) and the light chain variable domain (referred to as the "VL domain" or VL in this disclosure) present in antibodies of a general tetrapeptide chain structure. The VHH domain specifically binds to an epitope without other antigen-binding domains; This binding behavior differs from the VH or VL domains in typical tetrapeptide-chain antibodies, where 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. Terms "heavy-chain single-domain antibody," "VHH domain," "VHH," "V H"H domain," "VHH antibody fragment," "VHH antibody," "Nanobody®," and "Nanobody® domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) may be used interchangeably. VHH comprises, but is not limited to, natural antibodies produced by camelid animals, may also be antibodies produced by humanized camelid animals, or may be obtained through phage display technology screening. The total number of amino acid residues in VHH is typically 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 this disclosure. Methods for obtaining VHH that binds to a specific antigen or epitope have been previously disclosed in the following literature: 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 and WO94 / 04678.

[0261] As is widely known in the art regarding VH domains and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This generally means that 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 rules include Chothia, IMGT, and AbM.

[0262] A "humanized antibody," also referred to as a CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable domain framework. This can overcome the potent immune response induced by chimeric antibodies containing large amounts of non-human protein components. To prevent a decrease in activity from occurring simultaneously with a decrease in immunogenicity, activity may be maintained by performing minimal reversal mutations on the fully human antibody variable domain. Examples of "humanization" include converting a camelid-derived VHH domain by substituting the amino acid sequence of the original VHH sequence with one or more amino acid residues located at corresponding positions on the VH domain of a human general tetrapeptide chain structure antibody (also referred to herein as "sequence optimization," and in addition to humanization, "sequence optimization" may encompass other modifications of the sequence, such as removing potential post-translational modification sites through one or more mutations that provide VHH modification properties). The humanized VHH domain may contain one or more complete human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods, such as protein surface amino acid resurfacing and antibody humanization, and CDR grafting to a universal framework, involve "grafting" the CDR to other "scaffolds" (including, but not limited to, human stents or non-immunoglobulin stents). Stents and techniques suitable for said CDR grafting are known in the art. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the VBase human germline sequence database and Kabat, EAIt can be found in Sequences of Proteins of Immunological Interest, 5th edition, et al., 1991. The humanized antibody of the present disclosure also includes a humanized antibody after performing affinity maturation for CDR additionally by phage display. Additionally, to prevent a decrease in activity from occurring simultaneously with a decrease in immunogenicity, activity may be maintained by performing at least a reversal mutation or reverse mutation on the human antibody variable region framework sequence.

[0263] "Affinity-maturing" antibodies refer to antibodies having one or more changes in one or more hypervariable regions (HVR) compared to parent antibodies that do not have such changes, and such changes improve the affinity of the antibody for the antigen. For example, an "affinity-maturing" TRGV9 binding protein or anti-TRGV9 antibody has one or more changes in one or more CDRs, and said changes increase the affinity for the antigen compared to its parent antibody. Affinity-maturing antibodies may be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc.Nat.Acad.Sci, USA 91:3809-3813.; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J.MoI.Biol.226(3):889896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0264] Typically, the GPC3 / TRGV9 binding protein of the present disclosure, the TRGV9 binding protein, is preferably 10 as measured in Biacore, KinExA, or Fortibio assays. -7 to 10 -10 mole / liter (M), more preferably 10 -8 to 10 -10 mol / liter, even more preferably 10 -9 to 10 -10 or a lower dissociation constant (K D ) and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 It binds to the antigen or target protein (i.e., GPC3, TRGV9) to which it intends to bind using the binding constant (KA) of M. 10 -4 Any K exceeding M D The value is generally considered to indicate non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope may be measured by any known suitable method, including, for example, surface plasmon resonance (SPR) measurements, Scatchard measurements, and / or competitive binding measurements as described in this disclosure (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive analysis).

[0265] "Binding affinity" or "affinity" is used herein as a measure of the strength of non-covalent interaction between two molecules (e.g., an antibody or part thereof and an antigen). The binding affinity between two molecules is given by the dissociation constant (K D It can be quantified through the determination of ). For example, K can be determined through pharmacokinetic measurements of complex formation and dissociation using the Surface Plasmon Resonance (SPR) method (Biacore). DIt can be determined. The bundle rate constants corresponding to the binding and dissociation of the monovalent complex are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K D is equation K D It is related to ka and kd through =kd / ka. The value of the dissociation constant can be determined directly by well-known methods, and can even be calculated for complex mixtures, for example, by the method described by Caceci et al. (1984, Byte 9:340-362). For example, K D This can be determined using a double-filtered nitrocellulose filter binding assay, such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). Other standard measurements for evaluating the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry, and other measurements exemplified elsewhere in this disclosure. The binding pharmacokinetics and binding affinity of the antibody can also be measured using standards known in the art, such as Biacore, for example, surface plasmon resonance (SPR). TM It can be evaluated via the system or KinExA. K of the antibody / antigen complex, respectively. D By comparing values, binding affinities associated with different molecular interactions can be compared; for example, the binding affinities of different antibodies for a given antigen can be compared. Similarly, the specificity of an interaction is K of a target interaction (e.g., a specific interaction between an antibody and an antigen). D K of the value and non-purpose interaction (e.g., control antibody known not to bind to IGF-1R or TRGV9) D Values ​​can be determined and evaluated by comparison.

[0266] "Conservative substitution" refers to substitution with another single amino acid residue having properties similar to those of the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they possess basic side chains, and aspartic acid and glutamic acid have similar properties in that they possess acidic side chains. Additionally, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they possess uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they possess nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they possess aromatic side chains. Therefore, it will be obvious to those skilled in the art that even if amino acid residues from the group exhibiting similar properties as described above are substituted, no specific change in properties will be observed.

[0267] "Homology," "identity," or "sequence identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. If the positions of the two sequences being compared are all occupied by the same nucleotide or amino acid monomer, for example, if each position of 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 being compared × 100%. For example, if the sequences are optimally aligned, and 6 out of 10 positions of the two sequences are matching or homologous, then the two sequences are 60% homologous. Generally, a comparison is performed when the two sequences are aligned to obtain the highest percentage of homology.

[0268] "Titin-T chain" or "T chain" refers to a peptide fragment of a Titin protein of 78 to 118 amino acids in length containing a Titin Ig-like 152 domain or a functional variant thereof, said Titin-T chain may bind to an Obscurin Ig-like 1 domain to form a dimeric complex. said T chain functional variant has a polypeptide that mutates some amino acids of the wild-type T chain but still binds to an Obscurin Ig-like 1 domain to form a dimeric complex. For example, an appropriate length of amino acids may be added or cleaved at the C end and / or N end of the Titin Ig-like 152 domain; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues may be added or cleaved; For example, "KAGIR," which is five amino acids adjacent to the N-terminus of the Titin Ig-like 152 domain in the wild-type Titin protein, is added to the N-terminus of the Titin Ig-like 152 domain, and this still functions to bind with the Obscurin Ig-like 1 domain to form a complex. Other mutations can also be performed on the amino acids of the Titin Ig-like 152 domain; for example, it is convenient to mutate specific amino acids to improve interchain disulfide bonds and enhance the stability of the complex.

[0269] "Obscurin-O chain" or "O chain" refers to a peptide fragment of an Obscurin protein of 87 to 117 amino acids in length containing an Obscurin Ig-like 1 domain or a functional variant thereof, wherein the Obscurin-O chain may bind to a Titin Ig-like 152 domain to form a dimeric complex. The Obscurin-O chain functional variant has a polypeptide that mutates some amino acids of the wild-type O chain but still binds to a Titin Ig-like 152 domain to form a dimeric complex. For example, an appropriate length of amino acids is added or cleaved at the C end and / or N end of the Obscurin-O domain, for example, by adding or cleaving 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; For example, five 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, and this still functions to bind with the Titin Ig-like 152 domain to form a dimer complex. Other mutations can also be performed on some amino acids of the Obscurin Ig-like 1 domain; for example, it is convenient to mutate specific amino acids to improve interchain disulfide bonds or enhance antibody stability.

[0270] "Nucleic acid" or "polynucleotide" may be used interchangeably in this disclosure and refers to any single-stranded or double-stranded DNA molecule or RNA molecule and, in the case of a single strand, a molecule with a sequence complementary thereto, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or amplifier influences the transcription of a coding sequence, the promoter or amplifier is effectively linked to said coding sequence.

[0271] "Host cell" includes each cell or cell culture that may or is a receptor of a vector for incorporating a polynucleotide insertion fragment. Host cells include progeny of a single host cell, and due to natural, accidental, or intentional mutations, the progeny may not necessarily be exactly identical to the original parent cell (morphologically or in genomic DNA complement). Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. It should also be understood that due to significant or unintended mutations, not all progeny are likely to be exactly identical in terms of DNA content. It includes mutant progeny having the same function or biological activity as the cells screened from the original transformed cell.

[0272] "Inhibition" or "blocking" may be used interchangeably and encompasses both partial and complete inhibition / blocking. "Growth inhibition (e.g., cell-related)" is intended to include any measurable reduction in cell growth.

[0273] "Administration," "to administer," and "treatment" refer to the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biological fluids when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, e.g., therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact between a reagent and a cell, and contact between a reagent and a fluid, said fluid being in contact with the cell. "Administration," "to administer," and "treatment" also mean treating cells, e.g., in vitro and ex vivo, through a reagent, diagnostic, conjugated composition, or other cell. When applied to humans, veterinary, or research subjects, they refer to therapeutic treatment, prophylactic or preventive measures, and research and diagnostic applications.

[0274] “Therapeutics” means administering an internal or external therapeutic agent to a subject, for example, comprising any one of the binding proteins of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, wherein the subject has suffered, is suspected of suffering, or is likely to suffer from one or more proliferative diseases or symptoms thereof, and said therapeutic agent is known to have a therapeutic effect on such symptoms. Typically, the therapeutic agent is administered to a subject or group to be treated in an amount effective for alleviating one or more disease symptoms by inducing regression of such symptoms or inhibiting the progression of such symptoms to a clinically measurable degree. The amount of therapeutic agent effective for alleviating the symptoms of any specific disease (also referred to as the “therapeutic effective dose”) may vary depending on various factors, such as the subject’s disease status, age and weight, and the ability of the drug to produce the desired therapeutic effect on the subject. Whether disease symptoms have been alleviated may be assessed using any clinical detection method typically used by a physician or other healthcare professional to evaluate the severity or progression of said symptoms. Although an embodiment of the present disclosure (e.g., a treatment or product) may not be effective in alleviating symptoms of a target disease in a particular subject, it was determined that symptoms of the target disease should be alleviated in a statistically significant number of subjects according to any statistical test method known in the art, such as the Student t-test, the chi-square test, the U test according to Mann and Whitney, the Kruskal-Wallis test (H test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0275] "Effective dose" includes an amount sufficient to improve or prevent the symptoms or signs of a medical condition. The effective dose also refers to an amount sufficient to allow for or facilitate diagnosis. The effective dose used for a subject may vary depending on factors such as the condition to be treated, the subject's overall health status, the method or route of administration, the dosage, and the severity of side effects. The effective dose may be the maximum dose or administration method that avoids significant side effects or toxic effects. The subjects of this disclosure may be animal or human subjects.

[0276] “Optional” or “optional” means that the event or environment subsequently described may occur but is not necessarily so, and such description includes situations in which the event or environment occurs or does not occur. “And / or” should be construed as specifically disclosing that each of two specified features or components has or does not have the other. Accordingly, the term “And / or” used in phrases such as “A and / or B” in this disclosure includes “A and B,” “A or B,” and “A (alone) and “B (alone).” Unless otherwise clearly required by the context, words such as “include,” “have,” and “containing” throughout the specification and claims have an inclusive rather than exclusive or strict meaning; that is, they should be understood to mean “include but not limited thereto.” In the context of mutations included in the Fc region of the present disclosure, " / " indicates "and", for example, "354C / 366W" indicates "354C and 366W", that is, Fc includes 354C and 366W mutations; and all amino acid positions of mutations in the Fc region of the present disclosure are numbered according to the EU numbering system.

[0277] In the present disclosure, "subject" and "patient" mean mammals, particularly primates, particularly humans.

[0278] Examples

[0279] The following examples will be used to further explain the present disclosure by combining them, but these examples do not limit the scope of the present disclosure.

[0280] Experimental methods for which specific conditions are not specified in the examples or test examples of the present disclosure shall, ordinarily, follow general conditions or conditions recommended by the manufacturer of the raw material or product. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents for which specific sources are not specified are commercially available general reagents.

[0281] Example 1. Design and preparation of antigen

[0282] The TCR (γδ T cell receptor) on the surface of γδ T cells is a heteromeric membrane protein formed by the complexation of a γ chain and a δ chain. Amino acids at positions 1–242 of the human γδTCR extracellular domain protein γ9 chain (Protein data bank, number 1HXM, chain B) are selected, and a 3C enzymatic degradation site, a leucine zipper, and a FLAG label are sequentially added to the C end; amino acids at positions 1–229 of the human γδTCR extracellular domain protein δ2 chain (Protein data bank, number 1HXM, chain A) are selected, and a 3C enzymatic degradation site, a leucine zipper, and a His8 label are sequentially added to the C end. Additionally, Q180C and V168C mutations are introduced into the constant regions of the γ chain and δ chain, respectively, to form interchain disulfide bonds.

[0283] The recombinant monkey (Macaca mulatta) γ9δ2 TCR protein is formed by selecting amino acids at positions 1–241 of the monkey γ9 chain (sequences refer to Public Patent No.: US2019144540A1 SEQ ID NO: 42), whereby a 3C enzymatic degradation site, a leucine zipper, and a FLAG label are sequentially added to the C end; selecting amino acids at positions 1–229 of the monkey δ2 chain (sequences refer to Public Patent No.: US20190144540A1 SEQ ID NO: 37), whereby a 3C enzymatic degradation site, a leucine zipper, and a His8 label are sequentially added to the C end; and also by introducing Q179C and V168C mutations into the constant regions of the γ chain and δ chain, respectively, to form interchain disulfide bonds.

[0284] Recombinant human γ9 chain

[0285] AGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWYRERPGEVIQFLVSISYDGTVRKESGIPSGKFEVDRIPETSTTSTLTIHNVEKQDIATYYCALWEAQQELGKKIKVFGPGTKLII TDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWEEKKSNTILGSCEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN GSGLEVLFQGPGSGAQLEKELQALEKENAQLEWELQALEKELAQKGSGDYKDDDDK (SEQ ID NO: 1)

[0286] Recombinant human δ2 chain

[0287] AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDTLGMGGEYTDKLIFGKGTR VTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAICISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKS GSGLEVLFQGPGSGAQLKKKLQALKKKNAQLKWKLQALKKKLAQKGSGHHHHHHHHH (SEQ ID NO: 2)

[0288] Recombinant monkey γ9 chain

[0289] AGHLEQPQISSTKMLSKTARLECVVSGVTISETSIYWYRERPGEVIQFLVCIFYDGTVKKESSIPSGKFEVDRIPKTSTSTLTIHNVEKQDIATYYCALWEVQQFGRKVKLFGPGTKLII TDKHLDADVSPKPTIFLPSIAETNLHKAGTYLCLLENFFPDVIKIHWQEKKSNTILGSCEGNTVKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVTTMDPKDN GSGLEVLFQGPGSGAQLEKELQALEKENAQLEWELQALEKELAQKGSGDYKDDDDK (SEQ ID NO: 3)

[0290] Recombinant monkey δ2 chain

[0291] AVELVPEHQTVIVSVGDPATLKCSMKGEAISNYYINWYRKTQGNTMTFIYREKGIYGPGFKDNFQGDIDTEENQAVLKILAPSERDEGSYYCASDILSWVDSYTDKLIFGKGTR VTVEPKRQPHTKPSVFVMKNGTNVACLVKDFYGKDIRINLESSKKITEFDPAICVSPSGKYNAVKLGQYADSNSVTCSVQHNKEVVYSTDFEVKTNSTDHLKPTETENTKQPSKS GSGLEVLFQGPGSGAQLKKKLQALKKKNAQLKWKLQALKKKLAQKGSGHHHHHHHHH (SEQ ID NO: 4)

[0292] Plasmids carrying the target protein-coding genes were synthesized, human γ9 chain (SEQ ID NO: 1) and human δ2 chain (SEQ ID NO: 2) plasmids were mixed in a 1:1 ratio, and monkey γ9 chain (SEQ ID NO: 3) and monkey δ2 chain (SEQ ID NO: 4) plasmids were mixed in a 1:1 ratio. Each was transiently transfected and expressed in Expi293 cells (purchased from Thermo) for 7 days, isolated and purified to obtain human γ9δ2 TCR protein and monkey γ9δ2 TCR protein, which were stored at -80°C for later use.

[0293] Example 2. Screening of anti-γδTCR antibodies

[0294] 1. Anti-γδTCR single-domain antibody screening

[0295] Healthy adult alpacas were immunized using in vitro amplified human γ9δ2 T cells as an immunogen, and specific antibodies were screened using the recombinant human γ9δ2 TCR protein of Example 1 by adopting a phage display method.

[0296] Specifically, the primary immunization dose was 2E7 cells per alpaca. Three weeks after the primary immunization, an invigorating immunization was performed, with a dose of 2E7 cells per alpaca. Subsequently, invigorating immunizations were performed at three-week intervals. Serum samples were collected one week after each invigorating immunization, and antibody titers in the alpaca serum were detected using protein ELISA and FACS.

[0297] The specific detection procedure for the protein ELISA is as follows: recombinant human γ9δ2 TCR protein is diluted to 2 μg / mL using 0.05 M carbonate buffer (pH 9.6) and coated overnight at 4°C at 100 μL / well; the plates are blocked for 1 hour with PBST buffer containing 5% skim milk and washed 3 times; alpaca serum is diluted in a 2-fold gradient starting at 1:2000 in the blocking buffer, incubated at 37°C for 45 minutes, and then washed 5 times; 100 μL of horseradish peroxidase-labeled sheep anti-alpaca secondary antibody (AlpVHHs, 053-404-005, diluted 1:10000 with PBS) was added to each well, and after incubation at 37°C for 45 minutes, the plate was washed 5 times. Finally, 100 μL of TMB chromogenic solution was added to each well to induce color development, and after 5 minutes, 50 μL of stop solution was added to terminate the reaction. The absorbance value at 450 nm was read using a microplate reader.

[0298] The specific detection procedure by FACS is as follows: Cells were collected and resuspended at 4 E6 / mL, and gradient-diluted alpaca serum was added to 50 μL of cells and incubated at 4°C for 1 hour. After washing twice with 1% BSA / PBS buffer, the supernatant was discarded, and anti-alpaca IgG iFluor647 (AlpVHHs, 053-404-009) diluted 1:200 was added and incubated at 4°C under light protection for 45 minutes. After washing twice with 1% BSA / PBS buffer and resuspending in 200 μL of buffer, detection was performed by FACS.

[0299] Through detection, the alpaca serum titer after immunization was 128k or higher.

[0300] After three rounds of immunization, 50 mL of peripheral blood was collected one week after each round of immunization to isolate lymphocytes (PBMCs), and total RNA from PBMCs was extracted using RNAiso Plus reagent to construct a phage library, and a phage library with a titer of 3.08 × E13 cfu / mL was obtained.

[0301] A phage display method was adopted to perform panning on antibodies against recombinant human γ9δ2 TCR protein, antibodies cross-binding to monkey γ9δ2 TCR protein were evaluated by ELISA, and binding activity to in vitro amplified human γ9δ2 T cells was determined using FACS. Positive monoclonal single-domain antibodies were screened using the aforementioned ELISA and FACS detection methods, and their sequences are as follows.

[0302] SDP01346 Variable Area (Clone Number ac-025)

[0303] QVQLVESGGGLVQAGGSLRLSCTVSGSTFS DFAMG WLRQAPGKEREFVA AISWTGGRTYYADSVKG RFAISRDNGENTVYLQMNSLKSEDTAIYYCAA SRDCSGPGCRVHEYDY WGQGTQVTVSS (SEQ ID NO: 5)

[0304] Table 1. CDR sequences of anti-human γδTCR single-domain antibodies (Kabat numbering rules)

[0305]

[0306] 2. Screening of mouse-derived anti-γδTCR antibodies

[0307] 6-8 week old Balb / c and SJL mice were immunized using in vitro amplified human γ9δ2 T cells as an immunogen, and specific antibodies were screened using the recombinant human γ9δ2 TCR protein of Example 1 by adopting a hybridoma fusion method.

[0308] Specifically, the primary immunization dose was 1E7 per mouse. Two weeks after the primary immunization, booster immunization was performed, with a dose of 1E7 per mouse. Subsequently, booster immunization was performed at intervals of 2 to 3 weeks. Serum samples were collected one week after each booster immunization, and antibody activity in the mouse serum was detected using protein ELISA and FACS.

[0309] The specific detection procedure for the protein ELISA is as follows: 1 μg / mL recombinant human γ9δ2 TCR protein was coated onto a plate and incubated overnight at 4°C. The plate was then blocked for 1 hour with PBST buffer containing 1% BSA and washed three times. Mouse serum was diluted in a 3-fold gradient starting at 1:200 in the blocking buffer and incubated at 37°C for 1 hour. Afterward, the plate was washed three times and incubated for 1 hour with a secondary antibody against mouse IgG-Fc-HRP (Sigma, AP127P) diluted to 1:10000. The plate was washed three times with PBST, 100 μL of TMB chromogenic solution was added to each well for color development, and the reaction was terminated with a stop solution after 15 minutes. The absorbance value at 450 nm was read using a microplate reader.

[0310] The specific detection procedure by FACS is as follows: Cells were plated at 1 E5 cells per well, centrifuged, and the supernatant was discarded, after which gradient-diluted mouse serum was added. The cells were incubated at 4°C for 1 hour. After washing twice with 1% BSA / PBS buffer and discarding the supernatant, Alexa Flour 488 sheep anti-mouse secondary antibody (Jackson immuno research. 115-545-071) diluted 1:500 was added, and the cells were incubated at 4°C under light protection for 30 minutes. The cells were washed twice with 1% BSA / PBS buffer, resuspended in 200 μL of buffer, and detected by FACS. The mouse serum titer after immunization was 72,900 or higher.

[0311] For the final immunization, 50 μg of recombinant human γ9δ2 TCR protein was injected intraperitoneally. Four days later, the mice were euthanized, the spleens were removed and pulverized to collect splenocytes. These cells were then mixed with mouse myeloma cells SP2 / 0 and electrofused to obtain hybridoma cells. FACS detection was performed on positive clones with an OD of 450 nm > 0.2, and subcloning was performed on positive clones with an MFI value of > 5000 that bind to human γ9δ2 T cells. Following primary screening and re-detection of the subcloning, mouse-derived anti-γδTCR antibodies were screened using the aforementioned ELISA and FACS detection methods. The sequence of SDP01315 (clone number bph-003) is as follows.

[0312] SDP01315(Clone number bph-003) VH

[0313] EVKLVESGGGLVKPGGSLKLSCAASGFTFS SYAMS WVRQTPEKRLEWVA SISSGGSTYYPDSVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR DGYPPFDY WGQGTTLTVSS (SEQ ID NO: 9)

[0314] SDP01315(Clone number bph-003) VL

[0315] DIVMTQSQKFMSTSVGDRVSVTC KASQNVGTNVA WYQQKPGQSPKALIY SASYRYS GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYNSFPLT FGSGTKLEIK

[0316] (SEQ ID NO: 10)

[0317] Table 2. CDR sequences of mouse-derived anti-γδTCR antibodies (Kabat numbering rules)

[0318]

[0319] Example 3. Construction of anti-γδTCR chimeric antibody and verification of functional effect

[0320] 1. Expression of Chimeric Antibodies

[0321] A single-domain antibody was obtained by cloning the nucleotide sequence encoding the antibody into a pTT5 vector, transfecting ExpiCHO cells, removing the cells by centrifugation after 8 days, collecting and filtering the cell culture medium, purifying the harvested cell culture medium using a nickel-affinity column (HisTrap excel, GE), eluting the conjugated antibody using 300 mM imidazole, desalting, and liquid exchange with PBS to obtain the target antibody.

[0322] SDP01346 Battlefield

[0323] QVQLVESGGGLVQAGGSLRLSCTVSGSTFSDFAMGWLRQAPGKEREFVAAISWTGGRTYYADSVKGRFAISRDNGENTVYLQMNSLKSEDTAIYYCAASRDCSGPGCRVHEYDYWGQGTQVTVSS GGGGSHHHHHH

[0324] (SEQ ID NO: 17)

[0325] Mouse-derived antibodies were obtained by cloning the nucleotide sequence encoding the antibody into a pTT5 vector, transfecting ExpiCHO cells, removing the cells by centrifugation after 8 days, collecting and filtering the cell culture medium, purifying the harvested cell culture medium using a Protein A affinity column (MabSelect SuRe, GE), eluting the conjugated antibody with glycine, neutralizing the eluent with 1M Tris, and desalting to obtain the target antibody.

[0326] SDP01315 Heavy Chain Battlefield

[0327] EVKLVESGGGLVKPGGSLKLSCAASGFTFS SYAMS WVRQTPEKRLEWVA SISSGGSTYYPDSVKG RFTISRDNARNILYLQMSSLRSEDTAMYYCAR DGYPPFDY WGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0328] (SEQ ID NO: 18)

[0329] SDP01315 Light Chain Battlefield

[0330] DIVMTQSQKFMSTSVGDRVSVTC KASQNVGTNVA WYQQKPGQSPKALIY SASYRYS GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYNSFPLT FGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0331] (SEQ ID NO: 19)

[0332] 2. Binding of chimeric antibodies and γ9δ2 T cells

[0333] The process for detecting cell-level affinity of anti-γδTCR single-domain antibodies is as follows: gradient-diluted antibody molecules 1×10 5 γ9δ2 T cells were incubated with the cells at 4°C for 1 hour to wash away excess antibodies, and DyLight 405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) was added and incubated at 4°C for 30 minutes to wash away excess antibodies. After resuspending the cells in 200 μL of 2% FBS / PBS buffer, the fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. The results are shown in Table 3.

[0334] The process for detecting the cellular affinity of mouse-derived anti-γδTCR antibodies is as follows: gradient-diluted antibody molecules at a level of 1×10 5γ9δ2 T cells were incubated with 1 γ9δ2 T cell at 4°C for 1 hour, and excess antibodies were washed off. Then, mouse-derived Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added and incubated at 4°C for 30 minutes, and excess antibodies were washed off. After resuspending in 200 μL of 2% FBS / PBS buffer, the fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. The results are shown in Table 3.

[0335] Table 3. Binding ability of anti-γδTCR chimeric antibodies

[0336]

[0337] According to the results, SDP01346 and SDP01315 have good binding activity on γ9δ2 T cells.

[0338] Example 4. Detection of anti-γδTCR antibody and antigen-binding domain

[0339] In this example, a protein-based ELISA was employed to detect the binding regions of the anti-γδTCR antibody and the antigen. By employing a method similar to that of Example 1, recombinant human γ9δ1 TCR protein (dimeric protein of SEQ ID NO: 1 and SEQ ID NO: 20), human γ8δ2 TCR protein (dimeric protein of SEQ ID NO: 21 and SEQ ID NO: 2), human VγδCαβ chimeric TCR protein (dimeric protein of SEQ ID NO: 22 and SEQ ID NO: 23), and human VαβCγδ chimeric TCR protein (dimeric protein of SEQ ID NO: 24 and SEQ ID NO: 25) were prepared.

[0340] ELISA was adopted to evaluate the binding activity of the anti-γδTCR antibody against the aforementioned antigen, and to determine the approximate binding region of the antibody. The specific detection procedure for the protein ELISA is as follows: 1 μg / mL of recombinant TCR protein was coated onto each plate and incubated overnight at 4°C. After blocking for 1 hour with PBST buffer containing 1% BSA, the plates were washed three times. The antibody was diluted to 30 nM using blocking buffer and incubated at 37°C for 1 hour. After washing the plates three times, they were incubated for 1 hour with a secondary antibody against anti-mouse IgG-Fc-HRP (Sigma, AP127P) diluted 1:10000. After washing three times with PBST, 100 μL of TMB chromogenic solution was added to each well to induce color development, and the reaction was terminated with a stop solution after 15 minutes. The absorbance value at 450 nm was read using a microplate reader.

[0341] Recombinant human δ1 chain

[0342] AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGVRAFLRDWGIRVLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLV KEFYPKDIRINLVSSKKITEFDPAICISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSGSGLEVLFQGPGSGAQLKKKLQALKKKNAQLKWKLQALKKKLAQKGSGHHHHHHHH (SEQ ID NO: 20)

[0343] Recombinant human γ8 chain

[0344] ADLSSNLEGRTKSVTRPTGSSAVITCDLPVENAVYTHWYLHQEGKAPQRLLYYDSYNSRVVLESGISREKYHTYASTGKSLKFILENLIERDSGVYYCATWASSDWIKTFAKGTRLIVTSPDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLE KFFPDIIKIHWQEKKSNTILGSCEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNGSGLEVLFQGPGSGAQLEKELQALEKENAQLEWELQALEKELAQKGSGDYKDDDDK (SEQ ID NO: 21)

[0345] Recombinant human Vγ9Cβ chain

[0346] AGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWYRERPGEVIQFLVSISYDGTVRKESGIPSGKFEVDRIPETSTTSTLTIHNVEKQDIATYYCALWEVHELGKKIKVFGPGTKLIITEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYSLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADSGSGLEVLFQGPGSGAQLEKELQALEKENAQLEWELQALEKELAQKGSGDYKDDDDK (SEQ ID NO: 22)

[0347] Recombinant human Vδ2Cα chain

[0348] AIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYRKTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDPVQVTGGYKVDKLIFGKGTRVTVEPNIQNPDPAVYQLRDS KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSSGSGLEVLFQGPGSGAQLKKKLQALKKKNAQLKWKLQALKKKLAQKGSGHHHHHHHH (SEQ ID NO: 23)

[0349] Recombinant human Vβ7Cγ chain

[0350] GVSQTPSNKVTEKGKYVELRCDPISGHTALYWYRQSLGQGPEFLIYFQGTGAADDSGLPNDRFFAVRPEGSVSTLKIQRTERGDSAVYLCATSALGDTQYFGPGTRLTVLDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCL LEKFFPDVIKIHWEEKKSNTILGSCEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNGSGLEVLFQGPGSGAQLEKELQALEKENAQLEWELQALEKELAQKGSGDYKDDDDK (SEQ ID NO: 24)

[0351] Recombinant human Vα13Cδ chain

[0352] ENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASSFGNEKLTFGTGTRLTIIPSQPHTKPSVFVMKNGTNNVACLVKEFYP KDIRINLVSSKKITEFDPAICISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSGSGLEVLFQGPGSGAQLKKKLQALKKKNAQLKWKLQALKKKLAQKGSGHHHHHHHH (SEQ ID NO: 25)

[0353] The ELISA results are shown in Table 4, and the binding absorbance values ​​of antibodies SDP01346 and SDP01315 to different antigens are different. Both of these antibodies bind to the γ9δ1 TCR protein but not to the γ8δ2 TCR protein, which explains that the antibodies bind to the γ9 chain; both of these antibodies bind to the VγδCαβ chimeric TCR protein but not to the VαβCγδ chimeric TCR, which explains that the antibodies bind to the variable region (V region) of the TCR. In summary, it can be clarified that antibodies SDP01346 and SDP01315 bind to TRGV9.

[0354] Table 4. Analysis results of chimeric antibodies and antigen binding regions

[0355]

[0356] Example 5. Humanization modification of anti-γδTCR antibody

[0357] By comparing variable domain sequences with antibody germline databases, human germline templates with high homology were obtained. Here, the human germline heavy chain template used for SDP01346 was IGHV3-30; the human germline heavy chain template used for SDP01315 was IGHV3-21, and the human germline light chain template was IGKV1-12. After predicting the structure of monoclonal antibodies through homology modeling, the VH, VL of mouse-derived antibodies or the CDR of single-domain antibodies were chimeric with an appropriate human GermLine framework (Bioinformation. 2014; 10(4): 180-186; Methods Mol Biol. 2019;1904:213-230), and then reverse mutations were introduced.

[0358] The obtained humanization molecular sequence is as follows:

[0359] SDP01346 VH1

[0360] EVQLVESGGGVVQPGGSLRLSCAASGSTFS DFAMG WLRQAPGKGREFVA AISWTGGRTYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA SRDCSGPGCRVHEYDY WGQGTLVTVSS (SEQ ID NO: 26)

[0361] SDP01346 VH2

[0362] EVQLVESGGGVVQPGGSLRLSCAVSGSTFS DFAMG WLRQAPGKEREFVA AISWTGGRTYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA SRDCSGPGCRVHEYDY WGQGTLVTVSS (SEQ ID NO: 27)

[0363] SDP01346 VH3

[0364] EVQLVESGGGVVQPGGSLRLSCAVSGSTFS DFAMG WLRQAPGKEREFVA AISWTGGRTYYADSVKGRFTISRDNGKNTVYLQMNSLKSEDTAVYYCAA SRDCSGPGCRVHEYDY WGQGTLVTVSS (SEQ ID NO: 28)

[0365] > SDP01346 VH4

[0366] EVQLVESGGGLVQPGGSLRLSCSVSGSTFS DFAMG WLRQAPGKEREFVA AISWTGGRTYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA SRDCSGPGCRVHEYDY WGQGTLVTVSS (SEQ ID NO: 29)

[0367] > SDP01315 VH1

[0368] EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYAMS WVRQAPGKRLEWVS SISSGGSTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DGYPPFDY WGQGTTVTVSS (SEQ ID NO: 30)

[0369] > SDP01315 VH2

[0370] EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYAMS WVRQAPGKRLEWVA SISSGGSTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DGYPPFDY WGQGTTVTVSS (SEQ ID NO: 31)

[0371] > SDP01315 VH3

[0372] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKRLEWVA SISSGGSTYYPDSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR DGYPPFDY WGQGTLVTVSS (SEQ ID NO: 32)

[0373] > SDP01315 VL1

[0374] DIQMTQSPSSVSASVGDRVTITC KASQNVGTNVA WYQQKPGKAPKLLIY SASYRYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSFPLT FGQGTKLEIK

[0375] (SEQ ID NO: 33)

[0376] SDP01315 VL2

[0377] DIQMTQSPSSVSASVGDRVTITC KASQNVGTNVA WYQQKPGKAPKALIY SASYRYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSFPLT FGQGTKLEIK

[0378] (SEQ ID NO: 34)

[0379] SDP01315 VL3

[0380] DIQMTQSPSSVSASVGDRVTVTC KASQNVGTNVA WYQQKPGKSPKALIY SASYRYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYFC QQYNSFPLT FGQGTKLEIK

[0381] (SEQ ID NO: 35)

[0382] After humanizing the single-domain antibody, a full-length antibody was constructed, and GGGGSHHHHHH (SEQ ID NO: 72) was added after the corresponding VHH. Alternatively, after humanizing the single-domain antibody, a full-length antibody was constructed, and a linker and Fc were added after the corresponding VHH.

[0383] After humanizing mouse-derived antibodies, full-length antibodies were constructed; Cκ was used for the light chain constant region, and IgG1 with L234F and L235E mutations was used for the heavy chain constant region.

[0384] Table 5. Correspondence between antibody names and antibody structures.

[0385]

[0386] The following presents the full-length sequences of SDP01378, SDP05576, and SDP01369 as examples.

[0387] SDP01378 Battlefield

[0388] EVQLVESGGGLVQPGGSLRLSCSVSGSTFS DFAMG WLRQAPGKEREFVA AISWTGGRTYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA SRDCSGPGCRVHEYDY WGQGTLVTVSS GGGGSHHHHHH

[0389] (SEQ ID NO: 36)

[0390] SDP05576

[0391] EVQLVESGGGLVQPGGSLRLSCSVSGSTFSDFAMGWLRQAPGKEREFVAAISWTGGRTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAASRDCSGPGCRVHEYDYWGQGTLVTVSSAAASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0392] (SEQ ID NO: 73)

[0393] SDP01369 Heavy Chain Battlefield

[0394] EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYAMS WVRQAPGKRLEWVA SISSGGSTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DGYPPFDY WGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0395] (SEQ ID NO: 37)

[0396] SDP01369 Light Chain Battlefield

[0397] DIQMTQSPSSVSASVGDRVTITC KASQNVGTNVA WYQQKPGKAPKALIY SASYRYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSFPLT FGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0398] (SEQ ID NO: 38)

[0399] FACS detection of humanized monoclonal antibody binding to γ9δ2 T cells was performed below.

[0400] Detection of affinity of single-domain antibody SDP01346 after humanization: 1×10⁶ gradient-diluted antibody molecules 5 γ9δ2 T cells were incubated with 1 γ9δ2 T cell at 4°C for 1 hour, and excess antibodies were washed off. Then, DyLight 405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) was added and incubated at 4°C for 30 minutes, and excess antibodies were washed off. After resuspending in 200 μL of 2% FBS / PBS buffer, the fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. The results are shown in Table 6.

[0401] Detection of affinity of mouse-derived chimeric antibody SDP01315 after humanization: 1×10⁻¹⁰ gradient-diluted antibody molecules 5γ9δ2 T cells were incubated with 1 γ9δ2 T cell at 4°C for 1 hour, and excess antibodies were washed off. Then, mouse-derived Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added and incubated at 4°C for 30 minutes, and excess antibodies were washed off. After resuspending in 200 μL of 2% FBS / PBS buffer, the fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. The results are shown in Table 6.

[0402] Table 6. Detection of humanized molecular affinity of SDP01346 and SDP01315

[0403]

[0404] According to the results, the single-domain antibody humanization molecule SDP01378 has the strongest affinity, and the affinities of SDP01346 humanization molecules SDP01365-SDP01373 are similar, so SDP01369, which has an intermediate number of reverse mutations, was selected.

[0405] 실시예 6. 항-GPC3 / γδTCR 이중특이성 항체의 설계 및 제조

[0406] The sequences of antibody G, heavy chain variable region, and light chain variable region having nanomolar affinity selected from the GPC3 stage are as follows. Antibody G binds to the C-terminal subunit and does not bind to soluble GPC3.

[0407] GPC3 VH

[0408] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSS (SEQ ID NO: 39)

[0409] GPC3 VL

[0410] DVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIK (SEQ ID NO: 40)

[0411] Table 7. VH and VL of Antibody G (Kabat numbering rules)

[0412]

[0413] Various GPC3 / γδTCR bispecific antibodies were designed, and finally, a 1+1 asymmetric structure was selected with SDP01346H4 (i.e., SDP01378) and a KIH (knob into hole) was adopted to prevent heavy chain mismatch; a 2+1 asymmetric structure was selected with SDP01315H2L2 and a KIH was adopted to prevent heavy chain mismatch, and a HOT was adopted to prevent light chain mismatch. Refer to Figure 1 for the structural schematic.

[0414] To prevent and reduce heavy and light chain mismatches, refer to SEQ ID NO: 47–48 for the Obscurin-O and Titin-T used, and refer to SEQ ID NO: 49–50 for CH1 and Cκ. To prevent and reduce mismatches between the two strands of heavy chains, IgG mutations were used. The full-length sequences of the dual 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 Cκ, the italicized part is the IgG1 Fc region, the bold italicized part is a dot mutation in the Fc region, and the bold font is the linker.

[0415] Obscurin-O

[0416] SGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA

[0417] (SEQ ID NO: 47)

[0418] >Titin-T

[0419] GIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI (SEQ ID NO: 48)

[0420] > CH1

[0421] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0422] (SEQ ID NO: 49)

[0423] > Cκ

[0424] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0425] (SEQ ID NO: 50)

[0426] > IgG1 Fc(WT)

[0427] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51)

[0428] > IgG1 Fc1(S354C / T366W / L234F / L235E로 돌연변이됨)

[0429] DKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 52)

[0430] > IgG1 Fc2(Y349C / T366S / L368A / Y407V / L234F / L235E로 돌연변이됨)

[0431] DKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53)

[0432] > SDP01716 H1

[0433] EVQLVESGGGLVQPGGSLRLSCSVSGSTFS DFAMG WLRQAPGKEREFVA AISWTGGRTYYADSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA SRDCSGPGCRVHEYDY WGQGTLVTVSS AAAS DKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54)

[0434] > SDP01716 H2

[0435] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD (SEQ ID NO: 55)

[0436] > SDP01716 L

[0437] DVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 56)

[0438] > SDP01696 H1

[0439] EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYAMS WVRQAPGKRLEWVA SISSGGSTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DGYPPFDY WGQGTTVTVSS GGGGS SGAPRFLTRPKASVVSVGKDATLSCQIVGNPFPQVSWEKDKQPVTAGVRFRLAQDGDLYRLKILDLQLSDSGQYVCRARNAHGEAFACLGLQVDAEA DKTHTCPPCPAPEFEGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 57)

[0440] > SDP01696 L1

[0441] DIQMTQSPSSVSASVGDRVTITC KASQNVGTNVA WYQQKPGKAPKALIY SASYRYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSFPLT FGQGTKLEIK GGGGS GIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNHIRSI (SEQ ID NO: 58)

[0442] > SDP01696 H2

[0443] QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSEDTAVYYCTR PHYSICAL WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEFEGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS(SEQ ID NO: 59)

[0444] > SDP01696 L2

[0445] SDP01716 L과 동일함(SEQ ID NO: 56).

[0446] SDP01704 H1

[0447] SDP01696 H1과 동일함(SEQ ID NO: 57).

[0448] > SDP01704 L1

[0449] SDP01696 L1과 동일함(SEQ ID NO: 58).

[0450] > SDP01704 H2

[0451] QVQLVQSGAEVKKPGASVKVSKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSEDTAVYYCTR PHYSICAL WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC D GGGGSGGGGGS QVQLVQSGAEVKKPGASVKVSKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSEDTAVYYCTR PHYSICAL WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60)

[0452] > SDP01704 L2

[0453] DVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 61)

[0454] After cloning the nucleotide sequence encoding the antibody into the pTT5 vector, the cells were transfected into 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 Protein A affinity column (MabSelect SuRe, GE), the bound antibody was eluted with glycine, the eluent was neutralized with 1M Tris, desalted, and the target antibodies SDP01716, SDP01696, and SDP01704 were obtained through detection.

[0455] Example 7. Detection of binding affinity between anti-GPC3 / γδTCR bispecific antibody and human and monkey antigens

[0456] A Protein A biosensor chip (Cat. # 29139121-AB, Cytiva) was used. Each test antibody was conjugated with HBS-EP + buffer solution to perform capture of Protein A on the chip channel as a ligand. Human GPC3 (Sino Biological, Cat# 10088-H08H), cynomolgus monkey GPC3 (Acrobiosystems, Cat# GP3-C5225), human γ9δ2, and cynomolgus monkey γ9δ2 antigens were conjugated with HBS-EP + buffer solution as analytes, and the analytes were diluted by a 2-fold gradient. The diluted antigens were passed through the experimental channel and the reference channel at a flow rate of 30 µL / min, bound for 80 seconds, and dissociated for 300 seconds. 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was selected as the regeneration buffer and the test was run for 30 seconds at a flow rate of 10 L / min. Data were analyzed using Biacore 8K evaluation software.

[0457] The results are shown in Tables 8 and 9, and the antigen-binding affinities of the three antibodies against human and cynomolgus monkey GPC3 proteins were all approximately 2 nM, similar to the affinity of antibody G; the binding K of the three antibodies to the cynomolgus monkey γ9δ2 TCR protein D All are approximately 50 nM.

[0458] Table 8. Binding pharmacokinetic parameters of antibodies and human / cynomolgus monkey GPC3 proteins

[0459]

[0460] Table 9. Binding pharmacokinetic parameters of antibodies and human / cynomolgus monkey γ9δ2 proteins

[0461]

[0462] Example 8. Detection of binding activity of anti-GPC3 / γδTCR bispecific antibody with GPC3-positive cells and human γ9δ2 T cells

[0463] The binding activity of the γδ T bispecific antibody against HepG2 cells (purchased from ATCC) naturally high-expressing human GPC3 and DLD-1 cells (purchased from ATCC) not expressing human GPC3 was detected using FACS. The GPC3 binding activity of the anti-GPC3 / γδTCR bispecific antibody in cells was evaluated by detecting the fluorescent signal of the antibody bound to the cell surface and assessing the binding strength of the antibody based on the intensity of the fluorescence signal. Specifically, gradient-diluted antibody molecules and control molecules were 1 × 10⁶ 5 The cells were incubated with the cells at 4°C for 1 hour, the excess antibodies were washed off, mouse-derived Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added and incubated at 4°C for 30 minutes, the excess antibodies were washed off, the cells were resuspended in 200 μL of 2% FBS / PBS buffer, and the fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. The cells used were the HepG2 cell line.

[0464] The binding activity of the anti-GPC3 / γδTCR bispecific antibody against human γ9δ2 T cells and human PBMCs was detected using FACS. Human γ9δ2 T cells were obtained via induction using zoledronic acid and IL-2. The binding activity of the anti-GPC3 / γδTCR bispecific antibody on γδ T cells was evaluated by detecting the fluorescent signal of the antibody bound to the cell surface and assessing the binding strength based on the intensity of the fluorescence signal. Specifically, gradient-diluted antibody molecules and control molecules were 1 × 10⁶ 5 The cells were incubated with the mouse at 4°C for 1 hour, the excess antibody was washed off, mouse-derived Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat#209-605-098) was added and incubated at 4°C for 30 minutes, the excess antibody was washed off, the cells were resuspended in 200 μL of 2% FBS / PBS buffer, and the fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0465] The results are as illustrated in FIGS. 2A–2D, where, as illustrated in FIGS. 2A and 2B, the GPC3 affinity of SDP01716 and SDP01696 is similar, and EC 50 They are 4.853 nM and 5.166 nM, respectively. SDP01704 exhibits divalent bonding characteristics, and EC 50 The affinity was 2.466 nM; none of the tested antibodies showed non-specific binding in GPC3-negative cells. As shown in Figures 2C and 2D, the affinity of SDP01716 for γ9δ2 T cells was 0.9064 nM, SDP01696 was 2.676 nM, and SDP01704 was 4.289 nM; none of the tested antibodies showed non-specific binding in PBMCs.

[0466] Example 9. Effect of anti-GPC3 / γδTCR bispecific antibody on BTN2A / BTN3A-TCR natural signal

[0467] Under natural conditions, intracellular phosphorylated antigens bind to BTN3A and alter the molecular tension of BTN3A; two BTN3A molecules and two BTN2A molecules bind to form a heterotetramer, which further interacts with the TCR to activate the TCR and induce functions such as activation, proliferation, and apoptosis of γδ T cells. Since anti-γδTCR antibodies also bind to the TCR, this example evaluates the effect of antibodies on the natural BTN2A / BTN3A-TCR signal of the present disclosure. SDP01378 is a monovalent molecule of SDP01716 of γδ T, and SDP01315 is a monovalent molecule of SDP01696 and SDP01704.

[0468] A375-Luc cell medium is RPMI1640 containing 10% inactivated fetal bovine serum. γδ T cells are obtained by amplifying in PBMCs. Adjust the A375-Luc density, seed at 25 μL / well per well in a 96-well plate, and the cell mass is 1 × 10⁶ 4 / well; 25μL / well of γδ T cells (1×10⁶ 5 A well was taken and mixed with A375-Luc; then, 25 μL / well of SDP01378 or SDP01315 (final concentration 10 nM) and 25 μL / well of gradient-diluted BTN3A-active antibody were added. Incubation was performed for 24 hours in a 37°C, 5% CO2 incubator. After 24 hours, 100 μL / well of Bright-Glo™ Luciferase Assay System (Promega, E2650) reagent was added to perform detection. Refer to the reagent instructions for specific procedures.

[0469] The results are as shown in FIGS. 3A and 3B, and both SDP01378 and SDP01315 do not block the natural BTN2A / BTN3A-TCR signal. Therefore, the anti-GPC3 / γδTCR bispecific antibodies of the present disclosure can be considered not to block the natural BTN2A / BTN3A-TCR signal.

[0470] Example 10. Detection of γδ T cell apoptotic activity against tumor cells mediated in vitro by an anti-GPC3 / γδTCR bispecific antibody

[0471] In this example, the apoptotic activity of γδ T cells against target cells with different antibody-mediated GPC3 expression levels was evaluated using the lactate dehydrogenase (LDH) detection method.

[0472] HepG2 cells naturally high-expressed GPC3, and the cell medium was DMEM (Gibco, Cat#11995-065, hereinafter the same) containing 15% inactivated fetal bovine serum; Huh-7 cells expressed intermediate levels of GPC3, and the cell medium was DMEM; MKN-45 cells expressed low levels of GPC3, and the cell medium was RPMI 1640 (Gibco, Cat#10491A-01, hereinafter the same); and DLD-1 cells did not express GPC3, and the cell medium was RPMI 1640. After digestion, the target cells were resuspended in RPMI 1640 medium containing 2% serum, and the density was set to 7×10⁶ 4 The cell density was adjusted to cells / mL; subsequently, 50 μL / well was inoculated into a 96-well plate, and 50 μL of gradient-diluted test antibody was added. 50 μL of medium was added to each well. γδ T cells were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust cell density. They were inoculated into the aforementioned experimental plates at 50 μL / well and incubated for 24 hours in a 37°C, 5% CO2 incubator. The cell culture plates were removed, centrifuged (400g, 5 min), and the cell culture supernatant was collected. LDH levels were detected using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). Refer to the reagent instructions for specific procedures.

[0473] As shown in FIGS. 4A to 4D and Table 10, the apoptotic activity of the dual antibody of the present disclosure against tumor cells is weakened as the antigen expression amount decreases; the strength of the antibody γδ T affinity is positively correlated with the apoptotic activity against tumor cells, and the GPC3 monovalent molecule is stronger than the monovalent molecule.

[0474] Table 10. Killing of anti-GPC3 / γδTCR bispecific antibodies against cells expressing different antigens

[0475]

[0476] Note: - indicates that no killing activity was detected.

[0477] Example 11. Effect of anti-GPC3 / γδTCR bispecific antibody on γδ T cell death derived from different PBMC donors

[0478] In this example, the apoptotic activity of γδ T cells against GPC3-expressing target cells mediated by antibodies was evaluated using the lactate dehydrogenase (LDH) detection method.

[0479] After digesting HepG2 cells, they were resuspended in RPMI1640 medium containing 2% serum, and the cell density was set to 7×10 4 The concentration was adjusted to cells / mL; subsequently, 50 μL / well was inoculated into a 96-well plate, and 50 μL of gradient-diluted test antibody was added. 50 μL of medium was added to each well. γδ T cells amplified from different donors were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust cell density. The cells were inoculated into the aforementioned experimental plates at 50 μL / well and incubated for 24 hours in a 37°C, 5% CO2 incubator. The cell culture plates were removed and centrifuged (400g, 5 min) to collect the cell culture supernatant, and LDH levels were detected using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780).

[0480] The results of SDP01716 are presented as an example. The results are as shown in Figures 5A to 5D and Table 11, and SDP01716 can mediate γδ T cells from different donors to kill tumor cells, and EC 50 and there is no significant difference in the maximum mortality value.

[0481] Table 11. Antibody-mediated apoptotic activity of different donor-derived γδ T cells against HepG2

[0482]

[0483] Note: #SC12004, #SC12392, #XC11053, #XC11061 are γδ T cells amplified from different donors (healthy allogeneic human peripheral blood), and - indicates that no death was detected.

[0484] Example 12. Detection of apoptotic activity of anti-GPC3 / γδTCR bispecific antibodies against antigen-low expression cells under different effector-target ratios

[0485] In this example, the apoptotic activity of γδ T cells against antibody-mediated GPC3 low-expression target cells was evaluated using the lactate dehydrogenase (LDH) detection method.

[0486] MKN-45 cells naturally underexpress GPC3. After digestion, MKN-45 cells were resuspended in RPMI1640 medium containing 2% serum, and the cell density was set to 7×10⁶ 4The concentration was adjusted to cells / mL; subsequently, 50 μL / well was inoculated into a 96-well plate, and 50 μL of gradient-diluted test antibody was added. 50 μL of medium was added to each well. γδ T cells were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust cell density. They were inoculated into the aforementioned experimental plates at 50 μL / well, and the final effector-target ratio (γδ T cells:MKN-45 cells) was 1:1; 10:1; 30:1. Incubation was performed for 24 hours in a 37°C, 5% CO2 incubator. The cell culture plates were removed and centrifuged (400g, 5 min) to collect the cell culture supernatant, and LDH levels were detected using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). Refer to the reagent instructions for specific procedures.

[0487] The results for SDP01716 are presented as an example. As shown in Fig. 6A, the apoptotic activity of SDP01716 against antigen-low expression cells is weak at a low effector-target ratio of 1:1, with a maximum apoptotic value of 17%; as shown in Fig. 6B, the apoptotic activity of SDP01716 against antigen-low expression cells can be enhanced after increasing the effector-target ratio to 10:1, with a maximum apoptotic value reaching 64%; and as shown in Fig. 6C, nearly 100% apoptosis is reached when the effector-target ratio is increased to 30:1. That is, the anti-GPC3 / γδTCR bispecific antibody of the present disclosure can kill tumor target cells in a manner dependent on the effector-target ratio.

[0488] Example 13. Enhancement of apoptotic activity of anti-GPC3 / γδTCR bispecific antibody and detection of cytokine release by increasing the ratio of γδ T cells in PBMCs

[0489] In this example, the apoptotic activity of γδ T cells against GPC3-expressing target cells mediated by antibodies was evaluated using the lactate dehydrogenase (LDH) detection method.

[0490] HepG2 cells naturally highly express GPC3, and after digestion, the HepG2 cells were resuspended in 2% serum RPMI1640 medium, with a cell density of 7×10 4 The concentration was adjusted to cells / mL; then, 50 μL / well was inoculated into a 96-well plate, and 50 μL of gradient-diluted test antibody was added. 50 μL of medium was added to each well. PBMC cells were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust the cell density. The cells were inoculated into the aforementioned experimental plate at 50 μL / well; or 30% γδ T cells were added to the PBMCs and inoculated into the aforementioned experimental plate at 50 μL / well, and incubated for 24 hours in a 37°C, 5% CO2 incubator. The cell culture plates were removed, centrifuged (400g, 5 min), and the cell culture supernatant was collected; LDH levels were detected using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). At the same time, the corresponding cytokine secretion levels are detected using the kit detection IFNγ (Cisbio, S62HIFNGPEG) and TNFα (Cisbio, 62HTNFAPEH), and specific operations are referred to in the reagent instructions.

[0491] The results of SDP01716 are presented as an example. The results are as shown in FIGS. 7A to 7D, where FIG. 7A is the PBMC cell death of donor #XC11053; FIG. 7B is the apoptotic activity of donor #XC11053 with 30% γδ T cells incorporated into PBMC; FIG. 7C is the PBMC cell death of donor #SC12392; and FIG. 7D is the apoptotic activity of donor #SC12392 with 30% γδ T cells incorporated into PBMC.

[0492] According to Figs. 7A and 7C, the tumor cell killing ability of SDP01716 in a pure PBMC system is weaker than that of the same-target CD3 bispecific antibody BMK-029 (which is ERY974 of US20220348658), which is because the CD3 bispecific antibody can kill tumors by using more T cells as effector cells; according to Figs. 7B and 7D, it is explained that increasing the ratio of γδ T cells in PBMC can enhance its killing activity. At the same time, the cytokine release detection results corresponding to Figs. 7A to 7D are as shown in Figs. 8A to 8D, and SDP01716 induces less IFNγ and TNFα release when killing tumor cells.

[0493] Example 14. Detection of γδ T cell proliferation-promoting activity in PBMCs by anti-GPC3 / γδTCR bispecific antibody

[0494] In this example, the activity of an anti-GPC3 / γδTCR bispecific antibody inducing γδ T cell proliferation in PBMCs was evaluated using a flow cytometry detection method.

[0495] PBMC 800μL(1×10 6 Take cells and add them to a 24-well plate; after washing the HepG2 cells twice with PBS, set the cell density to 5×10⁶ 5 The cell / mL ratio was adjusted, 1 mL was taken and added to the corresponding well; the test antibody was mixed with the medium at a corresponding concentration, and 200 μL was taken and added to the well. The cells were incubated in a 37°C, 5% CO2 incubator, and the plate liquid was changed every 3 days. After 6 to 7 days, the cells were washed twice with PBS and stained with CD3 antibody (BD, Cat#564713), Vδ2 TCR antibody (BD, Cat#555739), and Vγ9 TCR antibody (BD, Cat#555732) to assess changes in the γδ T cell ratio.

[0496] The results are as shown in Figure 9, and among the PBMC cells derived from three donors #XC11211, #XC11061, and #XC11251, after 6 to 7 days of stimulation, SDP01716, SDP01696, and SDP01704 can all significantly improve the γδ T cell ratio.

[0497] Example 15. Validation of the mouse human liver cancer Huh-7 transplanted tumor model

[0498] 1. Human liver cancer Huh-7 cells (Cell Bank of the Chinese Academy of Sciences) 5×10 6 NSG mice (female, 6–8 weeks old, provided by Weitong Lihua) were inoculated subcutaneously at 100 μL / mice and randomly grouped into a total of 4 groups (G1–G4) of 7 mice per group. On the day of grouping, tumor cells were inoculated simultaneously with the injection of γδ T cells and drugs, and each group was administered the drugs according to the following plan:

[0499] G1: Mediator G2: γδ T cell + PBS

[0500] G3: γδ T cells + SDP01716 (3 mg / kg)

[0501] G4: γδ T+ cells SDP01716(0.3mg / kg)

[0502] Subsequently, γδ T cell injections and medication were administered once a week. During the medication and observation period, tumor volume was measured twice a week, and the measurements were recorded. The calculation formulas are as follows (hereinafter the same):

[0503] Tumor volume (TV) = 1 / 2 × a × b 2 It was calculated as follows, where a and b represent the measured long and short diameters of the tumor, respectively;

[0504] Relative tumor proliferation rate T / C%=(T-T0) / (C-C0)×100;

[0505] Tumor suppression rate TGI%=1-T / C%.

[0506] The results are as shown in Figures 10A, 10B and Table 12, with the TGI of the γδ T cell monotherapy group (G2) being 11% and the tumor growth inhibitory effect being limited; SDP01716 can inhibit tumor growth in a dose-dependent manner, with the TGI reaching 58% at a dose of 3 mpk, and each therapy group having no significant effect on mouse body weight.

[0507] Table 12. Antitumor activity of SDP01716 in Huh-7 transplanted tumor model

[0508]

[0509] Note: * indicates P<0.05, and ** indicates P<0.01.

[0510] 2. Huh-7 cells 5×10 6 NSG mice (female, 6–8 weeks old, supplied by GemPharmatech) were inoculated subcutaneously at 100 μL / mouse and randomly grouped into a total of 6 groups of 7 mice each. On the day of grouping, tumor cells were inoculated simultaneously with the injection of γδ T cells and drugs, and each group was administered the drugs according to the following plan:

[0511] G1: Medium

[0512] G2: γδ T cells

[0513] G3: γδ T cells + SDP01716 (3 mg / kg)

[0514] G4: γδ T cells + SDP01696 (3.9mg / kg)

[0515] G5: γδ T cells + SDP01704 (5.1 mg / kg).

[0516] Subsequently, γδ T cell injections and administration were performed once a week, and simultaneous administration was performed in cases where γδ T cells and antibodies were used in combination. During the administration and observation period, tumor volume was measured twice a week and the measurements were recorded.

[0517] The results are as shown in Figures 11A, 11B and Table 13, and both antibodies of the present disclosure can effectively suppress tumors, and no significant change in the body weight of mice in each dosing group was observed.

[0518] Table 13. Antitumor activity of dual antibodies in a Huh-7 transplanted tumor model

[0519]

[0520] Note: ** indicates P<0.01.

Claims

Claim 1 As a GPC3 / TRGV9 binding protein, it comprises: a first antigen-binding domain that specifically binds to GPC3; and comprises a second antigen-binding domain that specifically binds to TRGV9, wherein, where: the first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 among the amino acid sequences presented in SEQ ID NO: 39, and VL1 comprises LCDR1, LCDR2, and LCDR3 among the amino acid sequences presented in SEQ ID NO: 40, and the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; preferably, the CDR is defined according to the Kabat numbering system; preferably, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as presented in SEQ ID NO: 41–43, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as presented in SEQ ID NO: 44–46, respectively Same, GPC3 / TRGV9 binding protein. Claim 2 In claim 1, the second antigen-binding domain comprises an immunoglobulin single variable domain or comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 among the amino acid sequences presented in any one of SEQ ID NO: 29, 26–28, 5; the VH2 comprises HCDR1, HCDR2, and HCDR3 among the amino acid sequences presented in any one of SEQ ID NO: 31, 9, 30, 32; and the VL2 comprises LCDR1, LCDR2, and LCDR3 among the amino acid sequences presented in any one of SEQ ID NO: 34, 10, 33, 35; and the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; preferably, the CDR is defined according to the Kabat numbering system A GPC3 / TRGV9 binding protein defined; preferably, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 having amino acid sequences as presented in SEQ ID NO: 6–8, respectively, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of VH2 as presented in SEQ ID NO: 11–13, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of VL2 as presented in SEQ ID NO: 14–16, respectively. Claim 3 In claim 1 or 2, wherein the immunoglobulin single variable domain, the heavy chain variable region and / or the light chain variable region are modified by humanization, reverse mutation, affinity maturation, T cell epitope removal, reduction of antibody deamidation and / or reduction of antibody isomerization; preferably, the framework region of the human germline template used in the humanization modification process for the immunoglobulin single variable domain is derived from IGHV3-64; preferably, the framework region of the human germline template used in the humanization modification process for VH2 is derived from IGHV3-21, and / or the framework region of the human germline template used in the humanization modification process for VL2 is derived from IGKV1-12, GPC3 / TRGV9 binding protein. Claim 4 A GPC3 / TRGV9 binding protein according to any one of claims 1 to 3, wherein VH1 of the first antigen-binding domain comprises an amino acid sequence identical to that presented in SEQ ID NO: 39 or having at least 80% to 90% identity therewith, and VL1 comprises an amino acid sequence identical to that presented in SEQ ID NO: 40 or having at least 80% to 90% identity therewith. Claim 5 A GPC3 / TRGV9 binding protein according to any one of claims 1 to 4, wherein the immunoglobulin single variable domain comprises an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 29, 26–28, 5; and VH2 of the second antigen binding domain comprises an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 9, 30–32, and VL2 comprises an amino acid sequence identical to or having at least 80% to 90% identity with any one of SEQ ID NO: 10, 33–35. Claim 6 A GPC3 / TRGV9 binding protein according to any one of claims 1 to 5, wherein the Fc region of the immunoglobulin is further included; preferably, said Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; more preferably, said Fc region is the Fc region of human IgG1; and most preferably, said Fc region is the Fc region of IgG1 containing L234F and / or L235E mutations. Claim 7 In claim 6, the Fc region comprises a first subunit and a second subunit; preferably, the first subunit and the second subunit of the Fc region comprise a nop-into-hole mutation; preferably, the first subunit of the Fc region comprises a mutation at position 366, and the second subunit comprises a mutation selected from positions 366, 368, or 407 or any combination thereof; the first subunit of the Fc region comprises a mutation at position 354 or 356, and the second subunit comprises a mutation at position 349; or the first subunit of the Fc region contains a mutation at position 354 or 356, and the second subunit contains a mutation at position 349, 366, 368 or 407 or any combination thereof; more preferably, the first subunit of the Fc region contains a 366W mutation, and the second subunit contains a mutation selected from 366S, 368A, and 407V or any combination thereof; the first subunit of the Fc region contains a 354C or 356C mutation, and the second subunit contains a 349C mutation; or the first subunit of the Fc region contains a 354C / 366W mutation, and the second subunit contains a 349C / 366S / 368A / 407V mutation; and the coding rule of the mutation site is Eu coding, GPC3 / TRGV9 binding protein. Claim 8 In any one of claims 1 to 7, the linker further comprises a linker; preferably, the linker is (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h or (A m S n ) h As presented in [the document], wherein m and n are each independently selected from integers 1 to 8, and h is independently selected from integers 1 to 20; more preferably, the linker is an A3S or G4S, a GPC3 / TRGV9 binding protein. Claim 9 In any one of claims 1 to 8, the polypeptide chain is selected from any group of (1) to (3): (1) a first heavy chain, a second heavy chain and a light chain, wherein the first heavy chain is sequentially [immunoglobulin monovariable domain]-[linker 1]-[Fc1] from the N end to the C end, the second heavy chain is sequentially [VH1]-[linker 2]-[CH1]-[linker 3]-[Fc2] from the N end to the C end, and the light chain is sequentially [VL1]-[linker 4]-[CL] from the N end to the C end; said linker 1, linker 2, linker 3 and linker 4 may be the same or different, may exist independently or not exist, and may be independently selected from the linkers defined in claim 8; preferably, linker 1 is AAAS, and linker 2, linker 3 and linker 4 do not exist; (2) a first heavy chain, a first light chain, a second heavy chain and a second light chain, wherein, in the first heavy chain, sequentially from N to C, [VH2]-[linker 1]-[Obscurin-O chain]-[linker 2]-[Fc1], in the first light chain, sequentially from N to C, [VL2]-[linker 3]-[Titin-T chain], in the second heavy chain, sequentially from N to C, [VH1]-[linker 4]-[CH1]-[linker 5]-[Fc2], and in the second light chain, sequentially from N to C, [VL1]-[linker 6]-[CL]; or, in the first heavy chain, sequentially from N to C, [VH1]-[linker 1]-[Obscurin-O [Chain]-[Linker 2]-[Fc1], and in the first light chain, sequentially from the N-stage to the C-stage are [VL1]-[Linker 3]-[Titin-T chain], in the second heavy chain, sequentially from the N-stage to the C-stage are [VH2]-[Linker 4]-[CH1]-[Linker 5]-[Fc2], and in the second light chain, sequentially from the N-stage to the C-stage are [VL2]-[Linker 6]-[CL];The above linkers 1, 2, 3, 4, 5, and 6 may be identical or different, may exist independently or not exist independently, and may be independently selected from the linkers limited to claim 8; preferably, linkers 1 and 3 are G4S, and linkers 2, 4, 5, and 6 do not exist; (3) a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein in the first heavy chain, sequentially from N to C are [VH2]-[linker 1]-[Obscurin-O chain]-[linker 2]-[Fc1], in the first light chain, sequentially from N to C are [VL2]-[linker 3]-[Titin-T chain], and in the second heavy chain, sequentially from N to C are [VH1]-[linker 4]-[CH1]-[Linker 5]-[VH1]-[Linker 6]-[CH1]-[Linker 7]-[Fc2], and in the second light chain, sequentially from the N end to the C end is [VL1]-[Linker 8]-[CL]; or, in the first heavy chain, sequentially from the N end to the C end is [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], in the first light chain, sequentially from the N end to the C end is [VL1]-[Linker 3]-[Titin-T chain], and in the second heavy chain, sequentially from the N end to the C end is [VH2]-[Linker 4]-[CH1]-[Linker 5]-[VH2]-[Linker 6]-[CH1]-[Linker 7]-[Fc2], and the second In the light chain, from the N stage to the C stage, sequentially [VL2]-[Linker 8]-[CL]; said Linker 1, Linker 2, Linker 3, Linker 4, Linker 5, Linker 6, Linker 7, Linker 8, Linker 9, and Linker 10 may be identical or different, may exist independently or not exist, and may be independently selected from the linkers according to claim 8; preferably, Linker 1 and Linker 3 are G4S, Linker 5 is (G4S)2, and Linker 2, Linker 4, Linker 6, Linker 7, and Linker 8 do not exist;In (1) to (3) above, - indicates a peptide bond, and preferably, the amino acid sequences of the Obscurin-O chain and the Titin-T chain are as presented in SEQ ID NO: 47, 48, respectively; preferably, CL is Cκ, and the amino acid sequences of CH1 and Cκ are as presented in SEQ ID NO: 49, 50, respectively; preferably, the amino acid sequences of Fc1 and Fc2 are as presented in SEQ ID NO: 52, 53, respectively, of the GPC3 / TRGV9 binding protein. Claim 10 In any one of claims 1 to 9, the amino acid sequence is as presented in SEQ ID NO: 54 or has at least 80% to 90% identity therewith, a first heavy chain having an amino acid sequence as presented in SEQ ID NO: 55 or has at least 80% to 90% identity therewith, and a light chain having an amino acid sequence as presented in SEQ ID NO: 56 or has at least 80% to 90% identity therewith; (2) an amino acid sequence is as presented in SEQ ID NO: 57 or has at least 80% to 90% identity therewith, a first light chain having an amino acid sequence as presented in SEQ ID NO: 58 or has at least 80% to 90% identity therewith, and a second amino acid sequence is as presented in SEQ ID NO: 59 or has at least 80% to 90% identity therewith A second light chain having a heavy chain, amino acid sequence identical to that presented in SEQ ID NO: 56 or having at least 80% to 90% identity therewith; Or, (3) a polypeptide chain selected from any group of a first heavy chain having an amino acid sequence as presented in SEQ ID NO: 57 or having at least 80% to 90% identity therewith, a first light chain having an amino acid sequence as presented in SEQ ID NO: 58 or having at least 80% to 90% identity therewith, a second heavy chain having an amino acid sequence as presented in SEQ ID NO: 60 or having at least 80% to 90% identity therewith, and a second light chain having an amino acid sequence as presented in SEQ ID NO: 61 or having at least 80% to 90% identity therewith; preferably, the GPC3 / TRGV9 binding protein comprises: (1) a polypeptide chain having an amino acid sequence as presented in SEQ ID NO: 54 to 56; (2) a polypeptide chain having an amino acid sequence as presented in SEQ ID NO: 56 to 59;(3) The amino acid sequence comprises a polypeptide chain selected from any group of polypeptide chains as presented in SEQ ID NO: 57, 58, 60, 61; more preferably, the GPC3 / TRGV9 binding protein comprises: (1) a polypeptide chain as presented in SEQ ID NO: 54–56 with an amino acid sequence having a molar ratio of 1:1:1; (2) a polypeptide chain as presented in SEQ ID NO: 56–59 with an amino acid sequence having a molar ratio of 1:1:1:1; (3) a polypeptide chain selected from any group of polypeptide chains as presented in SEQ ID NO: 57, 58, 60, 61 with an amino acid sequence having a molar ratio of 1:1:1:2; GPC3 / TRGV9 binding protein.; Claim 11 A GPC3 / TRGV9 binding protein according to any one of claims 1 to 10, wherein the antibody or its antigen-binding fragment is, preferably, an anti-GPC3 / TRGV9 antibody or its antigen-binding fragment. Claim 12 As a TRGV9 binding protein, the immunoglobulin monovariable domain comprising: (1) CDR1, CDR2, and CDR3 in the amino acid sequence presented in any one of SEQ ID NO: 29, 26–28, 5, and preferably, CDR1, CDR2, and CDR3 in the amino acid sequence presented in SEQ ID NO: 6–8; or, (2) a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 in an amino acid sequence presented in any one of SEQ ID NO: 9, 30–32, and / or a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 in an amino acid sequence presented in any one of SEQ ID NO: 10, 33–35; wherein the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, e.g., a TRGV9 binding protein defined according to the Kabat numbering system. Claim 13 In claim 12, the immunoglobulin monovariable domain, VH and / or VL is modified by humanization, reverse mutation, affinity maturation, T cell epitope removal, reduction of antibody deamidation and / or reduction of antibody isomerization; preferably, the framework region of the human germline template used in the humanization modification process for the immunoglobulin monovariable domain is derived from IGHV3-64; preferably, the framework region of the human germline template used in the humanization modification process for VH is derived from IGHV3-21, and / or the framework region of the human germline template used in the humanization modification process for VL is derived from IGKV1-12, TRGV9 binding protein. Claim 14 In claim 12 or 13, wherein, (1) the immunoglobulin single variable domain comprises an amino acid sequence that is the same as or has at least 80% or at least 90% identity with any one of SEQ ID NO: 29, 26–28, 5; Or, (2) in VH and VL, said VH comprises an amino acid sequence that is the same as or has at least 80% and at least 90% identity with any one of SEQ ID NO: 9, 30 to 32, and said VL comprises an amino acid sequence that is the same as or has at least 80% and at least 90% identity with any one of SEQ ID NO: 10, 33 to 35; preferably, said TRGV9 binding protein is an anti-TRGV9 antibody or an antigen-binding fragment thereof; more preferably, said TRGV9 binding protein, where the TRGV9 binding protein comprises an immunoglobulin single variable domain, is an anti-TRGV9 single domain antibody or VHH. Claim 15 A TRGV9 binding protein according to any one of claims 12 to 14, wherein the Fc region further comprises an immunoglobulin Fc region; preferably, said Fc region is an Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, said Fc region is more preferably an Fc region of human IgG1; and preferably, said Fc region is an Fc region of IgG1 comprising L234F and / or L235E mutations. Claim 16 A TRGV9 binding protein according to any one of claims 12 to 15, wherein the TRGV9 binding protein further comprises a tumor-associated antigen (TAA) binding domain or a tumor-specific antigen binding domain, preferably an antigen binding domain that specifically binds to GPC3. Claim 17 A GPC3 / TRGV9 binding protein or a TRGV9 binding protein having a property or function selected from any one or more of: (a) specifically binding to the γ9 chain of a TCR, preferably specifically binding to a γ9δ1 TCR or a γ9δ2 TCR; (b) not detectable binding to the γ8 chain of a TCR, preferably not detectable binding to a γ8δ2 TCR; (c) specifically binding to the variable region (TRGV9) of the γ9 chain of a TCR; and (d) specifically binding to a VγδCαβ chimeric TCR, and not detectable binding to a VαβCγδ chimeric TCR. Claim 18 A polynucleotide, wherein the polynucleotide codes for a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11 or a TRGV9 binding protein according to any one of claims 12 to 16, or a combination thereof; preferably, said polynucleotide is DNA or RNA. Claim 19 A vector comprising a polynucleotide according to claim 18, preferably, said vector expressing a polynucleotide according to claim 18. Claim 20 A host cell containing or expressing a polynucleotide according to paragraph 18 or a vector according to paragraph 19. Claim 21 A method for producing a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11 or a TRGV9 binding protein according to any one of claims 12 to 16, comprising: a step of expressing a polynucleotide according to claim 18 or a vector according to claim 19 in a host cell according to claim 20, and a step of isolating the expressed GPC3 / TRGV9 binding protein or TRGV9 binding protein from said host cell; optionally, a method further comprising a step of purifying said GPC3 / TRGV9 binding protein or TRGV9 binding protein. Claim 22 A pharmaceutical composition comprising a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11, a TRGV9 binding protein according to any one of claims 12 to 16, a polynucleotide according to claim 18, and / or a vector according to claim 19; preferably, the pharmaceutical composition further comprises T cells, said T cells are preferably γδ T cells; and preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents, or adjuvants. Claim 23 A combination or kit comprising: (1) a GPC3 / TRGV9 binding protein and a T cell according to any one of claims 1 to 11, or (2) a TRGV9 binding protein and a T cell according to any one of claims 12 to 16; wherein the T cell is preferably a γδ T cell. Claim 24 In the manufacture of a drug for treating a disease, the use of a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11, a TRGV9 binding protein according to any one of claims 12 to 16, a polynucleotide according to claim 18, a vector according to claim 19, a combination or kit of claim 23; wherein the disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, and most preferably GPC3-positive liver cancer. Claim 25 In the manufacture of a drug for treating a disease, the use of a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11, a TRGV9 binding protein according to any one of claims 12 to 16, a polynucleotide according to claim 18, or a vector according to claim 19, wherein: said GPC3 / TRGV9 binding protein, said TRGV9 binding protein, said polynucleotide, or said vector is used in combination with a T cell; said T cell is preferably a γδ T cell; said disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, and most preferably GPC3-positive liver cancer. Claim 26 In the manufacture of a drug for treating a disease, the use of T cells, wherein: said T cells are used in combination with a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11, a TRGV9 binding protein according to any one of claims 12 to 16, a polynucleotide according to claim 18, or a vector according to claim 19; said T cells are preferably γδ T cells; said disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, and most preferably GPC3-positive liver cancer. Claim 27 A method for treating or alleviating a disease, comprising the step of administering to a subject in need an effective amount of a GPC3 / TRGV9 binding protein according to any one of claims 1 to 11, a TRGV9 binding protein according to any one of claims 12 to 16, a polynucleotide according to claim 18, a vector according to claim 19, a pharmaceutical composition according to claim 22, or a combination or kit according to claim 23; wherein the disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, and most preferably GPC3-positive liver cancer.

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