TRGV9-BINDING PROTEINS AND THEIR MEDICAL APPLICATIONS

Abstract

The present invention relates to TRGV9-binding proteins and their medical uses. In particular, the present invention relates to a TRGV9-binding protein, a GPC3 / TRGV9-binding protein, a method for treating malignant neoplasms using these proteins, and pharmaceutical uses of these proteins.

Description

TRGV9 binding protein and its medical use

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

[0002] The present disclosure relates to the field of biomedicine, and in particular to TRGV9 binding protein, GPC3 / TRGV9 binding protein, and methods for treating cancer and pharmaceutical uses thereof. Background Art

[0003] γδT cells are a group of T cells that naturally recognize and kill tumor cells. They have both antigen presentation and killing functions, bridging innate immunity and adaptive immunity. Unlike αβTCR, the two chains that make up the TCR of γδT cells are γ chain and δ chain, and their recognition of antigens is not restricted by MHC (Exp Mol Med.2021Mar;53(3):318-327). The number of γδT cells is relatively small, accounting for about 1%-5% of PBMC. According to the difference in δ chain, they can be divided into four categories: δ1, δ2, δ3, and δ5 (Front Immunol.2022 Jun 16;13:915837), among which δ1 and δ2 account for the largest number. δ1 can pair with different γ chains to form different TCRs, which are mainly distributed in tissues such as the skin and mucous membranes; δ2 mainly pairs with γ9 to form the γ9δ2 subtype, which is mainly distributed in the peripheral blood and accounts for up to 95% of the total γδT cells in the peripheral blood (Front Immunol. 2022 Jun 16; 13:915837).

[0004] γδT cells play an important role in anti-tumor activities and have multiple mechanisms for killing tumor cells, including TCR recognition of the BTN2A / BTN3A complex activated by phosphorylated antigens on target cells, initiating killing signals and exerting killing functions. In addition, there is a killing pathway similar to NK cells, which express receptors such as NKG2D on their cell surface and exert tumor killing effects by binding to corresponding ligands on tumor cells. γδT cells have varying degrees of infiltration in various tumors. A large sample analysis of 25 tumor types and 5782 tumors found that the infiltration of γδT cells indicates a good prognosis for patients and that they are the most favorable cell population among all immune cells. An analysis of 3238 samples of 14 non-brain cancer solid tumors also showed that their infiltration in tumors is an indicator of good prognosis (Oncoimmunology. 2017 Feb 6; 6(3): e1284723). Early clinical studies on γδT cells mainly focused on two methods: in vivo activation and in vitro expansion followed by re-infusion. Although the anti-tumor effects shown by different studies were relatively limited, no serious side effects were reported, suggesting that γδT cells have good safety.

[0005] Glypican-3 (GPC3) is a potential target for the treatment of liver cancer. In 2020, there were 900,000 new cases of liver cancer and 830,000 deaths worldwide; in China, there were 410,000 new cases and 390,000 deaths in 2020; about half of the world's liver cancer cases occurred in China. In recent years, first-line or second-line treatments for advanced liver cancer have made great progress, but there is still a lack of effective treatments for later-line treatments. GPC3 is a heparan sulfate proteoglycan composed of 580 amino acids, which is anchored to the cell membrane through glycosylphosphatidylinositol. GPC3 is highly expressed in various tumor tissues. For example, the GPC3 positive rate in hepatocellular carcinoma is as high as 90%. GPC3 expression in normal tissues is very limited, only expressed in the placenta and endometrium; and the expression level in the endometrium is much lower than that in tumor tissues, making it an ideal anti-tumor target (Sci Transl Med. 2017 Oct 4; 9(410): eaal4291).

[0006] The present disclosure provides antibodies targeting γδTCR with novel sequence structures, and constructs bispecific antibodies with tumor-associated antigen (e.g., GPC3) binding domains (e.g., antibodies). The bispecific antibodies bind to TAA (e.g., GPC3) at one end and to γδTCR at the other end, thereby recruiting and activating γδT cells to specifically kill TAA (e.g., GPC3)-positive tumor cells, significantly improving the effectiveness of γδT cell therapy. The bispecific antibodies disclosed herein have good tumor killing activity, safety, and drugability. Summary of the Invention

[0007] The present disclosure provides T cell receptor (TCR) binding proteins, glypican-3 (GPC3) and T cell receptor (TCR) binding proteins, nucleic acids encoding them, methods for preparing them, and methods and uses thereof for treating diseases.

[0008] T cell receptor gamma variable region 9 (TRGV9) binding protein

[0009] The present disclosure provides TRGV9 binding proteins. In some embodiments, they are capable of binding to TCRs. In some embodiments, they are capable of binding to γδ TCRs. In some embodiments, they are capable of binding to the γ9 chain of TCRs. In some embodiments, they are capable of binding to γ9δ2 TCRs. In other embodiments, they are capable of binding to γ9δ1 TCRs.

[0010] In some embodiments, a TRGV9 binding protein is provided, comprising:

[0011] 1) an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and / or CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 26-29, for example, the immunoglobulin single variable domain comprises CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 26-29; or

[0012] 2) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 9 and 30-32, and the VL comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 10 and 33-35,

[0013] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, e.g., according to the Kabat numbering system.

[0014] In some embodiments, a TRGV9 binding protein is provided, comprising:

[0015] 1) an immunoglobulin single variable domain, the immunoglobulin single variable domain comprising CDR1, CDR2 and / or CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 6, CDR2 comprises the amino acid sequence of SEQ ID NO: 7, and CDR3 comprises the amino acid sequence of SEQ ID NO: 8; or,

[0016] 2) VH and / or VL, wherein the VH comprises HCDR1, HCDR2 and / or HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 13; and the VL comprises LCDR1, LCDR2 and / or LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0017] In some embodiments, a TRGV9 binding protein is provided, comprising:

[0018] 1) an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs: 6-8, respectively; or

[0019] 2) VH and VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 11-13, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 14-16, respectively.

[0020] In some embodiments, the immunoglobulin single variable domain or VH or VL in the aforementioned proteins are each independently humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced and / or antibody isomerization reduced.

[0021] In some embodiments, the heavy chain framework region of the human germline template used for humanization of the immunoglobulin single variable domain in the aforementioned protein is derived from IGHV3-64; the heavy chain framework region of the human germline template used in the humanization process of VH is derived from IGHV3-21, and the light chain framework region of the human germline template used in the humanization process of VL is derived from IGKV1-12.

[0022] In some embodiments, a TRGV9 binding protein is provided, comprising:

[0023] 1) an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; or

[0024] 2) VH and VL, wherein the VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-33, or at least 80% or at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or at least 80% or at least 90% identical thereto.

[0025] In some embodiments, the TRGV9 binding protein comprises the following VH and VL:

[0026] 2-1) VH comprises an amino acid sequence as set forth in SEQ ID NO: 9, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 10, or at least 80% or at least 90% identical thereto,

[0027] 2-2) VH comprises an amino acid sequence as set forth in SEQ ID NO: 30, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0028] 2-3) VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0029] 2-4) VH comprises an amino acid sequence as shown in SEQ ID NO: 32, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto.

[0030] 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% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0031] In some embodiments, the aforementioned TRGV9 binding protein comprises or is an anti-TRGV9 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-TRGV9 antibody or antigen-binding fragment thereof 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).

[0032] When the TRGV9-binding protein comprises 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 TRGV9-binding protein itself is a single domain antibody or VHH. In some specific embodiments, the antibody or antigen-binding fragment thereof is a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody and a diabody, a triabody and a tetrabody, a tandem di-scFv, or a tandem tri-scFv.

[0033] Wherein, when the aforementioned TRGV9 binding protein contains VH and VL, it can be a murine 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, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv. In some specific embodiments, the antigen-binding fragment includes Fab, Fv, sFv, Fab', F(ab')2.

[0034] In some embodiments, the TRGV9 binding protein may comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains. The immunoglobulin single variable domains may form dimers or multimers. The immunoglobulin single variable domains may be homodimers or heterodimers.

[0035] In some embodiments, the aforementioned TRGV9 binding protein may comprise one or more (eg, 2, 3, 4, 5, 6, 7, 8) aforementioned VH, VL or a combination thereof.

[0036] In some embodiments, the aforementioned TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1, for example, as shown in SEQ ID NO: 51, or having at least 80% or at least 90% sequence identity thereto. In some specific embodiments, the Fc region may be an Fc region with reduced effector function, for example, the Fc region may have a mutation, an exemplary IgG with reduced effector function The Fc region includes substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P2 38S (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 means that the sequence contains L234A and L235A.

[0037] In some embodiments, the immunoglobulin single variable domain or VH, VL in the aforementioned TRGV9 binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. 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 ) hAs shown, wherein m and n are each independently selected from an integer of 1 to 8, and h is independently selected from an integer of 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.;

[0038] In some embodiments, the aforementioned 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 anti-TCR γ9 chain antibody or an antigen-binding fragment thereof; a protein that specifically binds to the variable region (Vγ9) of the γ9 chain of TCR, or an anti-Vγ9TCR antibody or an antigen-binding fragment thereof; a protein that specifically binds to γ9δ2 TCR, or an anti-γ9δ2TCR 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.

[0039] In some embodiments, the aforementioned TRGV9 binding protein comprises:

[0040] 1) an amino acid sequence as shown in any one of SEQ ID NO: 36, or an amino acid sequence having at least 80% or at least 90% sequence identity thereto;

[0041] 2) a heavy chain and a light chain, wherein the heavy chain is as shown in any one of SEQ ID NO: 37 or has at least 80%, at least 90% sequence identity thereto, and the light chain is as shown in any one of SEQ ID NO: 38 or has at least 80%, at least 90% sequence identity thereto.

[0042] In some embodiments, the aforementioned TRGV9 binding protein has at least one function or property selected from the group consisting of:

[0043] (a) With EC ≤ 10 nM 50 Combined with γ9δ2 T cells, the EC 50 For example, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. 50 It is obtained by FACS detection. FACS is a commonly used affinity detection method in the art, such as described in Example 5 of the present disclosure.

[0044] (b) specifically binds to the γ9 chain of TCR, for example, specifically binds to γ9δ1 TCR, γ9δ2 TCR;

[0045] (c) does not bind (cannot be detected by the assay) to the γ8 chain of the TCR, e.g., does not bind to the γ8δ2 TCR;

[0046] (d) specifically binds to the variable region of the γ9 chain of TCR (TRGV9);

[0047] (e) specifically binds to VγδCαβ chimeric TCR but not to VαβCγδ chimeric TCR;

[0048] (f) binding to cynomolgus monkey γ9δ2 TCR;

[0049] Among them, (b)-(e) can be obtained by conventional detection methods in the art, such as described in Example 4 of the present disclosure.

[0050] In some embodiments, the aforementioned proteins bind to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0051] In some embodiments, the aforementioned TRGV9 binding protein encompasses a variant of an immunoglobulin single variable domain, wherein the variant has one or more amino acid mutations compared to any one of SEQ ID NOs: 5, 26-29. In some embodiments, the aforementioned TRGV9 binding protein encompasses a variant of VH and / or VL, wherein the VH variant has one or more amino acid mutations compared to any one of SEQ ID NOs: 9, 30-32, and the VL variant has one or more amino acid mutations compared to any one of SEQ ID NOs: 10, 33-35. "Multiple" encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in the CDR region and / or the FR region.

[0052] In some embodiments, a protein is provided that binds to or competes for binding to the same antigenic epitope as the immunoglobulin single variable domain in the TRGV9 binding protein of the present disclosure.

[0053] In some embodiments, proteins are provided that bind to or compete for binding to the same antigenic epitope as the VH and VL in the aforementioned TRGV9 binding proteins of the present disclosure.

[0054] In some embodiments, a protein is provided whose binding to TRGV9 is blocked by the immunoglobulin single variable domain or VH and VL in the TRGV9 binding protein of the present disclosure.

[0055] In some embodiments, a protein or molecule is provided that comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains disclosed herein, for example, the immunoglobulin single variable domain comprises the CDR1, CDR2, and CDR3 of SEQ ID NOs: 6-8, or comprises the sequence of any one of SEQ ID NOs: 5, 26-29. The protein or molecule may be a conjugate or fusion protein formed with other compounds or other polypeptides, and the conjugate may, for example, comprise any detectable label.

[0056] In some embodiments, a protein or molecule is provided that comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) VH and VL of the foregoing disclosure, for example, the VH comprises the HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 11-13, and the VL comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 14-16, respectively. The protein or molecule may be a conjugate, coupling, or fusion protein formed with other compounds or other polypeptides. For example, the conjugate may comprise any detectable label.

[0057] GPC3 / TRGV9 binding protein

[0058] The present disclosure provides proteins that bind to GPC3 and TCR. In some embodiments, proteins are provided that bind to GPC3 and γδTCR. In some embodiments, proteins are provided that bind to GPC3 and the γ9 chain of TCR. In some embodiments, proteins are provided that bind to GPC3 and the variable region of the γ9 chain of TCR (Vγ9). In some embodiments, proteins are provided that bind to GPC3 and γ9δ2 TCR. In some embodiments, proteins are provided that bind to GPC3 and γ9δ1 TCR. In some embodiments, proteins are provided that bind to GPC3 and TRGV9. In some embodiments, the "binding" is simultaneous or sequential binding.

[0059] The present disclosure provides binding proteins that bind to tumor-associated antigens (TAA), tumor-specific antigens, and TRGV9, comprising a binding domain that binds to 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 TAA (or tumor-specific antigen) and a second antigen-binding domain that specifically binds to TRGV9. In some embodiments, the second antigen-binding domain that specifically binds to TRGV9 is the aforementioned TRGV9 binding protein of the present disclosure. In some embodiments, the second antigen-binding domain that specifically binds to TRGV9 is an immunoglobulin single variable domain in the aforementioned TRGV9 binding protein of the present disclosure, and / or a combination of VH and VL.

[0060] 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.

[0061] In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH1) and / or a light chain variable region (VL1), wherein the VH1 comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 39, and the VL1 comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 39, and the VL1 comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO: 40, and the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system.

[0062] In some specific embodiments, the first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 41-43; the VL1 comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 44-46.

[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 the VH1 comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 41-43, respectively; and the VL1 comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 44-46, respectively.

[0064] In some specific embodiments, the amino acid sequence of the first antigen-binding domain comprises any one or more sequences selected from SEQ ID NOs: 41-46.

[0065] In some embodiments, the VH1 and / or VL1 are humanized, backmutated, affinity matured, engineered to remove / reduce T cell epitopes (TCEs), reduce antibody deamidation, and / or reduce antibody isomerization.

[0066] In some specific embodiments, the VH1 comprises an amino acid sequence as shown in SEQ ID NO: 39, or a sequence identity thereof of at least 80%, at least 90%, and / or the VL1 comprises an amino acid sequence as shown in SEQ ID NO: 40, or a sequence identity thereof of at least 80%, at least 90%.

[0067] In some specific embodiments, the amino acid sequence of the VH1 is shown in SEQ ID NO: 39, and the amino acid sequence of the VL1 is shown in SEQ ID NO: 40.

[0068] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising any of the aforementioned first antigen-binding domains that specifically bind to GPC3 and a second antigen-binding domain that specifically binds to TRGV9, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0069] 1) an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and / or CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 26-29, for example, the immunoglobulin single variable domain comprises CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 26-29; or

[0070] 2) a heavy chain variable region (VH2) and / or a light chain variable region (VL2), wherein the VH2 comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 9 and 30-32, and the VL2 comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 10 and 33-35,

[0071] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, e.g., according to the Kabat numbering system.

[0072] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising any of the aforementioned first antigen-binding domains that specifically bind to GPC3 and a second antigen-binding domain that specifically binds to TRGV9, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0073] 1) an immunoglobulin single variable domain, the immunoglobulin single variable domain comprising CDR1, CDR2 and / or CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 6, CDR2 comprises the amino acid sequence of SEQ ID NO: 7, and CDR3 comprises the amino acid sequence of SEQ ID NO: 8; or,

[0074] 2) VH2 and / or VL2, wherein the VH2 comprises HCDR1, HCDR2 and / or HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 13; and the VL comprises LCDR1, LCDR2 and / or LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0075] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising any of the aforementioned first antigen-binding domains that specifically bind to GPC3 and a second antigen-binding domain that specifically binds to TRGV9, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0076] 1) an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs: 6-8, respectively; and / or,

[0077] 2) VH2 and VL2, wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 11-13, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 14-16, respectively.

[0078] In some embodiments, the aforementioned immunoglobulin single variable domain or VH, VL is humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced and / or antibody isomerization reduced.

[0079] In some embodiments, the heavy chain framework region of the human germline template used for humanization of the aforementioned immunoglobulin single variable domain is derived from IGHV3-64; the heavy chain framework region of the human germline template used in the humanization process of VH is derived from IGHV3-21, and the light chain framework region of the human germline template used in the humanization process of VL is derived from IGKV1-12.

[0080] In some embodiments, a GPC3 / TRGV9 binding protein is provided, comprising any of the aforementioned first antigen-binding domains that specifically bind to GPC3 and a second antigen-binding domain that specifically binds to TRGV9, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0081] 1) an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; or

[0082] 2) VH2 and / or VL2, wherein the VH2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-32, or at least 80% or at least 90% identical thereto, and the VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or at least 80% or at least 90% identical thereto.

[0083] In some specific embodiments, the combination of VH2 and VL2 is selected from the following:

[0084] 2-1) VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 10, or at least 80% or at least 90% identical thereto,

[0085] 2-2) VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 30, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0086] 2-3) VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0087] 2-4) VH2 comprises an amino acid sequence as shown in SEQ ID NO: 32, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto.

[0088] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises or is 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, a multispecific antibody (e.g., a trispecific antibody). In some embodiments, the immunoglobulin single variable domain in the anti-GPC3 / TRGV9 antibody or its antigen-binding fragment is a single domain antibody or VHH. In some specific embodiments, the antigen-binding fragment includes but is not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, diabodies, triabodies and tetrabodies, tandem two-scFv, tandem three-scFv.

[0089] 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 single variable domains. In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned VH1 and VL1 combinations. 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 VH2 and VL2 combinations.

[0090] 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.

[0091] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is the Fc region of human IgG1, for example, as shown in SEQ ID NO: 51, or having at least 80% or at least 90% sequence identity thereto. In some specific embodiments, the Fc region may be an Fc region with reduced effector function, for example, the Fc region may have a mutation, an exemplary IgG with reduced effector function The Fc region includes substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P2 38S(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).

[0092] In some embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2). In some embodiments, a mutation is introduced that causes the two subunits (Fc1, Fc2) of the Fc region to pair and form a dimer, or a mutation that reduces homodimerization. In some embodiments, the first and second subunits contain knob-into-hole mutations. For example, within the CH3 / CH3 interface, one, two, or more amino acid residues in the CH3 domain of Fc1 are mutated to one or more amino acid residues with a larger side chain volume, thereby creating 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 to amino acid residues with a smaller side chain volume, thereby creating a depression (or hole) on the surface of the CH3 domain of Fc2. The first subunit (Fc1) and the second subunit (Fc2) differ only in that they are two different subunits, and therefore, the two are interchangeable.

[0093] In some embodiments, the Fc1 contains one or more amino acid substitutions at positions selected from 354, 356, 358, and 366, and the Fc2 contains one or more amino acid substitutions at positions selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the Fc1 contains a mutation at position 366, and the Fc2 contains a mutation at position 366, 368, and 407, or any combination thereof. In some embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349. In some embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains mutations at position 349, 366, 368, and 407.

[0094] In some embodiments, the Fc1 comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 comprises one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the Fc1 comprises a 366W mutation, and the Fc2 comprises a mutation selected from 366S, 368A, and 407V, or any combination thereof. In some embodiments, the Fc1 comprises a 354C or 356C mutation, and the Fc2 comprises a 349C mutation. Alternatively, in some embodiments, the Fc1 comprises 354C / 366W mutations, and the Fc2 comprises 349C / 366S / 368A / 407V mutations.

[0095] In some specific embodiments, the Fc1 comprises a T366W mutation, and the Fc2 comprises a mutation selected from T366S, L368A, and Y407V, or any combination thereof; the Fc1 comprises an S354C or E356C mutation, and the Fc2 comprises a Y349C mutation; or the Fc1 comprises S354C / T366W mutations, and the Fc2 comprises Y349C / T366S / L368A / Y407V mutations. In some specific embodiments, the amino acid sequence of Fc1 is set forth in SEQ ID NO: 52, and the amino acid sequence of Fc2 is set forth in SEQ ID NO: 53.

[0096] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises amino acid size and charge mutations of the heavy chain CH1 and light chain CL interface amino acids to reduce mismatching between the light and heavy chains. For example, Roche exchanged the CH1 and CL domains and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187–11192(2011)), MedImmune introduced disulfide bonds by mutating the heavy chain F126C and the light chain S121C (Mazor et al., mAbs, 7(2), pp.377–389(2015)), Amgen further modified the CH1-CL region with electrostatic interactions (Liu et al., Journal of Biological Chemistry, 290(12), pp.7535–7562(2015)), and Lilly (Lewis et al., Nature Biotechnology, 32(2), pp.191–198 (2014)) and Genentech (Dillon et al., mAbs, 9(2), pp.213–230 (2017)) introduced mutations in both the variable and constant domains. The constant region of the antibody was replaced with that of a TCR, as described in CN109535257A (cited and incorporated in its entirety).

[0097] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises an Obscurin-O chain (as shown in SEQ ID NO: 47) and a Titin-T chain (as shown in SEQ ID NO: 48) to prevent or reduce mispairing between different VH and VL.

[0098] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein comprises a linker.

[0099] In some embodiments, the immunoglobulin single variable domain or VH, VL in the aforementioned GPC3 / TRGV9 binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. For example, the linker is (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) hor (EPKSS) h or (A m S n ) h As shown, wherein m and n are each independently selected from an integer of 1 to 8, and h is independently selected from an integer of 1 to 20. For example, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, A3S, etc.

[0100] In some embodiments, the GPC3 / TRGV9 binding protein comprises a combination selected from the group consisting of:

[0101] 1) a first heavy chain, a second heavy chain and a light chain, wherein,

[0102] The first heavy chain, from N-terminus to C-terminus, consists of: [immunoglobulin single variable domain]-[linker 1]-[Fc1],

[0103] The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc2],

[0104] Light chain, from N-terminus to C-terminus: [VL1]-[Linker 4]-[CL];

[0105] or

[0106] The first heavy chain, from N-terminus to C-terminus, consists of: [immunoglobulin single variable domain]-[linker 1]-[Fc2],

[0107] The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc1],

[0108] Light chain, from N-terminus to C-terminus: [VL1]-[Linker 4]-[CL];

[0109] Wherein, - represents a peptide bond, and the linker 1, linker 2, linker 3 and linker 4 may be the same or different, may exist independently or not, and may be independently selected from the linkers disclosed above;

[0110] In some embodiments, Linker 1 is AAAS, and Linker 2, Linker 3, and Linker 4 are absent.

[0111] 2) a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein:

[0112] The first heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],

[0113] The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 3]-[Titin-T chain],

[0114] The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 4]-[CH1]-[Linker 5]-[Fc2],

[0115] The second light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 6]-[CL];

[0116] or,

[0117] The first heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],

[0118] The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 3]-[Titin-T chain],

[0119] The second heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 4]-[CH1]-[Linker 5]-[Fc2],

[0120] The second light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 6]-[CL]

[0121] Wherein, - represents a peptide bond, and the linker 1, linker 2, linker 3, linker 4, linker 5 and linker 6 may be the same or different, may exist independently or not, and may be independently selected from the linkers disclosed above;

[0122] In some specific embodiments, linker 1 and linker 3 are GGGGS, and linker 2, linker 4, linker 5, and linker 6 do not exist.

[0123] 3) a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein:

[0124] The first heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],

[0125] The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 3]-[Titin-T chain],

[0126] The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 4]-[CH1]-[Linker 5]-[VH1]-[Linker 6]-[CH1]-[Linker 7]-[Fc2],

[0127] The second light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 8]-[CL];

[0128] or,

[0129] The first heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1],

[0130] The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 3]-[Titin-T chain],

[0131] The second heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 4]-[CH1]-[Linker 5]-[VH2]-[Linker 6]-[CH1]-[Linker 7]-[Fc2],

[0132] The second light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 8]-[CL];

[0133] Wherein, - represents a peptide bond, and the linker 1, linker 2, linker 3, linker 4, linker 5, linker 6, linker 7, and linker 8 may be the same or different, may exist independently or not, and may be independently selected from the linkers disclosed above;

[0134] 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 are absent;

[0135] In some specific embodiments, the molar ratio of first heavy chain:first light chain:second heavy chain:second light chain is 1:1:1:2.

[0136] In some embodiments, the amino acid sequences of the Obscurin-O chain and the Titin-T chain are shown in SEQ ID NOs: 47 and 48, respectively. In some embodiments, CL is Cκ, and the amino acid sequences of CH1 and Cκ are shown in SEQ ID NOs: 49 and 50, respectively. In some embodiments, the amino acid sequences of Fc1 and Fc2 are shown in SEQ ID NOs: 52 and 53, respectively.

[0137] In some embodiments, the GPC3 / TRGV9 binding protein is selected from the group consisting of:

[0138] 1) comprising a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 54, or at least 80%, at least 90% identical thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 55, or at least 80%, at least 90% identical thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 56, or at least 80%, at least 90% identical thereto;

[0139] 2) comprising a first heavy chain having an amino acid sequence as set forth in SEQ ID NO:57, or at least 80%, at least 90% identical thereto, a first light chain having an amino acid sequence as set forth in SEQ ID NO:58, or at least 80%, at least 90% identical thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO:59, or at least 80%, at least 90% identical thereto, and a second light chain having an amino acid sequence as set forth in SEQ ID NO:56, or at least 80%, at least 90% identical thereto; or,

[0140] 3) comprising a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 57, or at least 80%, at least 90% identical thereto, a first light chain having an amino acid sequence as set forth in SEQ ID NO: 58, or at least 80%, at least 90% identical thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 60, or at least 80%, at least 90% identical thereto, and a second light chain having an amino acid sequence as set forth in SEQ ID NO: 61, or at least 80%, at least 90% identical thereto.

[0141] In some embodiments, the GPC3 / TRGV9 binding protein is selected from the group consisting of:

[0142] 1) A combination of polypeptides comprising amino acid sequences as set forth in SEQ ID NOs: 54-56; in some embodiments, the molar ratio of the polypeptide of SEQ ID NO: 54: the polypeptide of SEQ ID NO: 55: the polypeptide of SEQ ID NO: 56 in the GPC3 / TRGV9 binding protein is 1:1:1;

[0143] 2) a combination of polypeptides comprising amino acid sequences as set forth in SEQ ID NOs: 56-59; in some embodiments, the molar ratio of the polypeptide of SEQ ID NO: 56: the polypeptide of SEQ ID NO: 57: the polypeptide of SEQ ID NO: 58: the polypeptide of SEQ ID NO: 59 in the GPC3 / TRGV9 binding protein is 1:1:1:1;

[0144] 3) A combination of polypeptides comprising amino acid sequences as shown in SEQ ID NOs: 57, 58, 60, and 61; in some embodiments, the molar ratio of the polypeptide shown in SEQ ID NO: 57: the polypeptide shown in SEQ ID NO: 58: the polypeptide shown in SEQ ID NO: 60: the polypeptide shown in SEQ ID NO: 61 in the GPC3 / TRGV9 binding protein is 1:1:1:2.

[0145] 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 GPC3 and the γ9 chain of TCR, or an anti-GPC3 / TCRγ9 chain antibody or an antigen-binding fragment thereof; a protein that specifically binds to GPC3 and the variable region (Vγ9) of the γ9 chain of TCR, or an anti-GPC3 / Vγ9TCR 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; a protein that specifically binds to GPC3 and TRGV9, or an anti-GPC3 / TRGV9 antibody or an antigen-binding fragment thereof

[0146] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein has at least one function or property selected from the group consisting of:

[0147] (a) With EC ≤ 10 nM 50 Combined with γ9δ2 T cells, the EC 50 For example, ≤5nM, ≤4nM, ≤3nM, ≤2nM, ≤1nM, ≤0.5nM, ≤0.2nM, ≤0.1nM. 50 The affinity is obtained by FACS detection, which is a commonly used affinity detection method in the art, such as described in Example 5 of the present disclosure;

[0148] (b) specifically binds to the γ9 chain of TCR, for example, specifically binds to γ9δ1 TCR, specifically binds to γ9δ2 TCR;

[0149] (c) does not bind the γ8 chain of the TCR, e.g., does not bind the γ8δ2 TCR;

[0150] (d) specifically binds to the variable region of the γ9 chain of TCR (TRGV9);

[0151] (e) specifically binds to VγδCαβ chimeric TCR but not to VαβCγδ chimeric TCR;

[0152] Wherein, (b)-(e) can be obtained by conventional detection methods in the art, such as those described in Example 4 of the present disclosure;

[0153] (f) specifically binds to GPC3 protein or GPC3-positive cells, and does not bind or barely binds to GPC3-negative cells (e.g., PBMCs), for example, using the detection method of Example 8 of the present disclosure;

[0154] (g) binds to cynomolgus monkey GPC3 and γ9δ2 proteins;

[0155] (h) does not block the natural BTN2A / BTN3A-TCR signal, for example, using the detection method of Example 9 of the present disclosure;

[0156] (i) mediating γδT cells killing GPC3-positive tumor cells or mediating γδT cells killing GPC3-positive tumor cells in a GPC3 expression level-dependent manner, for example, using the detection method of Example 10 of the present disclosure; mediating γδT cells killing GPC3-positive tumor cells in an effector-target ratio-dependent manner, for example, using the detection method of Example 12 of the present disclosure; mediating γδT cells killing GPC3-positive tumor cells in a manner with low individual variability, for example, using the detection method of Example 11 of the present disclosure;

[0157] (j) γδT cells induce the release of fewer cytokines (such as IFNγ and TNFα) during the killing of GPC3-positive tumor cells, for example, using the detection method of Example 13 of the present disclosure;

[0158] (k) promoting the proliferation of γδT cells in PBMCs, for example, using the detection method of Example 14 of the present disclosure;

[0159] (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 in vivo mouse model is as described in the detection method of Example 15 of the present disclosure.

[0160] In some embodiments, the aforementioned GPC3 / TRGV9 binding protein binds to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

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

[0162] In some embodiments, the immunoglobulin single variable domain in the aforementioned GPC3 / TRGV9 binding protein encompasses a variant having one or more amino acid mutations compared to any one of SEQ ID NOs: 5, 26-29. In some embodiments, the VH1 and / or VL1 in the aforementioned GPC3 / TRGV9 binding protein encompasses a variant having one or more amino acid mutations compared to any one of SEQ ID NOs: 39, and the VL variant having one or more amino acid mutations compared to any one of SEQ ID NOs: 40. In some embodiments, the VH2 and / or VL2 in the aforementioned GPC3 / TRGV9 binding protein encompasses a variant having one or more amino acid mutations compared to any one of SEQ ID NOs: 9, 30-32, and the VL variant having one or more amino acid mutations compared to any one of SEQ ID NOs: 10, 33-35. "Multiple" includes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid mutation may be a conservative replacement, substitution or modification, and / or a deletion or addition that does not affect the function; the amino acid mutation may occur in the CDR region and / or the FR region.

[0163] In some embodiments, proteins are provided that bind to or compete for binding to the same TRGV9 and / or GPC3 antigenic epitopes as the aforementioned GPC3 / TRGV9 binding proteins of the present disclosure.

[0164] In some embodiments, a protein is provided whose binding to TRGV9 and / or GPC3 is blocked by the aforementioned GPC3 / TRGV9 binding protein of the present disclosure.

[0165] In some embodiments, a protein or molecule is provided that comprises any of the GPC3 / TRGV9 binding proteins disclosed herein. The protein or molecule may be a conjugate, coupling, or fusion protein formed with other compounds or other polypeptides. For example, the conjugate may comprise any detectable label.

[0166] Polynucleotides and vectors

[0167] The present disclosure provides polynucleotides encoding the TRGV9 binding protein and GPC3 / TRGV9 binding protein of the present disclosure. The nucleic acid of the present disclosure may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acid of the present disclosure is a substantially isolated nucleic acid.

[0168] The nucleic acid of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector may in particular be an expression vector, i.e., a vector that provides for expressing the 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). The expression vector typically comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a particular host. Regulatory elements and other elements that are useful or necessary for the expression of the TRGV9 binding protein, GPC3 / TRGV9 binding protein of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0169] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0170] In some embodiments, a polynucleotide is provided that encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises:

[0171] 1) an immunoglobulin single variable domain comprising a CDR1, a CDR2 and / or a CDR3 of the amino acid sequence of any one of SEQ ID NOs: 5 and 26-29, for example, an immunoglobulin single variable domain comprising a CDR3 of the amino acid sequence of any one of SEQ ID NOs: 5 and 26-29; and / or,

[0172] 2) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises the HCDR1, HCDR2 and / or HCDR3 of any one of the amino acid sequences shown in SEQ ID NOs: 9 and 30-32, and the VL comprises the LCDR1, LCDR2 and / or LCDR3 of any one of the amino acid sequences shown in SEQ ID NOs: 10 and 33-35,

[0173] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, e.g., according to the Kabat numbering system.

[0174] In some embodiments, a polynucleotide is provided that encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises:

[0175] 1) an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs: 6-8, respectively; and / or,

[0176] 2) VH and VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 11-13, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 14-16, respectively.

[0177] In some embodiments, a polynucleotide is provided that encodes a TRGV9 binding protein, the TRGV9 binding protein comprising: 1) an immunoglobulin single variable domain, the immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; and / or,

[0178] 2) VH and / or VL, wherein the VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-33, or at least 80% or at least 90% identical thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or at least 80% or at least 90% identical thereto.

[0179] In some embodiments, a polynucleotide is provided that encodes a TRGV9 binding protein comprising the following VH and VL:

[0180] 2-1) VH comprises an amino acid sequence as set forth in SEQ ID NO: 9, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO: 10, or at least 80% or at least 90% identical thereto,

[0181] 2-2) VH comprises an amino acid sequence as set forth in SEQ ID NO: 30, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0182] 2-3) VH comprises an amino acid sequence as set forth in SEQ ID NO: 31, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0183] 2-4) VH comprises an amino acid sequence as shown in SEQ ID NO: 32, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto.

[0184] In some embodiments, a polynucleotide is provided that encodes a TRGV9 binding protein, wherein the TRGV9 binding protein comprises:

[0185] 1) an amino acid sequence as shown in any one of SEQ ID NO: 36, or an amino acid sequence having at least 80% or at least 90% sequence identity thereto;

[0186] 2) a heavy chain and a light chain, wherein the heavy chain is as shown in any one of SEQ ID NO: 37 or has at least 80%, at least 90% sequence identity thereto, and the light chain is as shown in any one of SEQ ID NO: 38 or has at least 80%, at least 90% sequence identity thereto.

[0187] In some embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein comprising a first antigen-binding domain that specifically binds to GPC3 and a second antigen-binding domain that specifically binds to TRGV9.

[0188] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the aforementioned first antigen-binding domain comprises a heavy chain variable region (VH1) and / or a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 39, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 40.

[0189] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the aforementioned first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 41-43, respectively; and the VL1 comprises LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 44-46, respectively.

[0190] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the aforementioned first antigen-binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises an amino acid sequence as shown in SEQ ID NO: 39, or a sequence identity thereof of at least 80%, at least 90%, and / or the VL1 comprises an amino acid sequence as shown in SEQ ID NO: 40, or a sequence identity thereof of at least 80%, at least 90%.

[0191] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0192] 1) an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and / or CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 26-29, for example, the immunoglobulin single variable domain comprises CDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 5, 26-29; or

[0193] 2) a heavy chain variable region (VH2) and / or a light chain variable region (VL2), wherein the VH2 comprises the HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 9 and 30-32, and the VL2 comprises the LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 10 and 33-35.

[0194] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0195] 1) an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3 having amino acid sequences as shown in SEQ ID NOs: 6-8, respectively; and / or,

[0196] 2) VH2 and VL2, wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 11-13, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 14-16, respectively.

[0197] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises:

[0198] 1) an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 26-29, or having at least 80% or at least 90% identity thereto; or

[0199] 2) VH2 and / or VL2, wherein the VH2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-32, or at least 80% or at least 90% identical thereto, and the VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or at least 80% or at least 90% identical thereto.

[0200] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein, wherein the second antigen-binding domain that specifically binds to TRGV9 comprises the following VH2 and VL2:

[0201] 2-1) VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 9, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 10, or at least 80% or at least 90% identical thereto,

[0202] 2-2) VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 30, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0203] 2-3) VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 31, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto;

[0204] 2-4) VH2 comprises an amino acid sequence as shown in SEQ ID NO: 32, or at least 80% or at least 90% identical thereto, and VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 33-35, or at least 80% or at least 90% identical thereto.

[0205] In some specific embodiments, a polynucleotide is provided that encodes a GPC3 / TRGV9 binding protein selected from the group consisting of:

[0206] 1) comprising a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 54, or at least 80%, at least 90% identical thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 55, or at least 80%, at least 90% identical thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 56, or at least 80%, at least 90% identical thereto;

[0207] 2) comprising a first heavy chain having an amino acid sequence as set forth in SEQ ID NO:57, or at least 80%, at least 90% identical thereto, a first light chain having an amino acid sequence as set forth in SEQ ID NO:58, or at least 80%, at least 90% identical thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO:59, or at least 80%, at least 90% identical thereto, and a second light chain having an amino acid sequence as set forth in SEQ ID NO:56, or at least 80%, at least 90% identical thereto; or,

[0208] 3) comprising a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 57, or at least 80%, at least 90% identical thereto, a first light chain having an amino acid sequence as set forth in SEQ ID NO: 58, or at least 80%, at least 90% identical thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 60, or at least 80%, at least 90% identical thereto, and a second light chain having an amino acid sequence as set forth in SEQ ID NO: 61, or at least 80%, at least 90% identical thereto.

[0209] host cells

[0210] The present disclosure provides recombinant host cells that express or are capable of expressing one or more TRGV9 binding proteins, GPC3 / TRGV9 binding proteins, and / or polynucleotides or vectors containing the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0211] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0212] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0213] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0214] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0215] Preparation method

[0216] The present disclosure provides a method for preparing a TRGV9 binding protein or a GPC3 / TRGV9 binding protein, comprising: expressing the target protein in a host cell as described above, and isolating the target protein from the host cell. Optionally, a purification step may also be included, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, and then eluting the bound antibodies using a pH gradient method, detecting with SDS-PAGE, and collecting. Optionally, conventional methods are used for filtration and concentration. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or freeze-dried.

[0217] Methods for producing and purifying antibodies are well known in the art and can be found in, for example, the Cold Spring Harbor Manual of Antibody Laboratory Techniques (Chapters 5-8 and 15).

[0218] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0219] Composition

[0220] The present disclosure provides compositions comprising the aforementioned TRGV9-binding protein and / or GPC3 / TRGV9-binding protein of the present disclosure. For example, pharmaceutical compositions are provided, comprising an effective amount of the aforementioned TRGV9-binding protein and / or GPC3 / TRGV9-binding protein for treating, alleviating, or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent, or adjuvant.

[0221] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the TRGV9-binding protein and / or GPC3 / TRGV9-binding protein in a unit dose, or the amount of the TRGV9-binding protein and / or GPC3 / TRGV9-binding protein in a unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0222] In some embodiments, an article or product (e.g., a kit) is provided that includes the aforementioned TRGV9-binding protein and / or GPC3 / TRGV9-binding protein. Optionally, the article comprises a container and a label. Containers such as bottles, syringes, and test tubes contain a pharmaceutical composition effective for treating a condition. A label on or associated with the container indicates that the pharmaceutical composition is used to treat the selected condition.

[0223] In some embodiments, the aforementioned disease is a cell proliferative disease or cancer.

[0224] Methods of treatment and pharmaceutical uses

[0225] The present disclosure provides the aforementioned TRGV9 binding protein, GPC3 / TRGV9 binding protein, polynucleotides encoding the same, and compositions (including pharmaceutical compositions) for use in methods for treating, alleviating, preventing, and diagnosing diseases or disorders.

[0226] In some embodiments, a method for improving, alleviating, treating or preventing a disease is provided, comprising administering to a subject an improving, alleviating, treating or preventing effective amount of:

[0227] 1) the TRGV9 binding protein or its encoding polynucleotide, or pharmaceutical composition disclosed above;

[0228] 2) The GPC3 / TRGV9 binding protein or its encoding polynucleotide and pharmaceutical composition disclosed above.

[0229] In some embodiments, there is provided the use of the aforementioned 1)-2) for preparing a drug for improving, alleviating, treating or preventing a disease.

[0230] In some embodiments, methods for treating a disease by combining a TRGV9 binding protein of the present disclosure with γδT cells are provided. In some embodiments, methods for treating a disease by combining a GPC3 / TRGV9 binding protein of the present disclosure with γδT cells are provided. In some embodiments, the γδT cells are autologous or allogeneic.

[0231] In some embodiments, any of the aforementioned TRGV9-binding proteins disclosed herein are used to treat a disease, including in combination with γδ T cells. In some embodiments, γδ T cells are used to treat a disease, including in combination with any of the aforementioned TRGV9-binding proteins disclosed herein.

[0232] In some embodiments, any of the aforementioned GPC3 / TRGV9 binding proteins disclosed herein are used to treat a disease, including in combination with γδT cells. In some embodiments, γδT cells are used to treat a disease, including in combination with any of the aforementioned GPC3 / TRGV9 binding proteins disclosed herein.

[0233] In some embodiments, a method for treating a disease is provided, comprising administering a therapeutically effective amount of any of the aforementioned TRGV9 binding proteins of the present disclosure and γδT cells to a subject in need thereof. In some embodiments, a method for treating a disease is provided, comprising administering a therapeutically effective amount of any of the aforementioned GPC3 / TRGV9 binding proteins of the present disclosure and γδT cells to a subject in need thereof.

[0234] In some embodiments, the aforementioned disease is a disease or disorder caused by overexpression of GPC3.

[0235] In some embodiments, the aforementioned disease is a cell proliferative disease or cancer.

[0236] In some embodiments, the cancer is GPC3-positive.

[0237] In some embodiments, the aforementioned disease is liver cancer.

[0238] In some embodiments, the cancer is GPC3-positive liver cancer.

[0239] Detection

[0240] The present disclosure provides TRGV9 binding proteins, GPC3 / TRGV9 binding proteins, and detection uses of polynucleotides and compositions encoding the same. The present disclosure also provides methods, systems, or devices for in vivo or in vitro detection of GPC3 and TRGV9, comprising treating a sample with the aforementioned binding proteins, polynucleotides, and compositions of the present disclosure.

[0241] In some embodiments, a kit is also provided, comprising the aforementioned TRGV9-binding protein, GPC3 / TRGV9-binding protein, polynucleotides encoding the same, and a composition, and instructions for diagnostic use. 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 polynucleotides encoding the same can be formulated as a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0242] FIG1 is a schematic diagram of the structure of an anti-GPC3 / γδTCR bispecific antibody.

[0243] Figures 2A to 2D show the FACS binding activity assay results of the anti-GPC3 / γδTCR bispecific antibody to GPC3-positive cells and human γ9δ2 T cells. Figure 2A shows the binding of the anti-GPC3 / γδTCR bispecific antibody to HepG2 cells; Figure 2B shows the binding of the anti-GPC3 / γδTCR bispecific antibody to DLD-1 cells; Figure 2C shows the binding of the anti-GPC3 / γδTCR bispecific antibody to human γδT cells; and Figure 2D shows the binding of the anti-GPC3 / γδTCR bispecific antibody to PBMCs.

[0244] Figures 3A and 3B show the effects of anti-γδTCR antibodies on native BTN2A / BTN3A-TCR signaling. Figure 3A shows the effects of SDP01378 on native BTN2A / BTN3A-TCR signaling; Figure 3B shows the effects of SDP01315 on native BTN2A / BTN3A-TCR signaling.

[0245] Figures 4A to 4D show the cytotoxicity of anti-γδTCR antibodies against tumor cells expressing different levels of GPC3. Figure 4A shows the cytotoxicity against HepG2 cells, Figure 4B shows the cytotoxicity against Huh-7 cells, Figure 4C shows the cytotoxicity against MKN-45 cells, and Figure 4D shows the cytotoxicity against DLD-1 cells.

[0246] Figures 5A to 5D show the cytotoxicity of γδT cells from different donors against HepG2 cells. The γδT cells used in Figures 5A to 5D were derived from the induced expansion of donors #SC12004, #SC12392, #XC11053, and #XC11061, respectively.

[0247] Figures 6A to 6C show the killing results of anti-GPC3 / γδTCR bispecific antibodies against low-antigen-expressing tumor cells at different effector-target ratios. Figure 6A shows an effector-target ratio of 1:1, Figure 6B shows an effector-target ratio of 10:1, and Figure 6C shows an effector-target ratio of 30:1.

[0248] Figures 7A to 7D show the results of increasing the proportion of PBMCs in the cytotoxicity of γδT cells. Figure 7A shows the cytotoxicity of PBMCs spiked with donor #XC11053; Figure 7B shows the cytotoxicity of 30% PBMCs spiked with donor #XC11053; Figure 7C shows the cytotoxicity of PBMCs spiked with donor #SC12392; Figure 7D shows the cytotoxicity of 30% PBMCs spiked with donor #SC12392.

[0249] Figures 8A to 8D show the results of cytokine release assays, wherein Figures 8A-8B show the release of IFNγ and TNFα upon killing donor #XC11053, and Figures 8C-8D show the release of IFNγ and TNFα upon killing donor #SC12392.

[0250] FIG9 shows the detection results of anti-GPC3 / γδTCR bispecific antibody promoting the proliferation of γδT cells in PBMCs.

[0251] 10A and 10B show the anti-tumor activity of SDP01716 in a Huh-7 xenograft tumor model, wherein FIG10A shows the changes in tumor volume in mice, and FIG10B shows the changes in body weight in mice.

[0252] Figures 11A and 11B show the anti-tumor activity of different anti-GPC3 / γδTCR bispecific antibodies in the Huh-7 xenograft tumor model, wherein Figure 11A shows the changes in mouse tumor volume, and Figure 11B shows the changes in mouse body weight. DETAILED DESCRIPTION

[0253] definition

[0254] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0255] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0256] "TRGV9" refers to a polypeptide that can form a T cell receptor when expressed on the surface of γδT cells. γδT cells expressing TRGV9 are one of the earliest T cells to develop in the human fetus and are the main γδT cell subset in the peripheral blood cells of healthy adults. "TRGV9" includes any TRGV9 variant, isoform, and species homolog that is naturally expressed by cells (including T cells) or can be expressed on cells transfected with a gene or cDNA encoding the polypeptide. In a specific embodiment, TRGV9 is human TRGV9. An exemplary human TRGV9 amino acid sequence is provided by GenBank Accession No. NG_001336.2.

[0257] "GPC3" refers to Glypican 3, which is anchored to the cell membrane by phosphatidylinositol and is a marker for hepatocellular carcinoma. The amino acid sequence of an exemplary human GPC3 is provided by Uniprot accession number P51654.

[0258] "TRGV9 binding protein" encompasses any protein that can specifically bind to TRGV9 or any molecule comprising the protein, including but not limited to antibodies, antigen-binding fragments thereof, or fusion proteins thereof as defined in the present disclosure for TRGV9. In some embodiments, the "TRGV9 binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) single-domain antibodies or VHHs that specifically bind to TRGV9 in the embodiments of the present disclosure. In some embodiments, the "TRGV9 binding protein" of the present disclosure may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) VH and VL combinations 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 an immunoglobulin single variable domain or a VH and VL combination of TRGV9, may also comprise a linker and / or a portion having effector function, such as a half-life extending portion (e.g., an immunoglobulin single variable 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, a "TRGV9 binding protein" encompasses the anti-γδTCR antibodies or antigen-binding fragments thereof in the embodiments of the present disclosure.

[0259] "GPC3 / TRGV9 binding protein" encompasses any protein that can specifically bind to GPC3 and TRGV9, or any molecule comprising such a 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 the anti-GPC3 / γδTCR bispecific antibodies of the disclosed embodiments.

[0260] "Antibodies" encompass various antibody structures, 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), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0261] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in a number of embodiments. In general, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0262] For the determination or definition of CDRs, the definitive depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used in this disclosure, CDRs can refer to CDRs defined by any method known in the art (including combinations of methods). The correspondence between the various numbering systems is well known to those skilled in the art.

[0263] The skilled person understands that although a specific numbering system (such as Kabat) is used in a specific embodiment or in a specific SEQ ID NO, the skilled person can determine the sequence corresponding to the sequence under other numbering systems, and such sequences are still considered to fall within the scope of the present disclosure.

[0264] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0265] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0266] An "immunoglobulin variable domain" is essentially composed of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," and three "complementarity determining regions" or "CDRs," respectively, "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3." The general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0267] "Antibody framework (FR)" refers to the portion of the variable domain that serves as a scaffold for the antigen binding loops (CDRs).

[0268] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0269] "VHH" is also called heavy chain single domain antibody, VHH, V H H domain, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an "antibody lacking a light chain") (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 "VH domain" or VH in this disclosure) and the light chain variable domain (referred to as "VL domain" or VL in this disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains; this binding behavior is different from the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the VL domain recognizes the epitope together with the VH domain. The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "V H H domain", "VHH antibody fragment", "VHH antibody", as well as" "Nanobody" is a trademark of Ablynx NV, Ghent, Belgium. VHHs include, but are not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes 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, 17 June, 2009 and WO94 / 04678.

[0270] As is well known in the art for VH 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 the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0271] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the human variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a simultaneous decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human variable region to maintain activity. Examples of "humanization" include "humanizing" a Camelidae-derived VHH domain by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain human framework region sequences from IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), i.e., CDR "grafting" onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies that have been further affinity-matured by phage display to CDRs. In addition, to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequences can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0272] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for the antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" TRGV9 binding protein or anti-TRGV9 antibody has one or more changes in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in 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):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0273] Typically, the GPC3 / TRGV9 binding proteins, TRGV9 binding proteins of the present disclosure will be expressed as measured in a Biacore or KinExA or Fortibio assay at a concentration of preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein to be bound (i.e., GPC3, TRGV9). Any -4 M's K DValues ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0274] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing 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 analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those for non-target interactions (e.g., control antibodies known not to bind IGF-1R or TRGV9). D The value is evaluated.

[0275] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0276] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0277] "Titin-T chain" or "T chain" refers to a 78-118 amino acid peptide segment of the titin protein comprising the titin Ig-like 152 domain, or a functional variant thereof. The titin-T chain is capable of binding to the obscurin Ig-like 1 domain to form a dimerization complex. Functional variants of the T chain are polypeptides in which some amino acids of the wild-type T chain are mutated, but which still bind to the obscurin Ig-like 1 domain to form a dimerization complex. For example, a suitable length of amino acids may be added or truncated at the C-terminus and / or N-terminus of the titin Ig-like 152 domain. One, two, three, four, five, six, seven, eight, nine, or ten amino acid residues may be added or truncated. For example, five amino acids, "KAGIR," located immediately adjacent to the N-terminus of the titin Ig-like 152 domain in the wild-type titin protein, may be added to the N-terminus of the titin Ig-like 152 domain, while still being able to associate with the obscurin Ig-like 1 domain to form a complex. Other mutations can also be made to the amino acids of the titin Ig-like 152 domain, for example, mutations to certain amino acids can be made to improve interchain disulfide bonds, enhance the stability of the complex, and the like.

[0278] "Obscurin-O chain" or "O chain" refers to a peptide segment of the obscurin protein containing the obscurin Ig-like 1 domain, or a functional variant thereof, that is 87-117 amino acids in length. The obscurin-O chain is capable of binding to the titin Ig-like 152 domain to form a dimerization complex. These functional obscurin-O chain variants are polypeptides that have had some amino acid mutations in the wild-type O chain but still bind to the titin Ig-like 152 domain to form a dimerization complex. For example, an appropriate length of amino acids can be added or truncated at the C-terminus and / or N-terminus of the obscurin-O domain, for example, by adding or truncating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. For example, the five amino acids "DQPQF" located immediately adjacent to the N-terminus of the obscurin Ig-like 1 domain in the wild-type obscurin protein can be added to the N-terminus of the obscurin-O domain, while still functioning to bind to the titin Ig-like 152 domain to form a dimerization complex. Other amino acid mutations can also be made to portions of the obscurin Ig-like 1 domain, for example, to improve interchain disulfide bonds or enhance antibody stability.

[0279] "Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0280] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0281] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0282] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0283] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0284] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0285] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" used in phrases such as "A and / or B" in the present disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to." In the context of mutations contained in the Fc region in the present disclosure, " / " means "and," for example, "354C / 366W" means "354C and 366W," that is, the Fc contains 354C and 366W mutations; the amino acid positions of the mutations in the Fc region of the present disclosure are all numbered according to the EU numbering system.

[0286] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0287] Example

[0288] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0289] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0290] Example 1. Design and preparation of antigens

[0291] The γδT cell receptor (TCR) on the surface of γδT cells is a heterodimeric membrane protein composed of a γ chain and a δ chain. Amino acids 1-242 of the human γδTCR extracellular domain protein γ9 chain (Protein data bank, accession number 1HXM, chain B) were selected, and a 3C restriction site, a Leucine zipper, and a FLAG tag were added to the C-terminus. Amino acids 1-229 of the human γδTCR extracellular domain protein δ2 chain (Protein data bank, accession number 1HXM, chain A) were selected, and a 3C restriction site, a Leucine zipper, and a His8 tag were added to the C-terminus. In addition, the Q180C and V168C mutations were introduced into the constant regions of the γ and δ chains, respectively, to form interchain disulfide bonds.

[0292] The recombinant monkey (Macaca mulatta) γ9δ2 TCR protein selected amino acids 1-241 of the monkey γ9 chain (sequence see published patent number: US2019144540A1 SEQ ID NO: 42), wherein a 3C restriction site, a Leucine zipper, and a FLAG tag were added to the C-terminus in sequence; amino acids 1-229 of the monkey δ2 chain (sequence see published patent number: US20190144540A1 SEQ ID NO: 37) were selected, wherein a 3C restriction site, a Leucine zipper, and a His8 tag were added to the C-terminus in sequence; in addition, Q179C and V168C mutations were introduced into the constant regions of the γ chain and δ chain, respectively, to form interchain disulfide bonds.

[0293] >Recombinant human γ9 chain

[0294] >Recombinant human δ2 chain

[0295] >Recombinant monkey γ9 chain

[0296] >Recombinant monkey δ2 chain

[0297] Plasmids carrying target protein encoding genes were synthesized separately. The human γ9 chain (SEQ ID NO: 1) and human δ2 chain (SEQ ID NO: 2) plasmids were mixed at a ratio of 1:1, and the monkey γ9 chain (SEQ ID NO: 3) and monkey δ2 chain (SEQ ID NO: 4) plasmids were mixed at a ratio of 1:1. The cells were transiently transfected into Expi293 cells (purchased from Thermo) and expressed for 7 days. The human γ9δ2 TCR protein and the monkey γ9δ2 TCR protein were isolated and purified and stored at -80°C for future use.

[0298] Example 2. Screening of anti-γδTCR antibodies

[0299] 1. Screening of anti-γδTCR single domain antibodies

[0300] Adult healthy alpacas (Alpaca) were immunized using in vitro expanded human γ9δ2 T cells as immunogens, and specific antibodies were screened using the recombinant human γ9δ2 TCR protein in Example 1 using the phage display method.

[0301] Specifically, the initial immunization dose was 2E7 cells per alpaca. Three weeks after the initial immunization, a booster immunization was administered with a dose of 2E7 cells per alpaca. Subsequent boosters were administered three weeks apart. Serum samples were collected one week after each booster immunization, and antibody titers in the alpaca serum were measured using protein ELISA and FACS.

[0302] The specific protein ELISA assay procedure was as follows: recombinant human γ9δ2 TCR protein was diluted to 2 μg / mL in 0.05 M carbonate buffer (pH 9.6), with 100 μL / well added, and coated overnight at 4°C. Plates were blocked with PBST buffer containing 5% skim milk for 1 hour and washed three times. Alpaca serum was serially diluted twofold, starting at 1:2000 in blocking buffer, incubated at 37°C for 45 minutes, and washed five times. 100 μL of horseradish peroxidase-conjugated goat anti-Alpaca secondary antibody (AlpVHHs, 053-404-005, diluted 1:10,000 in PBS) was added to each well, incubated at 37°C for 45 minutes, and washed five times. Finally, 100 μL of TMB colorimetric solution was added to each well for color development, and the reaction was terminated after 5 minutes by adding 50 μL of stop solution. The absorbance at 450 nm was read using a microplate reader.

[0303] The FACS analysis procedure was as follows: cells were harvested and resuspended at 4E6 / mL. 50 μL of cells were added with serially diluted alpaca serum and incubated at 4°C for 1 hour. The cells were washed twice with 1% BSA / PBS buffer, the supernatant discarded, and a 1:200 dilution of Anti-alpaca IgG iFluor647 (AlpVHHs, 053-404-009) was added. The cells were incubated at 4°C in the dark for 45 minutes. The cells were washed twice with 1% BSA / PBS buffer, and the cells were resuspended in 200 μL of buffer before FACS analysis.

[0304] After testing, the titer of alpaca serum after immunization was greater than 128k.

[0305] After three immunizations, 50 mL of peripheral blood was collected one week after each immunization to separate lymphocytes (PBMC). Total RNA of PBMC was extracted using RNAiso Plus reagent for phage library construction, and a phage library with a titer of 3.08×E13 cfu / mL was obtained.

[0306] Phage display was used to screen for antibodies targeting recombinant human γ9δ2 TCR protein. Antibodies that cross-bind to monkey γ9δ2 TCR protein were identified by ELISA, and binding activity to in vitro expanded human γ9δ2 T cells was determined by FACS. Positive monoclonal single-domain antibodies were screened using the ELISA and FACS assays described above, and the sequences are listed below.

[0307] >SDP01346 variable region (clone number ac-025)

[0308] Table 1. CDR sequences of anti-human γδTCR single domain antibodies (Kabat numbering convention)

[0309] 2. Screening of Mouse Anti-γδTCR Antibodies

[0310] In vitro expanded human γ9δ2 T cells were used as immunogens to immunize 6-8 week old Balb / c and SJL mice, and specific antibodies were screened using the recombinant human γ9δ2 TCR protein in Example 1 by hybridoma fusion.

[0311] Specifically, the initial immunization dose was 1E7 per mouse. Two weeks after the initial immunization, a booster immunization was administered at a dose of 1E7 per mouse. Subsequent boosters were administered every 2-3 weeks. Serum samples were collected one week after each booster immunization, and antibody activity in the mouse serum was assayed using protein ELISA and FACS.

[0312] Protein ELISA assay procedures are as follows: Plates were coated with 1 μg / mL recombinant human γ9δ2 TCR protein overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and washed three times. Mouse serum was serially diluted threefold, starting at 1:200, in blocking buffer, incubated at 37°C for 1 hour, washed three times, and incubated with a 1:10,000 dilution of anti-mouse IgG-Fc-HRP secondary antibody (Sigma, AP127P) for 1 hour. After washing three times with PBST, 100 μL of TMB colorimetric solution was added to each well for color development. The reaction was terminated after 15 minutes with stop solution. Absorbance was read at 450 nm using a microplate reader.

[0313] The FACS assay procedure was as follows: 1E5 cells were plated per well, centrifuged, and the supernatant discarded. A serial dilution of mouse serum was added. The cells were incubated at 4°C for 1 hour. The cells were washed twice with 1% BSA / PBS buffer, the supernatant discarded, and a 1:500 dilution of Alexa Flour 488 goat anti-mouse secondary antibody (Jackson Immuno Research, 115-545-071) was added. The cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with 1% BSA / PBS buffer, resuspended in 200 μL of buffer, and analyzed by FACS. The titer of the immunized mouse serum was greater than 72,900.

[0314] The final immunization was performed with an intraperitoneal injection of 50 μg of recombinant human γ9δ2 TCR protein. Four days later, the mice were sacrificed, and spleens were harvested. Splenocytes were ground and mixed with mouse myeloma SP2 / 0 cells, and electrofusion was performed to generate hybridoma cells. Positive clones with an OD 450nm > 0.2 were analyzed by FACS. Positive clones with an MFI binding to human γ9δ2 T cells > 5000 were subcloned. After initial and retesting of subclones, a murine anti-γδTCR antibody, SDP01315 (clone number bph-003), was identified using the aforementioned ELISA and FACS assays. The sequence is shown below.

[0315] >SDP01315 (clone bph-003) VH

[0316] >SDP01315 (clone number bph-003) VL

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

[0318] Example 3. Construction and functional effect verification of anti-γδTCR chimeric antibodies

[0319] 1. Expression of Chimeric Antibodies

[0320] For single-domain antibodies, the nucleotide sequence encoding the antibody was cloned into the pTT5 vector and then 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 nickel affinity column (HisTrap excel, GE), and the bound antibody was eluted with 300mM imidazole. The solution was desalted and exchanged with PBS to obtain the target antibody.

[0321] >SDP01346 full length

[0322] Mouse antibody: The nucleotide sequence encoding the antibody was cloned into the pTT5 vector and then 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, and the eluate was neutralized with 1M Tris and desalted to obtain the target antibody.

[0323] >SDP01315 full-length heavy chain

[0324] >SDP01315 full length light chain

[0325] 2. Binding of chimeric antibodies to γ9δ2 T cells

[0326] The affinity test process of anti-γδTCR single domain antibody at the cell level is as follows: the serially diluted antibody molecules are diluted to 1 x 10 5 γ9δ2 T cells were incubated at 4°C for 1 hour. Excess antibody was washed off, and DyLight405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) was added. The cells were incubated at 4°C for 30 minutes. Excess antibody was washed off, and the cells were resuspended in 200 μL of 2% FBS / PBS buffer. Cell surface fluorescence signals were measured using a Thermo Attune NxT flow cytometer. The results are shown in Table 3.

[0327] The affinity test process of mouse anti-γδTCR antibody at the cell level is as follows: the antibody molecules with gradient dilution are diluted to 1 x 10 5 γ9δ2 T cells were incubated at 4°C for 1 hour. Excess antibody was washed off, and mouse Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat# 209-605-098) was added. The cells were incubated at 4°C for 30 minutes. After washing off excess antibody, the cells were resuspended in 200 μL of 2% FBS / PBS buffer and the cell surface fluorescence signal was measured using a Thermo Attune NxT flow cytometer. The results are shown in Table 3.

[0328] Table 3. Binding ability of anti-γδTCR chimeric antibodies

[0329] The results showed that SDP01346 and SDP01315 had good binding activity on γ9δ2 T cells.

[0330] Example 4. Identification of Anti-γδ TCR Antibody-Antigen Binding Region

[0331] In this example, protein-based ELISA was used to detect the binding region of anti-γδTCR antibodies to antigens. Using methods similar to those used in Example 1, recombinant human γ9δ1 TCR protein (a dimer of SEQ ID NO: 1 and SEQ ID NO: 20), human γ8δ2 TCR protein (a dimer of SEQ ID NO: 21 and SEQ ID NO: 2), human VγδCαβ chimeric TCR protein (a dimer of SEQ ID NO: 22 and SEQ ID NO: 23), and human VαβCγδ chimeric TCR protein (a dimer of SEQ ID NO: 24 and SEQ ID NO: 25) were prepared.

[0332] ELISA was used to characterize the binding activity of the anti-γδTCR antibodies to the above antigens and to determine the approximate region of antibody binding. The specific protein ELISA assay procedure was as follows: Plates were coated with 1 μg / mL recombinant TCR protein, incubated overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and washed three times. The antibody was diluted to 30 nM in blocking buffer, incubated at 37°C for 1 hour, washed three times, and incubated with a 1:10,000 dilution of anti-mouse IgG-Fc-HRP secondary antibody (Sigma, AP127P) for 1 hour. Washed three times with PBST, 100 μL of TMB colorimetric solution was added to each well for color development, and the reaction was terminated after 15 minutes with stop solution. The absorbance at 450 nm was read using a microplate reader.

[0333] >Recombinant human δ1 chain

[0334] >Recombinant human γ8 chain

[0335] >Recombinant human Vγ9Cβ chain

[0336] >Recombinant human Vδ2Cα chain

[0337] >Recombinant human Vβ7Cγ chain

[0338] >Recombinant human Vα13Cδ chain

[0339] The ELISA results are shown in Table 4. The absorbance values ​​of antibodies SDP01346 and SDP01315 for different antigens are different. Both antibodies bind to the γ9δ1 TCR protein but not the γ8δ2 TCR protein, indicating that the antibodies bind to the γ9 chain; these two antibodies bind to the VγδCαβ chimeric TCR protein but not the VαβCγδ chimeric TCR, indicating that the antibodies bind to the TCR variable region (V region). In summary, it can be clearly seen that antibodies SDP01346 and SDP01315 bind to TRGV9.

[0340] Table 4. Identification results of chimeric antibody and antigen binding regions

[0341] Example 5. Humanized modification of anti-γδTCR antibodies

[0342] The variable region sequences were compared with the antibody germline database to obtain human germline templates with high homology. The human germline heavy chain template used in SDP01346 was IGHV3-30; the human germline heavy chain template used in SDP01315 was IGHV3-21, and the human germline light chain template was IGKV1-12. After predicting the structure of the monoclonal antibody through homology modeling, the VH, VL of the mouse antibody or the CDR of the single-domain antibody were chimerized into the appropriate human GermLine framework (Bioinformation. 2014; 10(4): 180-186; Methods Mol Biol. 2019; 1904: 213-230), and then back mutations were introduced.

[0343] The obtained humanized molecule sequence is as follows:

[0344] >SDP01346 VH1

[0345] >SDP01346 VH2

[0346] >SDP01346 VH3

[0347] >SDP01346 VH4

[0348] >SDP01315 VH1

[0349] >SDP01315 VH2

[0350] >SDP01315 VH3

[0351] >SDP01315 VL1

[0352] >SDP01315 VL2

[0353] >SDP01315 VL3

[0354] After humanization of a single domain antibody, a full-length antibody was constructed by adding GGGGSHHHHHH (SEQ ID NO: 72) after the corresponding VHH. Alternatively, after humanization of a single domain antibody, a full-length antibody was constructed by adding a linker and Fc after the corresponding VHH.

[0355] After humanization of the mouse antibody, a full-length antibody was constructed. The light chain constant region used Cκ, and the heavy chain constant region used IgG1 with L234F and L235E mutations.

[0356] Table 5. Correspondence between antibody names and antibody structures.

[0357] The full-length sequences of SDP01378, SDP05576, and SDP01369 are exemplified below.

[0358] >SDP01378 full length

[0359] >SDP05576

[0360] >SDP01369 full-length heavy chain

[0361] >SDP01369 full length light chain

[0362] The following is a FACS assay for the binding of humanized monoclonal antibodies to γ9δ2 T cells.

[0363] Affinity test of humanized single domain antibody SDP01346: the serially diluted antibody molecules were diluted to 1 x 10 5 γ9δ2 T cells were incubated at 4°C for 1 hour. Excess antibody was washed off, and DyLight 405-AffiniPure Goat Anti-Alpaca IgG, VHH domain antibody (Jackson, Cat#128-475-232) was added. The cells were incubated at 4°C for 30 minutes. Excess antibody was washed off, and the cells were resuspended in 200 μL of 2% FBS / PBS buffer. Cell surface fluorescence signals were measured using a Thermo Attune NxT flow cytometer. The results are shown in Table 6.

[0364] Affinity test of humanized mouse chimeric antibody SDP01315: dilute the antibody molecules in a gradient at 1 x10 5 γ9δ2 T cells were incubated at 4°C for 1 hour. Excess antibody was washed off, and mouse Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat# 209-605-098) was added. The cells were incubated at 4°C for 30 minutes. Excess antibody was washed off, and the cells were resuspended in 200 μL of 2% FBS / PBS buffer. Cell surface fluorescence signals were measured using a Thermo Attune NxT flow cytometer. The results are shown in Table 6.

[0365] Table 6. Affinity testing of humanized molecules SDP01346 and SDP01315

[0366] The results showed that the single-domain antibody humanized molecule SDP01378 had the strongest affinity, and the humanized molecules SDP01346 and SDP01365-SDP01373 had similar affinities. SDP01369, which had an intermediate number of back mutations, was selected.

[0367] Example 6. Design and preparation of anti-GPC3 / γδTCR bispecific antibodies

[0368] Antibody G, which has nanomolar affinity for GPC3, was selected. The sequences of the heavy and light chain variable regions are shown below. Antibody G binds to the C-terminal subunit and does not bind to soluble GPC3.

[0369] >GPC3 VH

[0370] >GPC3 VL

[0371] Table 7. VH and VL of Antibody G (Kabat numbering convention)

[0372] We designed various GPC3 / γδ TCR bispecific antibodies and ultimately selected a 1+1 asymmetric structure with SDP01346H4 (i.e., SDP01378), using a KIH (knob into hole) to prevent heavy chain mispairing; and a 2+1 asymmetric structure with SDP01315H2L2, using a KIH to prevent heavy chain mispairing and a HOT to prevent light chain mispairing. See Figure 1 for a schematic diagram of the structure.

[0373] To prevent or reduce mispairing between the heavy and light chains, the obscurin-O and titin-T sequences used are shown in SEQ ID NOs: 47-48, and the CHI and Cκ sequences are shown in SEQ ID NOs: 49-50. To prevent or reduce mispairing between the two heavy chains, IgG mutations were used. The full-length sequences of the bispecific antibodies are shown below, where the underlined portions represent CDR sequences, the wavy underlined portions represent the obscurin-O or titin-T sequences, the dotted underlined portions represent the CHI or Cκ sequences, the italicized portions represent the IgG1 Fc region, the bold italicized portions represent point mutations in the Fc region, and the bolded portions represent the linker.

[0374] >Obscurin-O

[0375] >Titin-T

[0376] >CH1

[0377] >Cκ

[0378] >IgG1 Fc(WT)

[0379] >IgG1 Fc1 (mutations S354C / T366W / L234F / L235E)

[0380] >IgG1 Fc2 (mutations: Y349C / T366S / L368A / Y407V / L234F / L235E)

[0381] >SDP01716 H1

[0382] >SDP01716 H2

[0383] >SDP01716 L

[0384] >SDP01696 H1

[0385] >SDP01696 L1

[0386] >SDP01696 H2

[0387] >SDP01696 L2

[0388] Same as SDP01716 L (SEQ ID NO: 56).

[0389] >SDP01704 H1

[0390] Same as SDP01696 H1 (SEQ ID NO: 57).

[0391] >SDP01704 L1

[0392] Same as SDP01696 L1 (SEQ ID NO: 58).

[0393] >SDP01704 H2

[0394] >SDP01704 L2

[0395] The nucleotide sequence encoding the antibody was cloned into the pTT5 vector and transfected into ExpiCHO cells. After 8 days, the cells were removed by centrifugation, 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 antibodies were eluted with glycine, and the eluate was neutralized with 1M Tris and desalted. After detection, the target antibodies SDP01716, SDP01696, and SDP01704 were obtained.

[0396] Example 7. Binding affinity detection of anti-GPC3 / γδTCR bispecific antibody to human and monkey antigens

[0397] A Protein A biosensor chip (Cat. #29139121-AB, Cytiva) was used. Each test antibody was prepared in HBS-EP+ buffer as a ligand for capture with Protein A on the chip channel. Human GPC3 (Sino Biological, Cat. #10088-H08H), cynomolgus monkey GPC3 (Acrobiosystems, Cat. #GP3-C5225), human γ9δ2, and cynomolgus monkey γ9δ2 antigens were prepared in HBS-EP+ buffer and serially diluted two-fold. The diluted antigens were flowed through the experimental and reference channels at a flow rate of 30 μL / min, with binding for 80 seconds and dissociation for 300 seconds. The regeneration buffer was 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) at a flow rate of 10 μL / min for 30 seconds. Data were analyzed using Biacore 8K evaluation software.

[0398] The results are shown in Tables 8 and 9. The antigen binding affinities of the three antibodies to human and cynomolgus monkey GPC3 proteins are all around 2 nM, which is similar to the affinity of antibody G. The K values ​​of the three antibodies for the binding of cynomolgus monkey γ9δ2 TCR protein are D Both are around 50nM.

[0399] Table 8. Kinetic parameters of antibody binding to human / cynomolgus monkey GPC3 protein

[0400] Table 9. Kinetic parameters of antibody binding to human / cynomolgus monkey γ9δ2 protein

[0401] Example 8. Detection of Binding Activity of Anti-GPC3 / γδTCR Bispecific Antibodies to GPC3-Positive Cells and Human γ9δ2 T Cells

[0402] FACS experiments were used to detect the binding activity of γδT bispecific antibodies to HepG2 cells (purchased from ATCC) that naturally overexpress human GPC3 and DLD-1 cells (purchased from ATCC) that do not express human GPC3. The GPC3 binding activity of anti-GPC3 / γδTCR bispecific antibodies on cells was tested by detecting the fluorescence signal of the antibody bound to the cell surface, and the binding strength of the antibody was evaluated based on the intensity of the fluorescence signal. Specifically, the serially diluted antibody molecules and control molecules were diluted to 1 x 10 5 Cells were incubated at 4°C for 1 hour, excess antibodies were washed off, and mouse Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat# 209-605-098) was added. The cells were incubated at 4°C for 30 minutes. After washing off excess antibodies, the cells were resuspended in 200 μL of 2% FBS / PBS buffer and the cell surface fluorescence signal was read using a Thermo Attune NxT flow cytometer. The cells used were HepG2 cell lines.

[0403] FACS assays were used to test the binding activity of the anti-GPC3 / γδTCR bispecific antibody to human γ9δ2 T cells and human PBMCs. Human γ9δ2 T cells were induced with zoledronic acid and IL-2. The binding activity of the anti-GPC3 / γδTCR bispecific antibody on γδT cells was determined by detecting the fluorescence signal of the antibody bound to the cell surface. The binding strength of the antibody was evaluated based on the intensity of the fluorescence signal. Specifically, serially diluted antibody molecules and control molecules were added at 1 x 10 5Cells were incubated at 4°C for 1 hour, excess antibodies were washed off, and mouse Alexa Flour 647-labeled anti-human Fc antibody (Jackson, Cat# 209-605-098) was added. The cells were incubated at 4°C for 30 minutes. After washing off excess antibodies, the cells were resuspended in 200 μL of 2% FBS / PBS buffer and the cell surface fluorescence signal was read using a Thermo Attune NxT flow cytometer.

[0404] The results are shown in Figures 2A-2D. As shown in Figures 2A and 2B, the GPC3 affinities of SDP01716 and SDP01696 are comparable, and EC 50 SDP01704 showed bivalent binding characteristics, with an EC 50 The affinity of SDP01716 for γ9δ2 T cells was 0.9064 nM, that of SDP01696 was 2.676 nM, and that of SDP01704 was 4.289 nM; none of the antibodies tested showed nonspecific binding on PBMCs.

[0405] Example 9. Effect of anti-GPC3 / γδTCR bispecific antibody on BTN2A / BTN3A-TCR natural signaling

[0406] In the natural state, after the intracellular phosphorylated antigen binds to BTN3A, it changes the molecular tension of BTN3A; two BTN3A molecules and two BTN2A molecules combine to form a heterotetramer, which then interacts with the TCR, activates the TCR, and induces the activation, proliferation, and killing functions of γδT cells. Because anti-γδTCR antibodies also bind to TCR, this example evaluates the effect of the antibodies disclosed herein on the natural signaling of BTN2A / BTN3A-TCR. SDP01378 is a monovalent molecule at the γδT end of SDP01716, and SDP01315 is a monovalent molecule of SDP01696 and SDP01704.

[0407] A375-Luc cells were cultured in RPMI1640 supplemented with 10% inactivated fetal bovine serum. γδ T cells were obtained by expansion from PBMCs. A375-Luc was plated at a density of 25 μL / well in a 96-well plate, with a cell count of 1 × 10 4 / well; take 25 μL / well γδT cells (1×10 5100 μL / well of Bright-Glo™ Luciferase Assay System (Promega, E2650) was added for detection. For detailed procedures, refer to the reagent instructions.

[0408] As shown in Figures 3A and 3B , neither SDP01378 nor SDP01315 blocked the natural BTN2A / BTN3A-TCR signal. Therefore, it can be concluded that the anti-GPC3 / γδTCR bispecific antibody of the present disclosure does not block the natural BTN2A / BTN3A-TCR signal.

[0409] Example 10. Detection of the cytotoxicity of γδT cells against tumor cells mediated by anti-GPC3 / γδTCR bispecific antibodies in vitro

[0410] In this example, the lactate dehydrogenase (LDH) assay was used to evaluate the antibody-mediated cytotoxicity of γδT cells against target cells expressing different GPC3 levels.

[0411] HepG2 cells naturally express high levels of GPC3, and the cell culture medium was DMEM (Gibco, Cat#11995-065, the same below) containing 15% inactivated fetal bovine serum. Huh-7 cells express moderate levels of GPC3, and the cell culture medium was DMEM. MKN-45 cells express low levels of GPC3, and the cell culture medium was RPMI 1640 (Gibco, Cat#10491A-01, the same below). DLD-1 cells do not express GPC3, and the cell culture medium was RPMI 1640. After digestion, the target cells were resuspended in RPMI 1640 medium containing 2% serum, and the density was adjusted to 7×10 4 cells / mL; then 50 μL / well was inoculated into a 96-well plate, and 50 μL of the test antibody after gradient dilution was added. 50 μL of culture medium was added to each well. γδT cells were collected and resuspended in RPMI1640 containing 2% fetal bovine serum to adjust the cell density. 50 μL / well was inoculated into the above experimental plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. The cell culture plate was removed and centrifuged (400g, 5 minutes) to collect the cell culture supernatant. CytoTox LDH levels were measured using a Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). For detailed procedures, refer to the kit instructions.

[0412] The results are shown in Figures 4A to 4D and Table 10. As the antigen expression level decreases, the cytotoxic activity of the disclosed bispecific antibodies against tumor cells weakens. The affinity of the antibody γδT end is positively correlated with the cytotoxic activity against tumor cells, and the divalent molecule at the GPC3 end is stronger than the monovalent molecule.

[0413] Table 10. Anti-GPC3 / γδTCR bispecific antibodies kill cells expressing different antigens

[0414] Note: - means no killing activity was detected.

[0415] Example 11. Effect of anti-GPC3 / γδTCR bispecific antibody on γδT cell killing from different PBMC donors

[0416] In this example, lactate dehydrogenase (LDH) detection was used to evaluate the antibody-mediated cytotoxicity of γδT cells against target cells expressing GPC3.

[0417] HepG2 cells were digested and resuspended in RPMI1640 medium containing 2% serum, and the cell density was adjusted to 7×10 4 / mL; then 50μL / well was inoculated into a 96-well plate, and 50μL of the test antibody after gradient dilution was added. 50μL of culture medium was added to each well. γδT cells expanded from different donors were collected, resuspended in RPMI1640 containing 2% fetal bovine serum, and the cell density was adjusted. 50μL / well was inoculated into the above experimental plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. The cell culture plate was removed and centrifuged (400g, 5 minutes) to collect the cell culture supernatant. CytoTox The LDH level was detected using Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780).

[0418] The results of SDP01716 are shown in Figures 5A to 5D and Table 11. SDP01716 can mediate the killing of tumor cells by γδT cells from different donors. 50 There is no significant difference in the maximum damage value.

[0419] Table 11. Antibody-mediated cytotoxicity of γδT cells from different donors against HepG2

[0420] Note: #SC12004, #SC12392, #XC11053, and #XC11061 are γδT cells expanded from different donor sources (healthy allogeneic human peripheral blood). - means no killing was detected.

[0421] Example 12. Detection of the killing activity of anti-GPC3 / γδTCR bispecific antibody against low antigen-expressing cells at different effector-target ratios

[0422] In this example, lactate dehydrogenase (LDH) detection was used to evaluate the antibody-mediated cytotoxicity of γδT cells against target cells with low GPC3 expression.

[0423] MKN-45 cells naturally express low levels of GPC3. MKN-45 cells were digested and resuspended in RPMI1640 medium containing 2% serum. The cell density was adjusted to 7×10 4 / mL; then inoculate 50μL / well in a 96-well plate, and add 50μL of gradient diluted antibody to be tested. Fill each well with 50μL of culture medium. Collect γδT cells, resuspend in RPMI1640 containing 2% fetal bovine serum, and adjust the cell density. Inoculate 50μL / well in the above experimental plate, and the final effector-target ratio (γδT cells: MKN-45 cells) is 1:1; 10:1; 30:1. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Remove the cell culture plate, centrifuge (400g, 5 minutes) to collect the cell culture supernatant, and use CytoTox LDH levels were measured using a Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). For detailed procedures, refer to the kit instructions.

[0424] The results for SDP01716 are exemplified. As shown in Figure 6A, at a low 1:1 efficacy-target ratio, SDP01716 showed weak cytotoxicity against low-antigen-expressing cells, with a maximum killing rate of 17%. As shown in Figure 6B, increasing the efficacy-target ratio to 10:1 enhanced SDP01716's cytotoxicity against low-antigen-expressing cells, reaching a maximum killing rate of 64%. As shown in Figure 6C, when the efficacy-target ratio was increased to 30:1, cytotoxicity reached almost 100%. This demonstrates that the anti-GPC3 / γδTCR bispecific antibody disclosed herein is capable of killing tumor target cells in an efficacy-target ratio-dependent manner.

[0425] Example 13. Increasing the proportion of γδT cells in PBMCs to enhance the killing activity of anti-GPC3 / γδTCR bispecific antibodies and cytokine release detection

[0426] In this example, lactate dehydrogenase (LDH) detection was used to evaluate the antibody-mediated cytotoxicity of γδT cells against target cells expressing GPC3.

[0427] HepG2 cells naturally highly express GPC3. After digestion, HepG2 cells were resuspended in RPMI1640 medium containing 2% serum and the cell density was adjusted to 7×10 4 / mL; then inoculate 50μL / well in a 96-well plate, and add 50μL of gradient diluted antibody to be tested. Fill each well with 50μL of culture medium. Collect PBMC cells, resuspend in RPMI1640 containing 2% fetal bovine serum, and adjust the cell density. Inoculate 50μL / well in the above experimental plate; or add 30% γδT cells to PBMC, inoculate 50μL / well in the above experimental plate, and incubate in a 37°C, 5% CO2 incubator for 24 hours. Remove the cell culture plate, centrifuge (400g, 5 minutes) to collect the cell culture supernatant, and use CytoTox The LDH level was measured using a Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). IFNγ (Cisbio, S62HIFNGPEG) and TNFα (Cisbio, 62HTNFAPEH) were also measured using kits to detect the secretion levels of the corresponding cytokines. For specific procedures, refer to the reagent instructions.

[0428] The results of SDP01716 are shown in Figures 7A to 7D, where Figure 7A shows the cell killing activity of PBMCs from donor #XC11053; Figure 7B shows the cell killing activity of γδ T cells spiked with 30% of PBMCs from donor #XC11053; Figure 7C shows the cell killing activity of PBMCs from donor #SC12392; and Figure 7D shows the cell killing activity of γδ T cells spiked with 30% of PBMCs from donor #SC12392.

[0429] Figures 7A and 7C illustrate that SDP01716's ability to kill tumor cells in a pure PBMC system is weaker than that of the CD3 bispecific antibody BMK-029 (ERY974 in US20220348658) with the same target, because CD3 bispecific antibodies can mobilize more T cells as effector cells to kill tumors; Figures 7B and 7D illustrate that increasing the proportion of γδT cells in PBMCs can enhance their killing activity. At the same time, the cytokine release assay results corresponding to Figures 7A to 7D are shown in Figures 8A to 8D. SDP01716 induces less IFNγ and TNFα release when killing tumor cells.

[0430] Example 14. Detection of γδT cell proliferation activity promoted by anti-GPC3 / γδTCR bispecific antibody in PBMC

[0431] In this example, flow cytometry was used to assess the activity of anti-GPC3 / γδTCR bispecific antibodies in inducing γδT cell proliferation in PBMCs.

[0432] Take 800 μL of PBMC (1×10 6HepG2 cells were washed twice with PBS and the cell density was adjusted to 5×10 5 For each cell / mL, add 1 mL to the corresponding wells. Prepare the test antibody in culture medium to the appropriate concentration and add 200 μL to each well. Incubate in a 37°C, 5% CO2 incubator, changing the medium every 3 days. After 6 to 7 days, wash the cells twice with PBS and stain with antibodies to CD3 (BD, Cat#564713), Vδ2 TCR (BD, Cat#555739), and Vγ9 TCR (BD, Cat#555732) to identify changes in the proportion of γδ T cells.

[0433] The results are shown in Figure 9. In PBMC cells derived from three donors #XC11211, #XC11061, and #XC11251, after 6-7 days of stimulation, SDP01716, SDP01696, and SDP01704 were able to significantly increase the proportion of γδT cells.

[0434] Example 15. Validation of the Huh-7 Human Hepatocellular Carcinoma Transplantation Model in Mice

[0435] 1.Human liver cancer Huh-7 cells (cell bank of Chinese Academy of Sciences) were cultured at 5×10 6 NSG mice (female, 6-8 weeks old, provided by Weitonglihua) were subcutaneously inoculated with 100 μL of γδT cells / 100 μL and randomly divided into 4 groups (G1-G4) with 7 mice in each group. Tumor cells were inoculated on the day of grouping, and γδT cells and drugs were infused at the same time. Each group was administered according to the following regimen:

[0436] G1: Medium G2: γδ T cells + PBS

[0437] G3: γδ T cells + SDP01716 (3 mg / kg)

[0438] G4: γδ T cells + SDP01716 (0.3 mg / kg)

[0439] Afterwards, γδT cells were infused and the drug was administered once weekly. Tumor volume was measured twice weekly during the administration and observation period, and the measured values ​​were recorded. The calculation formula is as follows (the same below):

[0440] Calculate tumor volume (TV) = 1 / 2 × a × b 2 , where a and b represent the long diameter and short diameter of the measured tumor, respectively;

[0441] Relative tumor growth rate T / C% = (T-T0) / (C-C0) × 100;

[0442] Tumor inhibition rate TGI% = 1-T / C%.

[0443] The results are shown in Figures 10A and 10B and Table 12. The TGI of the γδT cell monotherapy group (G2) was 11%, and the tumor growth inhibition effect was limited. SDP01716 inhibited tumor growth in a dose-dependent manner, with the TGI reaching 58% at a dose of 3 mpk. In addition, none of the treatment groups had a significant effect on the weight of the mice.

[0444] Table 12. Antitumor activity of SDP01716 in Huh-7 xenograft tumor model

[0445] Note: * represents P < 0.05; ** represents P < 0.01.

[0446] 2. Huh-7 cells were cultured at 5×10 6 NSG mice (female, 6-8 weeks old, provided by Jicui Yaokang) were subcutaneously inoculated with 100 μL of γδT cells / 100 μL and randomly divided into 6 groups, 7 mice in each group. On the day of grouping, tumor cells were inoculated, and γδT cells and drugs were infused at the same time. Each group was administered according to the following regimen:

[0447] G1: Medium

[0448] G2: γδ T cells

[0449] G3: γδ T cells + SDP01716 (3 mg / kg)

[0450] G4: γδT cells + SDP01696 (3.9mg / kg)

[0451] G5: γδ T cells + SDP01704 (5.1 mg / kg).

[0452] Thereafter, γδT cells were infused and administered weekly. If γδT cells and antibodies were used in combination, they were administered simultaneously. Tumor volume was measured twice weekly during the dosing and observation period, and the measured values ​​were recorded.

[0453] The results are shown in FIG11A , FIG11B and Table 13 . The dual antibodies disclosed herein can effectively inhibit tumors, and no significant changes in body weight were observed in the mice in each administration group.

[0454] Table 13. Antitumor activity of dual antibodies in Huh-7 xenograft tumor model

[0455] Note: ** represents P < 0.01.

Claims

1. A GPC3 / TRGV9 binding protein comprising: A first antigen binding domain that specifically binds to GPC3; and A second antigen binding domain that specifically binds to TRGV9, wherein in: The first antigen binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), The VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 39, The VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 40, The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDRs are defined according to the Kabat numbering system; Preferably, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NOs: 41-43, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NOs: 44-46, respectively.

2. The GPC3 / TRGV9 binding protein of claim 1, wherein 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 in the amino acid sequence shown in any one of SEQ ID NOs: 29, 26-28, and 5, The VH2 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 31, 9, 30 and 32, and the VL2 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of any one of SEQ ID NOs: 34, 10, 33 and 35, The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDRs are defined according to the Kabat numbering system; Preferably, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 having amino acid sequences as shown in SEQ ID NOs: 6-8, respectively. The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH2 are shown in SEQ ID NOs: 11-13, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL2 are shown in SEQ ID NOs: 14-16, respectively.

3. The GPC3 / TRGV9 binding protein according to claim 1 or 2, wherein: The immunoglobulin single variable domain, the heavy chain variable region and / or the light chain variable region are humanized, backmutated, affinity matured, T cell epitopes removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the framework region of the human germline template used in the humanization process of the immunoglobulin single variable domain is derived from IGHV3-64; Preferably, the framework region of the human germline template used in the humanization process of the VH2 is derived from IGHV3-21, and / or the framework region of the human germline template used in the humanization process of the VL2 is derived from IGKV1-12.

4. The GPC3 / TRGV9 binding protein according to any one of claims 1 to 3, wherein The VH1 in the first antigen binding domain comprises an amino acid sequence as shown in SEQ ID NO: 39 or having at least 80% or at least 90% identity thereto, and the VL1 comprises an amino acid sequence as shown in SEQ ID NO: 40 or having at least 80% or at least 90% identity thereto.

5. The GPC3 / TRGV9 binding protein according to any one of claims 1 to 4, wherein The immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 29, 26-28, 5, or having at least 80%, at least 90% identity thereto; The VH2 in the second antigen-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-32, or having at least 80% or at least 90% identity thereto, and the VL2 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or having at least 80% or at least 90% identity thereto.

6. The GPC3 / TRGV9 binding protein according to any one of claims 1 to 5, further comprising an Fc region of an immunoglobulin; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4; More preferably, the Fc region is the Fc region of human IgG1; Most preferably, the Fc region is an Fc region of IgG1 comprising L234F and / or L235E mutations.

7. The GPC3 / TRGV9 binding protein according to claim 6, wherein 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 knob-into-hole mutation; Preferably, the first subunit of the Fc region contains a mutation at position 366, and the second subunit contains a mutation selected from positions 366, 368 or 407, or any combination thereof; 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; 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; The coding rule for mutation sites is Eu coding.

8. The GPC3 / TRGV9 binding protein according to any one of claims 1 to 7, further comprising 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 shown, m and n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20; More preferably, the linker is A3S or G4S.

9. The GPC3 / TRGV9 binding protein according to any one of claims 1 to 8, comprising a polypeptide chain selected from any one of the groups (1) to (3): (1) a first heavy chain, a second heavy chain and a light chain, wherein: The first heavy chain, from N-terminus to C-terminus, is: [immunoglobulin single variable domain]-[linker 1]-[Fc1], The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 2]-[CH1]-[Linker 3]-[Fc2], Light chain, from N-terminus to C-terminus: [VL1]-[Linker 4]-[CL]; Wherein, the linker 1, linker 2, linker 3 and linker 4 may be the same or different, may exist independently or not, and may be independently selected from the linkers defined in claim 8; Preferably, linker 1 is AAAS, and linkers 2, 3 and 4 are absent; (2) a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein: The first heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 3]-[Titin-T chain], The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 4]-[CH1]-[Linker 5]-[Fc2], The second light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 6]-[CL]; or, The first heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 3]-[Titin-T chain], The second heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 4]-[CH1]-[Linker 5]-[Fc2], The second light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 6]-[CL]; Wherein, the linker 1, linker 2, linker 3, linker 4, linker 5 and linker 6 may be the same or different, may exist independently or not, and may be independently selected from the linkers defined in claim 8; Preferably, linker 1 and linker 3 are G4S, and linker 2, linker 4, linker 5 and linker 6 do not exist; (3) a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein: The first heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 3]-[Titin-T chain], The second heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 4]-[CH1]-[Linker 5]-[VH1]-[Linker 6]-[CH1]-[Linker 7]-[Fc2], The second light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 8]-[CL]; or, The first heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 2]-[Fc1], The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 3]-[Titin-T chain], The second heavy chain, from N-terminus to C-terminus, is: [VH2]-[Linker 4]-[CH1]-[Linker 5]-[VH2]-[Linker 6]-[CH1]-[Linker 7]-[Fc2], The second light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 8]-[CL]; Wherein, the linker 1, linker 2, linker 3, linker 4, linker 5, linker 6, linker 7, linker 8, linker 9, linker 10 may be the same or different, may exist independently or not, and may be independently selected from the linker described in 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, - represents a peptide bond, Preferably, the amino acid sequences of the Obscurin-O chain and the Titin-T chain are shown in SEQ ID NOs: 47 and 48, respectively; Preferably, the CL is Cκ, and the amino acid sequences of CH1 and Cκ are shown in SEQ ID NOs: 49 and 50, respectively; Preferably, the amino acid sequences of Fc1 and Fc2 are shown in SEQ ID NOs: 52 and 53, respectively.

10. The GPC3 / TRGV9 binding protein according to any one of claims 1 to 9, comprising a polypeptide chain selected from any one of the groups (1) to (3): (1) a first heavy chain having an amino acid sequence as set forth in SEQ ID NO:54, or having at least 80%, at least 90% identity thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO:55, or having at least 80%, at least 90% identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO:56, or having at least 80%, at least 90% identity thereto; (2) a first heavy chain having an amino acid sequence as set forth in SEQ ID NO:57, or having at least 80%, at least 90% identity thereto, a first light chain having an amino acid sequence as set forth in SEQ ID NO:58, or having at least 80%, at least 90% identity thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO:59, or having at least 80%, at least 90% identity thereto, and a second light chain having an amino acid sequence as set forth in SEQ ID NO:56, or having at least 80%, at least 90% identity thereto; or, (3) a first heavy chain having an amino acid sequence as set forth in SEQ ID NO:57, or having at least 80%, at least 90% identity thereto, a first light chain having an amino acid sequence as set forth in SEQ ID NO:58, or having at least 80%, at least 90% identity thereto, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO:60, or having at least 80%, at least 90% identity thereto, and a second light chain having an amino acid sequence as set forth in SEQ ID NO:61, or having at least 80%, at least 90% identity thereto; Preferably, the GPC3 / TRGV9 binding protein comprises a polypeptide chain selected from any one of the groups (1) to (3): (1) a polypeptide chain with an amino acid sequence as shown in SEQ ID NOs: 54-56; (2) a polypeptide chain with an amino acid sequence as shown in SEQ ID NOs: 56-59; (3) a polypeptide chain with an amino acid sequence as shown in SEQ ID NOs: 57, 58, 60, and 61; More preferably, the GPC3 / TRGV9 binding protein comprises a polypeptide chain selected from any one of the groups (1) to (3): (1) a polypeptide chain having an amino acid sequence as shown in SEQ ID NOs: 54-56 in a molar ratio of 1:1:1; (2) a polypeptide chain having an amino acid sequence as shown in SEQ ID NOs: 56-59 in a molar ratio of 1:1:1:1; (3) A polypeptide chain having an amino acid sequence in a molar ratio of 1:1:1:2 as shown in SEQ ID NOs: 57, 58, 60, and 61. 11 . The GPC3 / TRGV9 binding protein according to any one of claims 1 to 10 , which is an antibody or an antigen-binding fragment thereof, preferably an anti-GPC3 / TRGV9 antibody or an antigen-binding fragment thereof.

12. A TRGV9 binding protein comprising: (1) an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 29, 26-28, and 5, preferably, comprises CDR1, CDR2 and CDR3 in the amino acid sequence of SEQ ID NOs: 6-8; or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 9 and 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of any one of SEQ ID NOs: 10 and 33-35; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, e.g., according to the Kabat numbering system.

13. The TRGV9 binding protein of claim 12, wherein the immunoglobulin single variable domain, VH and / or VL is humanized, backmutated, affinity matured, T cell epitope removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the framework region of the human germline template used in the humanization process of the immunoglobulin single variable domain is derived from IGHV3-64; Preferably, the framework region of the human germline template used in the humanization process of the VH is derived from IGHV3-21, and / or the framework region of the human germline template used in the humanization process of the VL is derived from IGKV1-12.

14. The TRGV9 binding protein according to claim 12 or 13, wherein (1) an immunoglobulin single variable domain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 29, 26-28, 5, or having at least 80% or at least 90% identity thereto; or (2) VH and VL, wherein the VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9, 30-32, or having at least 80% or at least 90% identity thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10, 33-35, or having at least 80% or at least 90% identity thereto; Preferably, the TRGV9 binding protein is an anti-TRGV9 antibody or an antigen-binding fragment thereof; More preferably, when the TRGV9 binding protein comprises an immunoglobulin single variable domain, it is an anti-TRGV9 single domain antibody or VHH.

15. The TRGV9 binding protein of any one of claims 12 to 14, further comprising an immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, and the Fc region is more preferably the Fc region of human IgG1; Preferably, the Fc region is an Fc region of IgG1 comprising L234F and / or L235E mutations.

16. The TRGV9 binding protein according to any one of claims 12 to 15, further comprising a tumor-associated antigen (TAA) binding domain, or a tumor-specific antigen binding domain, preferably an antigen binding domain that specifically binds to GPC3.

17. The GPC3 / TRGV9 binding protein of any one of claims 1 to 11 or the TRGV9 binding protein of any one of claims 12 to 16, which has any one or more properties or functions selected from (a) to (d): (a) specifically binds to the γ9 chain of TCR, preferably, specifically binds to γ9δ1TCR and γ9δ2TCR; (b) no detectable binding to the γ8 chain of the TCR, preferably, no detectable binding to the γ8δ2 TCR; (c) specifically binds to the variable region of the γ9 chain of TCR (TRGV9); (d) It specifically binds to VγδCαβ chimeric TCR, and no binding to VαβCγδ chimeric TCR is detected.

18. A polynucleotide encoding the GPC3 / TRGV9 binding protein of any one of claims 1 to 11 or the TRGV9 binding protein of any one of claims 12 to 16, or a combination thereof; Preferably, the polynucleotide is DNA or RNA.

19. A vector comprising the polynucleotide according to claim 18, Preferably, the vector expresses the polynucleotide of claim 18.

20. A host cell containing or expressing the polynucleotide of claim 18 or the vector of claim 19.

21. A method for preparing the GPC3 / TRGV9 binding protein of any one of claims 1 to 11 or the TRGV9 binding protein of any one of claims 12 to 16, comprising: expressing the polynucleotide of claim 18 or the vector of claim 19 in the host cell of claim 20, and isolating the expressed GPC3 / TRGV9 binding protein or TRGV9 binding protein from the host cell; Optionally, the method further comprises the step of purifying the GPC3 / TRGV9 binding protein or TRGV9 binding protein.

22. A pharmaceutical composition comprising the GPC3 / TRGV9 binding protein of any one of claims 1 to 11, the TRGV9 binding protein of any one of claims 12 to 16, the polynucleotide of claim 18 and / or the vector of claim 19; Preferably, the pharmaceutical composition further comprises T cells, and the T cells are preferably γδT cells; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or adjuvants.

23. A combination or kit comprising: (1) the GPC3 / TRGV9 binding protein and T cells according to any one of claims 1 to 11, or (2) the TRGV9 binding protein and T cell according to any one of claims 12 to 16; The T cells are preferably γδ T cells.

24. Use of the GPC3 / TRGV9 binding protein of any one of claims 1 to 11, the TRGV9 binding protein of any one of claims 12 to 16, the polynucleotide of claim 18, the vector of claim 19, the combination of claim 23 or the kit in the preparation of a drug for treating a disease; The disease is preferably cancer, More preferably, it is liver cancer or GPC3-positive cancer, and most preferably, it is GPC3-positive liver cancer.

25. Use of the GPC3 / TRGV9 binding protein according to any one of claims 1 to 11, the TRGV9 binding protein according to any one of claims 12 to 16, the polynucleotide according to claim 18, and the vector according to claim 19 in the preparation of a drug for treating a disease, wherein: The GPC3 / TRGV9 binding protein, the TRGV9 binding protein, the polynucleotide, or the vector is used in combination with a T cell; The T cells are preferably γδT cells; The disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, most preferably GPC3-positive liver cancer.

26. Use of T cells in the preparation of a drug for treating a disease, wherein: The T cell is used in combination with the GPC3 / TRGV9 binding protein of any one of claims 1 to 11, the TRGV9 binding protein of any one of claims 12 to 16, the polynucleotide of claim 18, or the vector of claim 19; The T cells are preferably γδT cells; The disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, most preferably GPC3-positive liver cancer.

27. A method for treating or alleviating a disease, comprising administering to a subject in need thereof a therapeutically or alleviatingly effective amount of the GPC3 / TRGV9 binding protein of any one of claims 1 to 11, the TRGV9 binding protein of any one of claims 12 to 16, the polynucleotide of claim 18, the vector of claim 19, the pharmaceutical composition of claim 22, or the combination or kit of claim 23; The disease is preferably cancer, more preferably liver cancer or GPC3-positive cancer, most preferably GPC3-positive liver cancer.

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