Anti-gamma delta TCR antibodies and their use

Abstract

Molecules containing antibodies that bind to T cell receptors (TCRs) are provided. In particular, antibodies that bind to human γδ TCRs are provided, in which human γδ T cells can be activated and regulated. Also provided are bispecific tumor-targeted immunomodulators that simultaneously bind to tumor-associated antigens and γδ T cell receptors and enhance cell-mediated immune responses in cancer treatment. Methods for producing these antibodies and methods for using these antibodies to kill cancer cells are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to International Application PCT / CN2023 / 116291, filed on 31 August 2023, and International Application PCT / CN2023 / 123953, filed on 11 October 2023, which are incorporated herein by reference in their entirety.

[0002] Presentation of the array The contents submitted in the following XML file are incorporated herein by reference in their entirety: a sequence listing in computer-readable format (CRF) (filename: IEC240451PCT_SEQUENCE LISTING.xml, date: August 26, 2024, size: 239,106 bytes).

[0003] Technical field This disclosure relates to molecules comprising antibodies that bind to T cell receptors (TCRs). In particular, this disclosure relates to antibodies that bind to human γδ TCRs, where human γδ T cells can be activated and regulated. This disclosure further relates to tumor-bispecific immunomodulators, which simultaneously bind to tumor-associated antigens and γδ T cell receptors and enhance cell-mediated immune responses in cancer treatment. Methods for preparing these antibodies and methods for killing cancer cells using these antibodies are further provided. [Background technology]

[0004] background Human γδ T cells are a unique subset of immune cells, accounting for 0.5% to 5% of lymphocytes in peripheral blood. Unlike αβ T cells, the T cell receptor (TCR) expressed on γδ T cells consists of γ and δ chains and recognizes antigens independently of MHC restriction. Typically, human γδ T cells can be divided into four main groups based on the TCR δ chain: Vδ1, Vδ2, Vδ3, and Vδ5 γδ T cells. Vδ1 can co-express with multiple types of Vγ chains (Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, and Vγ10) to form different Vδ1 γδ T cell subsets, which are mainly distributed in the skin, small intestine, and other mucosal tissues. Small amounts of Vδ1 γδ T cells have also been found in the liver and spleen. Vδ2 co-expresses almost exclusively with Vγ9 to form Vγ9Vδ2 T cells, which are the main γδ T cells in blood circulation. Vδ3 γδ T cells are mainly found in the liver and small intestinal epithelium. Vδ5 γδ T cells are mainly found in peripheral blood (Liu and Zhang, Cells (2020) 9(5):1206) (Non-patent Literature 1).

[0005] γδ T cells recognize tumor cells via the γδ TCR and natural killer cell receptor (NKR). The Vγ9Vδ2 TCR recognizes non-peptide phosphate antigens (e.g., isopentenyl pyrophosphate, IPP) that are dependent on butyrophyllin 3A1 (BTN3A1) and butyrophyllin 2A1 (BTN2A1). Furthermore, Vγ9Vδ2 T cells recognize tumor cells via NKG2D and DNAM-1 (which bind to their ligands (MICA / B, ULBP, Nectin-2, and PVR)) (Liu and Zhang, Cells (2020) 9(5):1206 (Non-patent Literature 1)). Vδ1 T cells recognize lipid antigens presented by CD1d via the Vδ1 TCR. NKG2D and innate cytotoxic receptors (NCR, NKp30, NKp44, NKp46) and their ligands are involved in tumor cell recognition by Vδ1 T cells. Under continuous stimulation with a TCR agonist, if IL-15 or IL-2 is present, Vδ1 T cells can express NCR (Mikulak et al., JCI Insight (2019) 19, 4(24):e125884 (Non-patent Literature 2), Almeida et al., Clin. Cancer Res (2016) 22:5795-5804 (Non-patent Literature 3)). These cells exhibit strong antitumor activity against tumor cells and have very high IFNγ secretion capacity. There are few studies on Vδ3 and Vδ5 TCR ligands, and little is known about the antitumor mechanisms of these cells.

[0006] Currently, the majority of clinical trials focus on autologous γδ T cells due to their unique characteristics (e.g., not subject to MHC restriction and without the risk of graft-versus-host disease (GVHD)), and autologous Vγ9Vδ2 T cell adoptive transfer has shown good resistance and can induce antitumor immunity. The majority of strategies currently being evaluated incorporate tumor-targeting mechanisms, such as chimeric antigen receptors (CARs) or bispecific T cell engagers (bsTCEs), which may be key to obtaining more robust and consistent clinical responses. Preliminary results of these targeting methods (simultaneously cell- and antibody-based) have shown enormous potential and demonstrated the safety of Vγ9Vδ2 and Vδ1 T cell-based strategies. However, cell-based products face challenges not present in antibody-based therapies, such as high cost, manufacturing difficulties or the need for specialized facilities, and the need for preliminary lymphodermectomy regimens. Therefore, this disclosure relates to the development of antibodies that can effectively activate and revitalize γδ T cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Liu and Zhang, Cells (2020)9(5):1206) [Non-Patent Document 2] Mikulak et al., JCI Insight (2019) 19, 4(24):e125884 [Non-Patent Document 3] Almeida et al., Clin. Cancer Res (2016) 22:5795-5804 [Overview of the project]

[0008] overview This specification describes V that binds to γδ TCR. H H provides, the V HH is CDR1 containing amino acid sequences of SEQ ID NO: 15, 19, 23, 27, 31, 35, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 85, 89, 93, 121, 125, 140, 150, 154, 158, 162, 178, 182 or 186, and CDR2 containing amino acid sequences of SEQ ID NO: 16, 20, 24, 28, 32, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 82, 86, 90, 94, 122, 126, 137, 141, 151, 155, 159, 179, 183 or 187, and SEQ ID NO: Includes CDR3 containing amino acid sequences 17, 21, 25, 29, 33, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 83, 87, 91, 95, 123, 127, 138, 142, 152, 156, 160, 163, 180, 184, or 188.

[0009] In some embodiments, V HH includes CDR1, CDR2 and CDR3, which are (1) SEQ ID NO: 15, 16 and 17, or (2) SEQ ID NO: 19, 20 and 21, or (3) SEQ ID NO: 23, 24 and 25, or (4) SEQ ID NO: 27, 28 and 29, or (5) SEQ ID NO: 31, 32 and 33, or (6) SEQ ID NO: 35, 24 and 36, or (7) SEQ ID NO: 38, 39 and 40, or (8) SEQ ID NO: 42, 43 and 44, or (9) SEQ ID NO: 46, 47 and 48, or (10) SEQ ID NO: 50, 51 and 52, or (11) SEQ ID NO: 54, 55 and 56, or (12) SEQ ID NO: 58, 59 and 60, or (13) SEQ ID NO:62, 63 and 64, or (14) SEQ ID NO:66, 67 and 68, or (15) SEQ ID NO:70, 71 and 72, or (16) SEQ ID NO:74, 75 and 76, or (17) SEQ ID NO:78, 79 and 80, or (18) SEQ ID NO:46, 82 and 83, or (19) SEQ ID NO:85, 86 and 87, or (20) SEQ ID NO:89, 90 and 91, or (21) SEQ ID NO:93, 94 and 95, or (22) SEQ ID NO:121, 122 and 123, or (23) SEQ ID NO:125, 126 and 127, or (24) SEQ ID NO:54, 137 and 138, or (25) SEQ ID Each contains the amino acid sequences of NO:140, 141 and 142, or (26)SEQ ID NO:150, 151 and 152, or (27)SEQ ID NO:154, 155 and 156, or (28)SEQ ID NO:158, 159 and 160, or (29)SEQ ID NO:162, 59 and 163, or (30)SEQ ID NO:178, 179 and 180, or (31)SEQ ID NO:182, 183 and 184, or (32)SEQ ID NO:186, 187 and 188.

[0010] In some embodiments, V HH includes the amino acid sequence of SEQ ID NO: 14, 18, 22, 26, 30, 34, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 84, 88, 92, 120, 124, 136, 139, 149, 153, 157, 161, 177, 181 or 185, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0011] In some embodiments, V H H is humanized. In some examples, V H H includes the amino acid sequence of SEQ ID NO: 199, 200, 201, 202, 203, or 204, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0012] This specification further provides polypeptide constructs that bind to γδ TCR, the polypeptide constructs being disclosed above. H It contains H and the immunoglobulin Fc region.

[0013] In some embodiments, the immunoglobulin Fc region is an IgG Fc region, such as IgG1, IgG2, IgG3, or IgG4 Fc region.

[0014] This specification further provides an antibody or antigen-binding fragment thereof that binds to the γδ TCR, and said antibody or antigen-binding fragment thereof a) i) HCDR1 containing sequences with SEQ ID NO: 97, 105, 113, 129, 144, 165, 171, 190, or 209, ii) HCDR2 containing sequences with SEQ ID NO: 98, 106, 114, 130, 145, 166, 172, 206, 191 or 210, iii) A heavy chain variable region (VH) containing HCDR3 containing the sequence of SEQ ID NO: 99, 107, 115, 131, 146, 167, 173, 192 or 211, and / or b) i) LCDR1 containing the sequence SEQ ID NO: 101, 109, 117, 133, 175, 194 or 213, ii) LCDR2 containing sequences with SEQ ID NO: 102, 110, 118, 134 or 214, iii) Includes a light chain variable region (VL) containing LCDR3 containing the sequence of SEQ ID NO: 103, 111, 119, 135, 148, 169, 176, 195, or 215.

[0015] In some examples, the antibody or its antigen-binding fragment is (1) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 97, 98, and 99, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 101, 102, and 103, respectively. (2) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 105, 106, and 107, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 111, respectively. (3) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 113, 114, and 115, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 117, 118, and 119, respectively. (4) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 129, 130, and 131, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively. (5) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 144, 145, and 146, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 148, respectively. (6) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 165, 166, and 167, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 169, respectively. (7) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 171, 172, and 173, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. (8) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 171, 206, and 173, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. (9) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 190, 191, and 192, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 194, 110, and 195, respectively, (10) Includes HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 209, 210, and 211, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 213, 214, and 215, respectively.

[0016] In some examples, the antibody or its antigen-binding fragment includes a VH containing an amino acid sequence with SEQ ID NO: 96, 104, 112, 128, 143, 164, 170, 189, 205, or 208, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or a VL containing an amino acid sequence with SEQ ID NO: 100, 108, 116, 132, 147, 168, 174, 193, 207, or 212, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0017] In some examples, the antibody or its antigen-binding fragment is (1) VH containing the amino acid sequence of SEQ ID NO:96 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:100 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (2) VH containing the amino acid sequence of SEQ ID NO:104 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:108 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (3) VH containing the amino acid sequence of SEQ ID NO:112 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:116 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (4) VH containing the amino acid sequence of SEQ ID NO:128 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:132 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (5) VH containing the amino acid sequence of SEQ ID NO:143 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:147 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (6) VH containing the amino acid sequence of SEQ ID NO:164 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:168 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (7) VH containing the amino acid sequence of SEQ ID NO:170 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:174 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. (8) VH containing the amino acid sequence of SEQ ID NO:189 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:193 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or (9) VH containing the amino acid sequence of SEQ ID NO:208 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:212 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0018] In some examples, the antibody or its antigen-binding fragment is humanized. In some examples, the antibody or its antigen-binding fragment includes VH, which contains the amino acid sequence of SEQ ID NO:205 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL, which contains the sequence of SEQ ID NO:207 or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0019] In some examples, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) containing an amino acid sequence derived from the human immunoglobulin heavy chain constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. Preferably, the antibody or its antigen-binding fragment further comprises a light chain constant region (CL) containing an amino acid sequence derived from the human immunoglobulin light chain constant region, such as a κ light chain constant region.

[0020] In some examples, antibodies or their antigen-binding fragments are selected from scFv, Fab, Fab', (Fab')2, Fv fragments, diabodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, or humanized antibodies.

[0021] A multispecific molecule is further provided, which comprises a γδ TCR binding domain and an additional binding domain, the γδ TCR binding domain being the V disclosed above. H The molecule contains H, and the additional binding domain binds to targets other than γδ TCR, and preferably the multispecific molecule is a bispecific antibody.

[0022] A multispecific molecule is further provided, wherein the multispecific molecule comprises a γδ TCR binding domain and an additional binding domain, the γδ TCR binding domain comprises the VH and VL regions of the antibody or an antigen-binding fragment thereof disclosed above, and the additional binding domain binds to a target other than γδ TCR, and preferably, the multispecific molecule is a bispecific antibody. In some embodiments, the γδ TCR binding domain is an scFv, for example, an scFv comprising the structure of VL-linker-VH or VH-linker-VL.

[0023] In some embodiments, the target other than γδ TCR is a cancer antigen, for example, a tumor-specific antigen or a tumor-associated antigen.

[0024] In some embodiments, the cancer antigen is an antigen of hematological cancer, and the hematological cancer is, for example, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma or multiple myeloma.

[0025] In some embodiments, the cancer antigen is an antigen of a solid tumor, and the solid tumor is, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumor, mesothelioma, melanoma, sarcoma or brain tumor (for example, glioma such as glioblastoma).

[0026] In some embodiments, the cancer antigen is selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, claudin 6, claudin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA or PSCA.

[0027] In some embodiments, the additional binding domain is a monovalent antibody fragment, such as Fab, Fv, scFv or V H H.

[0028] In some examples, the multispecific molecule further comprises an immunoglobulin Fc domain composed of a first subunit and a second subunit, preferably an IgG Fc domain, such as an IgG1 Fc domain or an IgG4 Fc domain.

[0029] In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding affinity to the Fc receptor and / or effector function, improve stability (e.g., prevent Fab arm exchange), and / or promote heterodimerization (e.g., form a knob-into-hole).

[0030] In some embodiments, the Fc domain includes a knob-into-hole structure having, for example, substitution T366W in a first subunit of the Fc domain and substitution T366S and L368A (numbered based on the Kabat EU index) in a second subunit of the Fc domain.

[0031] In some examples, the Fc domain is the Fc domain of a human IgG4 isotype having amino acid substitutions S228P, L234A, and L235A (numbered based on the Kabat EU index).

[0032] In some examples, the γδ TCR binding domain is V H H or scFv, and the additional binding domain is Fab, which consists of a light chain (VL-CL) and a heavy chain (VH-CH1), and here the heavy chain of Fab is fused with one of the Fc domain subunits, and V H H or scFv is fused with the other Fc domain subunit.

[0033] In some examples, the multispecific molecules are A first polypeptide chain comprising an additional binding domain (VL) and a light chain constant domain (CL), A second polypeptide chain comprising an additional binding domain VH, a first heavy chain constant domain 1 (CH1), and a first Fc subunit, A third polypeptide chain comprising a γδ TCR-binding domain and a second Fc subunit The system includes, preferably, a first Fc subunit and a second Fc subunit, which include a knob-into-hole structure.

[0034] In some examples, the amino acid sequences of the first CH1 and the first Fc subunit are shown in SEQ ID NO:197, and the amino acid sequence of the second Fc subunit is shown in SEQ ID NO:198.

[0035] In some examples, the amino acid sequence of CL is shown in SEQ ID NO:196.

[0036] In some embodiments, the additional binding domain is a Fab that binds to CD33, and the Fab comprises VH and VL. In some embodiments, VH comprises the sequence of SEQ ID NO:216 or an amino acid sequence having at least 80% sequence identity thereto, and VL comprises the sequence of SEQ ID NO:217 or an amino acid sequence having at least 80% sequence identity thereto.

[0037] Isolated nucleic acid molecules are further provided, and the isolated nucleic acid molecules are as disclosed above. H H, comprising a polypeptide construct, an antibody or its antigen-binding fragment, or a nucleotide sequence encoding a multispecific molecule.

[0038] A vector is further provided, which comprises the nucleic acid molecule disclosed above.

[0039] Further providing cells, which include nucleic acid molecules or vectors disclosed above.

[0040] The V disclosed above HFurther providing a method for producing H, polypeptide constructs, antibodies or antigen-binding fragments thereof, or multispecific molecules, the method comprising culturing a host cell containing the isolated nucleic acid molecule or vector disclosed herein, or the host cell disclosed herein, under conditions that enable protein expression, and obtaining V from the culture of the cultured host cell. H This includes recovering H, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules.

[0041] Further providing is a pharmaceutical composition which is the same as disclosed above. H The material comprises H, a polypeptide construct, an antibody or its antigen-binding fragment, or a multispecific molecule, and a pharmaceutically acceptable carrier and / or excipient.

[0042] In some embodiments, the pharmaceutical composition further comprises additional therapeutic agents.

[0043] In some embodiments, additional therapeutic agents include anticancer agents, such as immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, or anti-CTLA-4 antibody), cytotoxic agents (e.g., alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, or radionuclides), or cytokines (e.g., immune cell activating cytokines such as IL-2, IL-15, IL-7, IL-6, IL-12, IL-18, IFNα, IFNβ, or IFNγ).

[0044] Further providing a method for treating the target cancer, the method disclosed in an effective amount or more to a subject in need thereof. H This includes administering H, a polypeptide construct, an antibody or its antigen-binding fragment, a multispecific molecule, or a pharmaceutical composition.

[0045] In some embodiments, the cancer is a hematological cancer, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0046] In some embodiments, cancer is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (such as glioblastoma or other gliomas).

[0047] In some embodiments, the method comprises administering a multispecific molecule for cancer treatment, wherein the cancer is characterized by a cancer antigen targeted by the multispecific molecule.

[0048] In some examples, the cancer is characterized by a cancer antigen selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, Claudin 6, Claudin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, Mesothelin, HER2, PSMA, or PSCA.

[0049] In some examples, the subject is a mammal, such as a human.

[0050] In some embodiments, V H H, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules or pharmaceutical compositions are administered in combination with additional therapeutic agents or additional therapies. In some examples, the additional therapeutic agent or additional therapy is an anticancer agent or therapy, preferably an immune checkpoint inhibitor, a cytotoxic agent, or a cytokine, and preferably the additional therapy is a standard treatment for cancer, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0051] Further providing a method for activating γδ T cells or increasing γδ T cell proliferation, the method comprising T cells as disclosed above. HThis involves contacting H with a polypeptide construct, an antibody or its antigen-binding fragment, a multispecific molecule, or a pharmaceutical composition.

[0052] The V disclosed above H The present invention further provides the use of H, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules or pharmaceutical compositions in the manufacture of drugs for treating target cancers.

[0053] Further features and advantages of some embodiments of this disclosure will become more apparent from the following descriptions of the embodiments and their drawings, and from the claims. [Brief explanation of the drawing]

[0054] [Figure 1] This is a construct design and sequence ID for soluble human and cynomolgus monkey γ9δ2 TCR. [Figure 2] This document contains the construct designs and sequence IDs for five types of artificial human TCR proteins. [Figure 3] This is the construct design and sequence ID for CD33-targeting TCE-KIH. Anti-γδ TCR VHH (Figure 3a) or anti-γδ TCR scFv (Figure 3b) was incorporated into the CD33-targeting TCE-KIH BsAb molecule. The linker between VL and VH in scFv is (G4S)3. [Figure 4] This study involved the killing of AML-193 cells by CD33×γδ TCR TCE-KIH BsAb redirection. γ9δ2 T cells were used as effector cells, with effector-to-target cell ratios of 2:1 (Figures 4a, 4b, 4c, and 4d) or 1:1 (Figures 4e, 4f, and 4g). A total of 35 BsAbs were tested in the two experiments, and 5E7-KIH was used as a positive control. [Figure 5]CD33×γδ TCR TCE-KIH is a method of killing CD33+ AML-193 cells by BsAb redirection. AS281876-KIH and AS282152-KIH are shown in Figure 5a. AS288170-KIH and AS282067-KIH are shown in Figure 5b. AS282116-KIH and AS287963-KIH are shown in Figure 5c. 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls. [Figure 6] CD33×γδ TCR TCE-KIH is a method of killing CD33+ MOLM-13 cells by BsAb redirection. AS281876-KIH and AS282152-KIH are shown in Figure 6a. AS288170-KIH and AS282067-KIH are shown in Figure 6b. AS282116-KIH and AS287963-KIH are shown in Figure 6c. 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls. [Figure 7] CD33×γδ TCR TCE-KIH is a method of killing CD33+ KG-1A cells by BsAb redirection. AS281876-KIH and AS282152-KIH are shown in Figure 7a. AS288170-KIH and AS282067-KIH are shown in Figure 7b. AS282116-KIH and AS287963-KIH are shown in Figure 7c. 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls. [Figure 8] CD33×γδ TCR TCE-KIH is a method of killing CD33+ NALM6 cells by BsAb redirection. AS281876-KIH and AS282152-KIH are shown in Figure 8a. AS288170-KIH and AS282067-KIH are shown in Figure 8b. AS282116-KIH and AS287963-KIH are shown in Figure 8c. 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls. [Figure 9]CD33×γδ TCR TCE-KIH is a method of killing CD33-CCRF-CEM by BsAb redirection. AS281876-KIH and AS282152-KIH are shown in Figure 9a. AS288170-KIH and AS282067-KIH are shown in Figure 9b. AS282116-KIH and AS287963-KIH are shown in Figure 9c. 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls. [Figure 10] Figures a-e show the cell proliferation ratio (Figure 10a), viability (Figure 10b), purity (Figure 10c), cell subtype percentage (Figure 10d), and CAR positivity rate (Figure 10e) of γδ T cells produced using different antibodies. [Figure 11] Figures a-c show the cytotoxicity of γδ T cells produced using different antibodies (Figure 11a), total T cell proliferation (Figure 11b), and CAR+ T cell proliferation after tumor cell elimination (Figure 11c). [Modes for carrying out the invention]

[0055] Detailed explanation definition In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. For a better understanding of this disclosure, definitions and interpretations of terms are provided below.

[0056] As used herein, the term “antibody” refers to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Unless otherwise specified or evident from the context, the term “antibody” as used herein may include a complete antibody and any antigen-binding fragment thereof (i.e., “antigen-binding portion”) or a single chain. The term “antibody” may also refer to a typical antibody that typically contains at least one heavy chain and at least one light chain. The antibody light chain may be classified into κ light chains and λ light chains. The heavy chain may be classified into μ, δ, γ, α, or ε, and the antibody isotypes may be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region and the constant region are linked via a “J” region of about 12 or more amino acids, and the heavy chain further contains a “D” region of about 3 or more amino acids. Each heavy chain has a heavy chain variable region (V H ) and heavy chain constant region (C H ) consists of the following. The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3) consists of a light chain variable region (V L ) and the light chain steady region (C L ) consists of the following. The light chain constant region is one domain C L It consists of V. H and V L The region may be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), with relatively conserved regions called framework regions (FRs) inserted between them. H and V L Each of these consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy / light chain pair. H and V L These each form an antibody-binding site. The term "antibody" is not limited to any specific method for producing an antibody.

[0057] As used herein, "V H H" or "V" H The term "H domain" refers to the antigen-binding portion of a single-domain antibody (e.g., a camelid heavy chain antibody or a shark heavy chain antibody). H H may include three CDRs and four framework areas designated as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. H If H maintains its antigen binding and specificity substantially, V H H may be cleaved at the N-terminus or C-terminus to include only partial FR1 and / or FR4 or to lack one or two of those framework regions. As used herein, "Humanized V H "H" is a V in which one or more of the framework areas are substantially replaced by the human framework area. H This refers to H. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Note that humanized V H H is the original V H It may include residues not found in either H or the human framework sequence, but these residues are included in V. H Further refine and optimize the performance of H. As is understood, the humanized sequence may be identified by its primary sequence and does not necessarily represent the antibody production process.

[0058] As used herein, the terms “complementarity-determining region” or “CDR” refer to the amino acid residues responsible for antigen binding in the variable region of an antibody. The precise boundaries of these amino acid residues may be defined based on various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987) J.Mol.Biol.196:901-917, Chothia et al., (1989) Nature 342:878-883), the AbM numbering system (Martin, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag), or the IMGT numbering system (Lefranc et al., Dev.Comparat.Immunol.27:55-77, 2003). A person skilled in the art can readily recognize the CDR defined by each numbering system for a given antibody. The correspondences between different numbering systems are well known to those skilled in the art (see, for example, Lefranc et al., Dev.Comparat.Immunol.27:55-77, 2003). The CDR of the antibodies in this disclosure may be defined based on the Kabat, AbM, IMGT, or Chothia numbering system or any combination thereof. Unless otherwise specified or evident from the context, the CDR of the antibodies in this disclosure is preferably defined based on the AbM numbering system.

[0059] As used herein, the terms “framework region” or “FR” residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues defined above.

[0060] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide comprising a full-length antibody fragment, which retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, and the polypeptide is also called the “antigen-binding moiety.” Antigen-binding fragments of antibodies may be produced by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. Antigen-binding fragments may include fragments of Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining regions (CDRs), single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody (the at least portion being sufficient to confer specific antigen-binding ability to the polypeptide).

[0061] As used herein, the term "Fd fragment" is V H and C H The term "dAb fragment" refers to an antibody fragment consisting of one domain. H The term "Fab fragment" refers to an antibody fragment consisting of domains. L , V H , C L and C H The term "Fab fragment" refers to an antibody fragment consisting of one domain, while the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments, which are linked via disulfide crosslinks in the hinge region.

[0062] As used herein, the term "Fv fragment" refers to the V of a single arm of an antibody. L and V H This refers to an antibody fragment consisting of domains. Fv fragments are generally considered to be the smallest antibody fragments capable of forming a complete antigen-binding site. People believe that six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (for example, an Fd fragment containing only three CDRs specific to the antigen) can recognize and bind to the antigen, but its affinity is lower than the affinity of the entire binding site.

[0063] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, having a common structure of NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable linker in the prior art consists of a repeating G4S amino acid sequence or a variant thereof. In some cases, a disulfide bond may be present between the VH and VL domains of the scFv.

[0064] As used herein, the term “diabody” refers to a dimer of scFv, which consists of VH and VL domains linked via a short-chain peptide linker. The linker is too short to form intrachain pairing of the VH and VL domains. Conversely, two such scFv fragments co-express to form a multimer through interchain pairing (cross-pairing) of the VH and VL domains.

[0065] Each antigen-binding fragment retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to that antigen. Antigen-binding fragments can be obtained from a given antibody (e.g., a complete antibody as defined herein) using common techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the specificity of these antigen-binding fragments can be screened in the same manner as that used to screen the complete antibody.

[0066] As used herein, the term “Fc region” refers to a portion of the heavy chain constant region containing CH2 and CH3. The Fc region may include the hinge, CH2, and CH3. The Fc region may be any antibody heavy chain constant region isotype discussed herein. The Fc region may be IgG1, IgG2, IgG3, or IgG4.

[0067] As used herein, the term “chimeric antibody” refers to an antibody in which part of its light chain and / or heavy chain originates from one antibody (which may originate from a particular species or belong to a particular antibody type or subtype), and another part of its light chain and / or heavy chain originates from another antibody (which may originate from the same or different species or belong to the same or different antibody type or subtype), provided that the antibody still retains the activity to bind to the target antigen. For example, the term “chimeric antibody” may include an antibody in which the heavy chain and light chain variable regions originate from a first antibody (e.g., a camelid antibody), but the heavy chain and light chain constant regions originate from a second antibody (e.g., a human antibody) (e.g., a human-camelid chimeric antibody).

[0068] As used herein, the term “humanized antibody” refers to a genetically modified non-human antibody whose amino acid sequence has been modified to increase homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies typically retain the expected properties of the donor antibody, including, but not limited to, the ability to specifically bind to γδ TCRs and the ability to activate γδ T cells.

[0069] As used herein, the terms “specifically bind” or “specifically binding” refer to the binding of two molecules in a non-random manner, for example, a reaction between an antibody and its target antigen. Antibodies that specifically bind to an antigen (or antibodies that have specificity for an antigen) are approximately 10 -5 Less than M (for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K) less than or equal to M D ) may also refer to an antibody that binds to an antigen.

[0070] As used herein, "K D The term "AQU" refers to a specific antibody-antigen interaction dissociation constant used to describe the binding affinity of an antibody to an antigen. A smaller dissociation constant results in tighter antibody binding and higher affinity between the antibody and the antigen. Typically, an antibody (e.g., the antibody in this disclosure) has a dissociation constant of approximately 10. -5 Less than M (for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K (less than or equal to M) D It binds to the antigen (e.g., human γδ TCR), and the K D This is determined, for example, by surface plasmon resonance (SPR) in a BIACORE instrument.

[0071] As used herein, “isolated nucleic acid” refers to a nucleic acid (e.g., RNA, DNA, or mixed nucleic acid) that is substantially isolated from other genomic DNA sequences and proteins or complexes naturally associated with the sequence, such as ribosomes and polymerases. “Isolated” nucleic acid molecules are nucleic acid molecules that are isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. “Isolated” nucleic acid molecules, such as cDNA molecules, if produced by recombinant technology, may substantially contain no other cellular material or culture medium, or if chemically synthesized, they may substantially contain no chemical precursors or other chemicals. One or more nucleic acid molecules encoding the chimeric constructs described herein may be isolated or purified. The term includes nucleic acid sequences removed from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule may include an isolated form of the molecule.

[0072] As used herein, the term “vector” refers to a nucleic acid medium into which polynucleotides can be inserted. If a vector enables the expression of a protein encoded by the polynucleotide into which it is inserted, the vector is called an expression vector. A vector can cause a host cell to express genetic material elements that are delivered by transformation, transfection, or other means. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), phages (e.g., λ phage or M13 phage), and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papova viruses (e.g., SV40). The vector may contain multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. The vector may also contain an origin of replication.

[0073] As used herein, the term “host cell” refers to a cell into which a vector can be introduced or transformed, and includes, but is not limited to, prokaryotic cells (e.g., Escherichia coli or Bacillus subtilis) and eukaryotic cells (e.g., mammalian cells (e.g., mouse cells or human cells), insect cells or yeast cells). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells or MDCKII cells.

[0074] As used herein, the culture medium may include a basal medium. Any suitable mammalian cell medium (e.g., AIM-V®, X-VIVO, TexMACS, RPMI 1640, OPTMIZER CTS® (Gibco, Life Technologies), XVIVO-10, XVIVO-15, or XVIVO-20 (Lonza)) may be used as a basal medium. The cell medium may contain L-glutamine, streptomycin sulfate, and gentamicin sulfate. The cell medium may contain L-glutamine, 50 μg / mL streptomycin sulfate, and 10 μg / mL gentamicin sulfate. The mammalian cell medium may contain serum or plasma. The cell medium may contain about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the basal medium by volume. The cell culture medium may contain more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the basal culture medium by volume. The cell culture medium may also contain less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the basal culture medium by volume.

[0075] As used herein, the term “identity” refers to the degree of similarity between two polypeptides or two nucleic acids. Two sequences used for comparison are identical at a site if they have the same base or amino acid monomer subunit at a site (for example, each of two DNA molecules has adenine at a site, or each of two polypeptides has lysine at a site). The percentage of identity between two sequences is calculated by multiplying the ratio of the number of identical sites shared by the two sequences to the total number of sites used for comparison by 100. For example, if six out of ten sites of two sequences match, these two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six sites match). Typically, the comparison of two sequences is performed in a manner that produces maximum identity. Such comparisons may be performed using a computer program (e.g., the Align program (DNAstar, Inc.) based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970)). The percentage of identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has already been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percentage of identity between two amino acid sequences may also be determined using the algorithm of Needleman and Wunsch (J.Mol.Biol.48:444-453(1970)), which is already incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com), using a Blossum 62 matrix or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0076] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the target and active ingredient, and such carrier and / or excipient includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintenance reagents, absorption retardant reagents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffer solutions. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Osmotic pressure maintenance reagents include, but are not limited to, sugars and NaCl. Absorption retardant reagents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols, and polyols (e.g., glycerol). Stabilizers have a meaning generally understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug (including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysis products)). Pharmaceutically acceptable carriers or excipients may include sterile injectable liquids (e.g., aqueous or non-aqueous suspensions or solutions). Such sterile injectable liquids may be selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0077] As used herein, the term “treatment” refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease extent, stabilization of the disease state (i.e., no worsening), delay or mitigation of disease progression, and reduction of symptoms (partial or complete), whether detectable or undetectable. “Treatment” further means extending survival compared to the expected survival (without treatment). Beneficial or desired clinical outcomes described herein may include, but are not limited to, mitigation of tumor progression, regression of cancer, enhancement of the anti-tumor immune response, reduction of tumor growth or size, tumor necrosis, reduction of the severity of at least one disease symptom, increase in the frequency and duration of asymptomatic periods of the disease, prevention of disability or dysfunction from disease-related distress, or improvement of the patient’s disease symptoms in other ways.

[0078] As used herein, the term “subject” refers to any human or non-human animal receiving treatment. The term “non-human animal” includes all vertebrates, such as non-human primates, mammals and non-mammals such as sheep, dogs, cows, chickens, amphibians, and reptiles.

[0079] As used herein, the term “effective dose” refers to an amount sufficient to produce, or at least partially produce, the desired effect. For example, an effective dose for treating a disease refers to an amount that effectively cures, or at least partially blocks, the disease and its complications in a patient suffering from the disease. Determining such an effective dose is within the capabilities of those skilled in the art. For example, an effective dose used for therapeutic purposes depends on the severity of the disease to be treated, the general state of the patient’s immune system, the patient’s general circumstances (e.g., age, weight, and sex), the route of drug administration, and any additional therapies used in combination.

[0080] As used herein, the terms “cancer” and “tumor” are interchangeable and refer to a class of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells, which can invade adjacent tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers and dormant tumors or micrometastases. Cancer further includes hematological malignancies.

[0081] V that binds to γδ TCR H H antibody In one embodiment, V binds to the γδ TCR. H H, heavy chain antibody, or V H Provides polypeptide constructs containing H. H H" or "V" H The term “H domain” is interchangeable herein and is intended to refer to the antigen-binding portion of a single-domain antibody (e.g., a camelid heavy chain antibody or a shark heavy chain antibody). H H typically includes three CDRs and four framework areas designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some cases, V H If H maintains its antigen binding and specificity substantially, V H H may be truncated at its N-terminus or C-terminus to include only partial FR1 and / or FR4, or to lack one or two of those framework regions.

[0082] The term "heavy-chain only antibody" or "HCAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in four-chain antibodies. Camelids (e.g., camels, llamas, alpacas) are known to produce HCAbs. HCAbs may contain sdAbs fused to the Fc region. HCAbs may also contain sdAbs fused to the human IgG1 hinge and Fc region.

[0083] V according to this specification HH, heavy chain antibodies, or polypeptide constructs can be particularly conjugated to γ9δ2 TCRs (e.g., human γ9δ2 TCRs containing the γ chain of SEQ ID NO:1 and the δ chain of SEQ ID NO:2).

[0084] In one embodiment, this specification relates to V binding to γδ TCR (e.g., γ9δ2 TCR). H H provides, the V H H contains CDR1 containing the amino acid sequence of SEQ ID NO: 15, 19, 23, 27, 31, 35, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 85, 89, 93, 121, 125, 140, 150, 154, 158, 162, 178, 182 or 186, and CDR2 containing the amino acid sequence of SEQ ID NO: 16, 20, 24, 28, 32, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 82, 86, 90, 94, 122, 126, 137, 141, 151, 155, 159, 179, 183 or 187, and SEQ ID NO:Includes CDR3 containing amino acid sequences of 17, 21, 25, 29, 33, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 83, 87, 91, 95, 123, 127, 138, 142, 152, 156, 160, 163, 180, 184, or 188. The above-mentioned CDRs may be defined based on the AbM numbering system.

[0085] In one embodiment, this specification relates to V binding to γδ TCR (e.g., γ9δ2 TCR). H H provides, the V H H is a V that corresponds to any one of the following SEQ ID NO: 14, 18, 22, 26, 30, 34, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 84, 88, 92, 120, 124, 136, 139, 149, 153, 157, 161, 177, 181, 185, 199, 200, 201, 202, 203, or 204 H H contains three CDRs. CDRs may be defined based on the Kabat, AbM, IMGT, or Chothia numbering system or any combination thereof.

[0086] V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 15, 16 and 17, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 19, 20 and 21, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 23, 24 and 25, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 27, 28 and 29, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 31, 32 and 33, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 35, 24 and 36, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 38, 39 and 40, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 42, 43 and 44, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 46, 47 and 48, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 50, 51 and 52, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 54, 55 and 56, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 58, 59 and 60, respectively. V H V H may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 62, 63 and 64, respectively. V HVH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 66, 67 and 68, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 70, 71 and 72, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 74, 75 and 76, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 78, 79 and 80, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 46, 82 and 83, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 85, 86 and 87, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 89, 90 and 91, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 93, 94 and 95, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 121, 122 and 123, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 125, 126 and 127, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 54, 137 and 138, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 140, 141 and 142, respectively. H VH may comprise CDR1, CDR2 and CDR3 each comprising the amino acid sequences of SEQ ID NO: 150, 151 and 152, respectively. HH may include CDR1, CDR2, and CDR3, which contain the amino acid sequences of SEQ ID NO: 154, 155, and 156, respectively. H H may include CDR1, CDR2, and CDR3, which contain the amino acid sequences of SEQ ID NO: 158, 159, and 160, respectively. H H may include CDR1, CDR2, and CDR3, which contain the amino acid sequences of SEQ ID NO: 162, 59, and 163, respectively. H H may include CDR1, CDR2, and CDR3, which contain the amino acid sequences of SEQ ID NO: 178, 179, and 180, respectively. H H may include CDR1, CDR2, and CDR3, which contain the amino acid sequences of SEQ ID NO: 182, 183, and 184, respectively. H H may include CDR1, CDR2, and CDR3, which contain the amino acid sequences of SEQ ID NO: 186, 187, and 188, respectively.

[0087] V disclosed herein H H may further include the framework area (FR).

[0088] V H H may include a framework region (FR) derived from a heavy chain antibody of the camelid family.

[0089] V H H may include the amino acid sequence of SEQ ID NO: 14, 18, 22, 26, 30, 34, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 84, 88, 92, 120, 124, 136, 139, 149, 153, 157, 161, 177, 181 or 185, or an amino acid sequence having at least 80% (for example, at least 85%, 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%) sequence identity thereto.

[0090] Alternatively, V HH is humanized V H It may also be H, where one or more of the framework domains are substantially replaced by the human framework domain. H H may contain a framework region (FR) derived from the variable region of the human immunoglobulin heavy chain, and one or more of these framework residues can be mutated by returning them to the camelid control (i.e., a reversion mutation).

[0091] V H H may include the amino acid sequence of SEQ ID NO: 199, 200, 201, 202, 203, or 204, or an amino acid sequence having at least 80% (for example, at least 85%, 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%) sequence identity thereto.

[0092] V H H may be selected from AS282152 (containing CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 66, 67, and 68, respectively), AS281876 (containing CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively), or AS282067 (containing CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 50, 51, and 52, respectively), which recognizes epitopes located in Vγ9 and the constant region of the γδ TCR.

[0093] V H H may be selected from AS282116 (containing CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 58, 59, and 60, respectively) or AS287963 (containing CDR1, CDR2, and CDR3 containing amino acid sequences of SEQ ID NO: 162, 59, and 163, respectively), which recognizes an epitope located in the constant region of the γδ TCR.

[0094] In one embodiment, this specification further provides a heavy chain antibody that binds to a γδ TCR (e.g., γ9δ2 TCR), which is described herein as V H Contains H. The heavy chain antibody may also be a dimer of two polypeptides, where each polypeptide contains at least one V H It contains the H domain and Fc region. The heavy chain antibody has multiple V H It may cover polypeptides containing an H domain.

[0095] In one embodiment, this specification further provides polypeptide constructs that bind to γδ TCRs (e.g., γ9δ2 TCRs), which are described herein as V H Includes H and immunoglobulin Fc regions.

[0096] The immunoglobulin Fc region may also be an IgG Fc region, such as an IgG1, IgG2, IgG3, or IgG4 Fc region. The immunoglobulin Fc region may further be a human IgG Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0097] The immunoglobulin Fc region may be a natural sequence Fc region, which contains an amino acid sequence identical to that of an Fc region found in nature.

[0098] Alternatively, the immunoglobulin Fc region may be a mutant Fc region, which contains an amino acid sequence different from the native Fc region due to at least one amino acid mutation. Introducing one or more mutations (e.g., one or more amino acid substitutions) into the constant-region Fc region can alter (increase, decrease, or eliminate) one or more effector functions of the same antibody, such as ADCC, CDC, or ADCP, compared to an antibody without the one or more mutations. The immunoglobulin Fc region may have the decreased or eliminated one or more effector functions.

[0099] V HH can fuse with the N-terminus of an immunoglobulin Fc region without a linker (for example, at its C-terminus).

[0100] Alternatively, V H H can fuse with the N-terminus of the immunoglobulin Fc region if a linker is present (e.g., at its C-terminus). The linker may be selected from cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. The linker may be a peptide linker. The linker may include a (GmS)n sequence, where m is selected from an integer between 1 and 6 (e.g., 1, 2, 3, or 4) and n is selected from an integer between 1 and 6 (e.g., 1, 2, or 3).

[0101] In all of those embodiments described above, the V disclosed herein H H, heavy chain antibodies, or polypeptide constructs can activate γδ T cells and / or increase γδ T cell proliferation, potentially producing an effective antitumor response.

[0102] Common antibodies that bind to γδ TCR In one embodiment, a general antibody that binds to γδ TCR is provided. The term “general antibody” refers to an antibody that typically comprises at least one heavy chain and at least one light chain. In some cases, it refers to a general Y-type antibody consisting of two heavy chains and two light chains. In some cases, the general antibody according to this specification binds specifically to γ9δ2 TCR (e.g., human γ9δ2 TCR comprising the γ chain of SEQ ID NO:1 and the δ chain of SEQ ID NO:2).

[0103] CDR and variable area In one embodiment, this specification provides an antibody or antigen-binding fragment thereof that binds to a γδ TCR (e.g., γ9δ2 TCR), and the antibody or antigen-binding fragment thereof a) i) HCDR1 containing sequences with SEQ ID NO: 97, 105, 113, 129, 144, 165, 171, 190, or 209, ii) HCDR2 containing the sequence SEQ ID NO: 98, 106, 114, 130, 145, 166, 172, 206, 191 or 210, iii) A heavy chain variable region (VH) including HCDR3 containing the sequence of SEQ ID NO: 99, 107, 115, 131, 146, 167, 173, 192 or 211, and / or b) i) LCDR1 containing sequence SEQ ID NO: 101, 109, 117, 133, 175, 194 or 213, ii) LCDR2 containing a sequence with SEQ ID NO: 102, 110, 118, 134 or 214, iii) Includes a light chain variable region (VL) containing an LCDR3 containing the sequence of SEQ ID NO: 103, 111, 119, 135, 148, 169, 176, 195, or 215. The CDR may be defined based on the AbM numbering system.

[0104] In one embodiment, this specification provides an antibody or antigen-binding fragment thereof that binds to a γδ TCR (e.g., γ9δ2 TCR), the antibody or antigen-binding fragment comprising three heavy-chain CDRs contained in a VH shown as one of SEQ ID NO: 96, 104, 112, 128, 143, 164, 170, 205, 189, or 208, and / or three light-chain CDRs contained in a VL shown as one of SEQ ID NO: 100, 108, 116, 132, 147, 168, 174, 207, 193, or 212. CDRs may be defined based on the Kabat, AbM, IMGT, or Chothia numbering system or any combination thereof.

[0105] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 97, 98, and 99, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 101, 102, and 103, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 97, 98, 99, 101, 102, and 103, respectively.

[0106] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 105, 106, and 107, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 111, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 105, 106, 107, 109, 110, and 111, respectively.

[0107] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 113, 114, and 115, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 117, 118, and 119, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 113, 114, 115, 117, 118, and 119, respectively.

[0108] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 129, 130, and 131, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 129, 130, 131, 133, 134, and 135, respectively.

[0109] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 144, 145, and 146, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 148, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 144, 145, 146, 109, 110, and 148, respectively.

[0110] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 165, 166, and 167, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 169, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 165, 166, 167, 109, 110, and 169, respectively.

[0111] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 171, 172, and 173, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 171, 172, 173, 175, 118, and 176, respectively.

[0112] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 171, 206, and 173, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 171, 206, 173, 175, 118, and 176, respectively.

[0113] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 190, 191, and 192, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 194, 110, and 195, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 190, 191, 192, 194, 110, and 195, respectively.

[0114] The antibody or its antigen-binding fragment may include HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 209, 210, and 211, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 213, 214, and 215, respectively. The antibody or its antigen-binding fragment may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 209, 210, 211, 213, 214, and 215, respectively.

[0115] A typical antibody may be AS288170 (containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing amino acid sequences of SEQ ID NO: 171, 172, or 206, 173, 175, 118, and 176, respectively), which recognizes the Vγ9 and constant region epitopes of the γδ TCR.

[0116] The antibodies or antigen-binding fragments disclosed herein may further comprise a framework region (FR). The antibodies or antigen-binding fragments may comprise a framework region (FR) derived from mammalian (e.g., camelid or human) immunoglobulin.

[0117] The antibody or its antigen-binding fragment may include a framework region (FR) derived from a common antibody of a camelid animal.

[0118] The antibody or its antigen-binding fragment may contain VH, which contains an amino acid sequence of SEQ ID NO: 96, 104, 112, 128, 143, 164, 170, 189, or 208, or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto.

[0119] The antibody or its antigen-binding fragment may contain a VL, which contains an amino acid sequence of SEQ ID NO: 100, 108, 116, 132, 147, 168, 174, 193, or 212, or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto.

[0120] The antibody or its antigen-binding fragment may include VH containing the amino acid sequence of SEQ ID NO:96 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or VL containing the sequence of SEQ ID NO:100 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include VH containing the amino acid sequence of SEQ ID NO:96 and VL containing the sequence of SEQ ID NO:100.

[0121] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:104 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:108 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:104 and a VL containing the sequence of SEQ ID NO:108.

[0122] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:112 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:116 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:112 and a VL containing the sequence of SEQ ID NO:116.

[0123] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:128 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:132 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:128 and a VL containing the sequence of SEQ ID NO:132.

[0124] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:143 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:147 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:143 and a VL containing the sequence of SEQ ID NO:147.

[0125] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:164 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:168 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:164 and a VL containing the sequence of SEQ ID NO:168.

[0126] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:170 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:174 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:170 and a VL containing the sequence of SEQ ID NO:174.

[0127] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:189 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:193 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:189 and a VL containing the sequence of SEQ ID NO:193.

[0128] The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:208 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or a VL containing the sequence of SEQ ID NO:212 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may include a VH containing the amino acid sequence of SEQ ID NO:208 and a VL containing the sequence of SEQ ID NO:212.

[0129] Alternatively, the antibody or its antigen-binding fragment may be humanized, where the amino acid sequence is modified to increase homology with the human antibody sequence. Typically, humanized antibodies can be produced by transplanting a camelid CDR region onto a human framework sequence. If the affinity of these straight-graft antibodies is lost, some framework residues may be mutated back into the camelid control (i.e., reverse mutation) to restore the antibody binding affinity.

[0130] The antibody or its antigen-binding fragment may contain framework regions (FRs) derived from human immunoglobulins, and optionally contain one or more reverse mutations from human residues to camelid residues. The antibody or its antigen-binding fragment may contain heavy chain FRs derived from human immunoglobulin heavy chain variable region sequences. The antibody or its antigen-binding fragment may contain light chain FRs derived from human immunoglobulin light chain variable region sequences.

[0131] The antibody or its antigen-binding fragment may contain VH, which includes the sequence of SEQ ID NO:205 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto, and / or VL, which includes the sequence of SEQ ID NO:207 or an amino acid sequence having at least 80% (e.g., at least 85%, 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%) sequence identity thereto. The antibody or its antigen-binding fragment may contain VH containing the sequence of SEQ ID NO:205 and VL containing the sequence of SEQ ID NO:207.

[0132] Steady-state region The antibodies or antigen-binding fragments disclosed herein may further comprise constant region sequences derived from mammalian (e.g., camelid or human) immunoglobulins. The antibodies or antigen-binding fragments disclosed herein may comprise constant region sequences derived from human immunoglobulins.

[0133] Any immunoglobulin constant region can be used in the antibodies disclosed herein. The heavy chain constant region may be the heavy chain constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) immunoglobulin molecule. The light chain constant region may be λ or κ. The heavy chain constant region may further be an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. The light chain constant region may further be κ.

[0134] The selection of the constant region depends in part on whether antibody-dependent complement and / or cell-mediated cytotoxicity is desired. A heavy chain constant region with less or reduced effector function may be preferred. Alternatively, a heavy chain constant region with effector function (e.g., ADCC) or enhanced effector function may be preferred.

[0135] The heavy chains of antibodies or antigen-binding fragments disclosed herein may include a heavy chain constant region (CH), which comprises an amino acid sequence derived from the human immunoglobulin heavy chain constant region.

[0136] The antibodies or antigen-binding fragments disclosed herein may include a heavy chain constant region, which is a wild-type heavy chain constant region.

[0137] Alternatively, the antibodies or antigen-binding fragments disclosed herein may include a heavy chain constant region, which is a variant of the wild-type heavy chain constant region. One or more mutations (e.g., one or more amino acid substitutions) can be introduced into the Fc region of the constant region to alter (increase, decrease, or eliminate) one or more effector functions of the same antibody, such as ADCC, CDC, or ADCP, compared to an antibody without the one or more mutations.

[0138] The light chains of antibodies or antigen-binding fragments disclosed herein may include a light chain constant region (CL), which comprises an amino acid sequence derived from a human immunoglobulin light chain constant region.

[0139] antigen binding fragment The antigen-binding fragments disclosed herein may be any type of antibody fragment having a common "Y" structure, which substantially retains the ability to specifically bind to γδ TCRs and promote the activation or proliferation of γδ T cells.

[0140] The antibodies or antigen-binding fragments disclosed herein may be selected from the group consisting of scFv, Fab, Fab', (Fab')2, Fv fragments, diabodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, or humanized antibodies.

[0141] In all of the embodiments described above, the antibodies or antigen-binding fragments disclosed herein can activate γδ T cells and / or increase γδ T cell proliferation, potentially producing an effective antitumor response.

[0142] Antibody sequences according to this specification (V HFurther, we provide "conservative sequence modifications" for antibodies (including H, heavy chain antibodies, and general antibodies), i.e., nucleotide and amino acid sequence modifications that do not remove the binding of antibodies to antigens, either encoded by nucleotide sequences or containing amino acid sequences. For example, modifications may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). The resulting modified antibodies may be screened for their binding activity.

[0143] Conservative sequence modifications include conservative amino acid substitutions, where an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have already been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues predicted in the antibodies disclosed herein may preferably be replaced by other amino acid residues from the same side chain family. Methods for identifying nucleotide and amino acid conserved substitutions that do not remove antigen binding are well known in this field. See, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997).

[0144] Multiple specific molecules that bind to γδ TCR In another embodiment, a multispecific molecule that binds to γδ TCR is provided. As used herein, the term “multispecific” molecule refers to a molecule having at least two binding specificities, such as a polypeptide.

[0145] The multispecific molecule may also be a bispecific antibody that has two binding specificities.

[0146] Multispecific molecules include V as described herein. H It may also contain a γδ TCR-binding domain that includes H.

[0147] The multispecific molecule may include a γδ TCR binding domain, which includes the VH and VL regions of the antibody or antigen-binding fragment described herein. The γδ TCR binding domain may be scFv. The γδ TCR binding domain may have a VL-linker-VH structure. Alternatively, the γδ TCR binding domain may have a VH-linker-VL structure. The linker may include a (GmS)n sequence, where m is selected from an integer between 1 and 6 (e.g., 1, 2, 3, or 4) and n is selected from an integer between 1 and 6 (e.g., 1, 2, or 3). The linker may also include a (G4S)3 sequence.

[0148] The multispecific molecule may include additional binding domains that bind to targets other than the γδ TCR.

[0149] Cancer antigens Other targets besides the γδ TCR may be cancer antigens, such as tumor-specific antigens or tumor-associated antigens.

[0150] The cancer antigen may be an antigen of a blood cancer. The blood cancer may be selected from, for example, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0151] The cancer antigen may also be an antigen of a solid tumor. The solid tumor may be selected from, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (e.g., gliomas such as glioblastoma).

[0152] The cancer antigen may be selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, claudin 6, claudin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0153] The multispecific molecule may include a CD33-binding domain comprising VH and VL. The CD33-binding domain may include VH and VL, where VH comprises the sequence of SEQ ID NO:216 and VL comprises the sequence of SEQ ID NO:217.

[0154] structural form The multispecific molecules described herein may have some different structural forms known in the art.

[0155] Additional binding domains that bind to targets other than the γδ TCR may include monovalent antibody fragments. The monovalent antibody fragments may be Fab, Fv, scFv, or V H It may also be selected from H.

[0156] The multispecific molecules described herein may further comprise immunoglobulin Fc domains. Exemplary Fc domains may be selected from the heavy chain constant regions of IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and more particularly from the heavy chain constant regions of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0157] The Fc domain may consist of a first subunit and a second subunit. The γδ TCR binding domain and the additional binding domain may be fused to the first Fc subunit and the second Fc subunit, respectively. The γδ TCR binding domain may be fused to one N-terminus of the Fc subunit, with or without a linker (e.g., at its C-terminus). The additional binding domain may be fused to another N-terminus of the Fc subunit, with or without a linker (e.g., a peptide linker), at or without its C-terminus.

[0158] The first Fc subunit and the second Fc subunit may be manipulated to be used for heterodimerization.

[0159] Heterodimerization of different polypeptides in multispecific molecules may be facilitated by methods known in this field (including, but not limited to, knob-into-hole heterodimerization). Such techniques may be developed by introducing a "knob" (or protuberance) in the CH3 domain of one Fc by replacing a small amino acid residue with a large amino acid residue, and introducing a "hole" (or cavity) in the CH3 domain of another Fc by replacing one or more large amino acid residues with small amino acid residues. One strand of the Fc fragment in the fusion protein may contain the knob, and the second strand of the Fc fragment may contain the hole.

[0160] Preferred residues for forming the knob are typically naturally occurring amino acid residues with large side-chain volumes, and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferably, tryptophan (W) and tyrosine (Y). Exemplary amino acid substitutions in the CH3 domain for forming the knob include, but are not limited to, T366W, T366Y, or F405W substitutions.

[0161] Preferred residues for forming holes are typically naturally occurring amino acid residues with small side-chain volumes, and are preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Exemplary amino acid substitutions in the CH3 domain for hole production include, but are not limited to, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions.

[0162] In some cases, the knob includes a T366W replacement, and the hole includes a T366S / L368A replacement.

[0163] It should be understood that this disclosure further considers and covers other modifications to the Fc region that promote heterodimerization known in the art.

[0164] In some cases, the multispecific molecules include an Fc domain containing a knob-into-hole structure, for example, having a T366W substitution in the first subunit of the Fc domain and T366S and L368A substitutions in the second subunit of the Fc domain.

[0165] Typically, the numbering of residues in immunoglobulin heavy chains follows the EU index numbering in Kabat.

[0166] In other cases, the Fc domain is modified, for example, by mutation, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. In some cases, the Fc domain contains one or more amino acid substitutions that reduce binding affinity to the Fc receptor and / or effector function. In some cases, the Fc domain contains one or more amino acid substitutions that improve stability (e.g., prevent Fab arm replacement). In some cases, the Fc domain is the Fc domain of a human IgG4 isotype having the S228P, L234A, and L235A amino acid substitutions (numbered based on the Kabat EU index).

[0167] Furthermore, the multifunctional molecule may also contain a half-life extender, such as human serum albumin or an antibody against human serum albumin.

[0168] The Fc subunit containing the knob may also contain the sequence with SEQ ID NO:197.

[0169] The Fc subunit containing a hole may also contain the sequence SEQ ID NO:198.

[0170] The γδ TCR binding domain is V H The binding domain may be H or scFv, and the additional binding domain may be a Fab consisting of a light chain (VL-CL) and a heavy chain (VH-CH1). The heavy chain of the Fab may be fused with one of the Fc domain subunits (e.g., its N-terminus), and VHH or scFv may be fused with another of the Fc domain subunits (e.g., its N-terminus).

[0171] As described herein, multispecific molecules are A first polypeptide chain including an additional binding domain (VL) and a light chain constant domain (CL), A second polypeptide chain comprising an additional binding domain VH, a first heavy chain constant domain 1 (CH1), and a first Fc subunit, It may also include a γδ TCR-binding domain and a third polypeptide chain containing a second Fc subunit.

[0172] Those skilled in the art will understand that CH1 typically pairs with CL, and that the first and second Fc subunits act as dimerizing domains to form a dimer. The first and second Fc subunits may also contain a knob-into-hole structure.

[0173] The Fc domain may also contain CH2 and CH3. Alternatively, the Fc domain may contain a hinge, CH2, and CH3.

[0174] The VL of the additional binding domain may fuse with the N-terminus of the CL (for example, at its C-terminus).

[0175] The VH of the additional binding domain may fuse with the N-terminus of CH1 (for example, at its C-terminus) and further with the N-terminus of the first Fc subunit. Alternatively, the VH of the additional binding domain may fuse with the N-terminus of a full-length heavy chain constant domain (CH) containing CH1, hinge, CH2, and CH3 (for example, at its C-terminus).

[0176] γδ TCR binding domain (e.g., V H H or scFv) may fuse with the N-terminus of the second Fc subunit (for example, at its C-terminus).

[0177] The multispecific molecule may further include a linker between CL and an additional binding domain VL, a linker between CH1 and an additional binding domain VH, a linker between CH1 and a first Fc subunit, a linker between the γδ TCR binding domain and a second Fc subunit, or a combination thereof. The linkers may be selected from cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. The linker may also be a peptide linker. The linker may include a (GmS)n sequence, where m is selected from an integer between 1 and 6 (e.g., 1, 2, 3, or 4) and n is selected from an integer between 1 and 6 (e.g., 1, 2, or 3).

[0178] The multispecific molecule may include a first binding arm for γδ TCR and a second binding arm for targets other than γδ TCR (e.g., cancer antigens), and comprises chains 1, 2, and 3, where, Chain 1 is the light chain of the second arm and has a VL-CL structure. Chain 2 is the heavy chain of the second arm and has a VH-CH1-hinge-CH2-CH3 structure, where VH and VL form binding sites to targets other than the γδ TCR. Chain 3 is the γδ T cell binding arm, anti-γδ TCR V H It has an H / scFv-hinge-CH2-CH3 structure.

[0179] Chain 1 may include VL of the second binding arm and CL containing the sequence of SEQ ID NO:196. Chain 2 may include VH of the second binding arm and CH containing the sequence of SEQ ID NO:197. Chain 3 may include V of the first binding arm H It may also contain H or scFv and an Fc fragment containing the sequence of SEQ ID NO:198.

[0180] In all of the embodiments described above, the multispecific molecules disclosed herein may be T cell engagers that constitutively bind to the γδ TCR on T cells and to cancer antigens on cancer cells. By producing a physical link between T cells and cancer cells, T cell engagers redirect T cells to cancer cells, activate T cells against cancer cells, and potentially produce an effective antitumor response.

[0181] Antibody production In another aspect, V disclosed herein H The present invention provides nucleic acid molecules, vectors, and host cells for producing H, heavy chain antibodies, polypeptide constructs, general antibodies, or multispecific molecules.

[0182] We provide isolated nucleic acid molecules, which are V as described herein. H Contains a nucleotide sequence that codes for H.

[0183] Further providing is an isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain antibody as described herein.

[0184] Further providing is an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide construct described herein.

[0185] Further provided are isolated nucleic acid molecules comprising a nucleotide sequence encoding a general antibody or its antigen-binding fragment as described herein, or its heavy chain variable region and / or light chain variable region. The isolated nucleic acid molecule may also comprise a first nucleotide sequence and a second nucleotide sequence encoding the heavy chain variable region / heavy chain and the light chain variable region / light chain of the antibody or its antigen-binding fragment disclosed herein, respectively.

[0186] Further provided are isolated nucleic acid molecules comprising nucleotide sequences encoding the multispecific molecules described herein. The isolated nucleic acid molecules may also comprise different nucleotide sequences encoding different polypeptide chains of the multispecific molecules, respectively.

[0187] Further, vectors (e.g., cloning vectors or expression vectors) are provided, which include isolated nucleic acid molecules as disclosed herein.

[0188] The vectors disclosed herein may be, for example, plasmids, cosmids, or phages.

[0189] The vector is V disclosed herein. H It may include a nucleotide sequence that codes for H.

[0190] The vector may contain a nucleotide sequence encoding the heavy chain antibody described herein.

[0191] The vector may include a nucleotide sequence encoding a polypeptide construct disclosed herein.

[0192] The vector may include a first nucleotide sequence and a second nucleotide sequence encoding the heavy chain variable region / heavy chain and the light chain variable region / light chain of a common antibody or its antigen-binding fragment disclosed herein, respectively. The first and second nucleotide sequences may be located in the same or different vectors.

[0193] The vector may contain different nucleotide sequences encoding different polypeptide chains of the multispecific molecule. The different nucleotide sequences may be located in the same or different vectors.

[0194] The present invention further provides host cells which include or undergo transformation using isolated nucleic acid molecules or vectors disclosed herein. Such host cells include, but are not limited to, prokaryotic cells (e.g., Escherichia coli cells) and eukaryotic cells (e.g., yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells and human cells)).

[0195] V disclosed herein H Further providing a method for producing H, heavy chain antibodies, polypeptide constructs, general antibodies or their antigen-binding fragments, or multispecific molecules, the method comprising culturing a host cell containing an isolated nucleic acid molecule or vector disclosed herein or a host cell disclosed herein under conditions that enable protein expression, and from the culture of the cultured host cell V H This includes recovering H, heavy chain antibodies, polypeptide constructs, general antibodies or their antigen-binding fragments, or multispecific molecules.

[0196] Pharmaceutical composition In another embodiment, a pharmaceutical composition is provided, the pharmaceutical composition being the V disclosed herein. H This includes H, heavy chain antibodies, polypeptide constructs, general antibodies or their antigen-binding fragments, or multispecific molecules.

[0197] The pharmaceutical composition may further contain pharmaceutically acceptable carriers and / or excipients.

[0198] V as described in this specification H The present invention provides a pharmaceutical composition containing H. The pharmaceutical composition contains an effective amount of V H It may include H. H H may be the sole active ingredient in the pharmaceutical composition.

[0199] Further information is provided herein, comprising a pharmaceutical composition containing the heavy chain antibody described herein. The pharmaceutical composition may contain an effective amount of the heavy chain antibody. The heavy chain antibody may be the sole active ingredient in the pharmaceutical composition.

[0200] Further information is provided herein, comprising a polypeptide construct described herein. The pharmaceutical composition may contain an effective amount of the polypeptide construct. The polypeptide construct may be the sole active ingredient in the pharmaceutical composition.

[0201] Further information is provided herein, comprising a common antibody or its antigen-binding fragment. The pharmaceutical composition may contain an effective amount of the common antibody or its antigen-binding fragment. The common antibody or its antigen-binding fragment may be the sole active ingredient in the pharmaceutical composition.

[0202] Further information is provided regarding pharmaceutical compositions containing the multispecific molecules described herein. The pharmaceutical composition may contain an effective amount of the multispecific molecule. The multispecific molecule may be the sole active ingredient in the pharmaceutical composition.

[0203] The pharmaceutical compositions disclosed herein may further comprise additional therapeutic agents. The additional therapeutic agents may be anticancer agents. Alternatively, the additional therapeutic agents may be immune checkpoint inhibitors, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, or anti-CTLA-4 antibodies. Alternatively, the additional therapeutic agents may be cytotoxic agents, such as alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, or radionuclides. Alternatively, the additional therapeutic agents may be cytokines (e.g., immune cell activating cytokines such as IL-2, IL-15, IL-7, IL-6, IL-12, IL-18, IFNα, IFNβ, or IFNγ).

[0204] V disclosed herein HH, heavy chain antibodies, polypeptide constructs, conventional antibodies or antigen-binding fragments thereof, or multispecific molecules and additional therapeutic agents may be provided as separate components or as multiple components in a single composition. The V disclosed herein H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or antigen-binding fragments thereof, or multispecific molecules may be used in combination with other agents simultaneously, separately or sequentially.

[0205] The pharmaceutical composition may be provided in unit dosage form (i.e., a dose for single administration).

[0206] The pharmaceutical composition may be prepared using one or more pharmaceutically acceptable carriers and / or excipients. The formulation depends on the route of administration selected. For parenteral administration, the pharmaceutical composition is preferably sterile, substantially isotonic, and manufactured under GMP conditions. For example, the V disclosed herein H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or antigen-binding fragments thereof, or multispecific molecules are prepared into aqueous solutions for injection, preferably physiologically compatible buffers such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% Polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution. The solution may contain formulation agents such as suspending aids, stabilizers and / or dispersants. Alternatively, the V disclosed herein H H, polypeptide constructs, conventional antibodies or antigen-binding fragments thereof, or multispecific molecules may be in lyophilized form, so that they can be reconstituted with a suitable medium (e.g., sterile, pyrogen-free water) before use.

[0207] The pharmaceutical composition described herein can be used to promote activation and / or proliferation of γδ T cells and / or to treat a disease (e.g., cancer).

[0208] Methods or uses for treating a disease In another embodiment, V as described herein H The present invention provides methods or uses for treating diseases using H, heavy chain antibodies, polypeptide constructs, general antibodies or their antigen-binding fragments, or multispecific molecules.

[0209] In one embodiment, a method is provided for treating a target disease, wherein the method provides an effective amount of the V described herein to a target that requires it. H This includes administering H, heavy chain antibodies, polypeptide constructs, common antibodies or their antigen-binding fragments, or multispecific molecules. Such diseases include any disease that benefits from increased T cell (e.g., γδ T cell) proliferation and activation.

[0210] As described herein, the disease may be cancer.

[0211] A method for treating cancer is provided herein, the method which targets the γδ TCR. H This includes increasing the proliferation and / or activation of γδ T cells by administering H, heavy chain antibodies, polypeptide constructs, or common antibodies or their antigen-binding fragments.

[0212] Further methods for treating cancer are provided herein, which include increasing the proliferation and / or activation of γδ T cells by administering a multispecific molecule that binds to γδ TCRs and cancer antigens, as disclosed herein. The cancer may be characterized by a cancer antigen targeted by the multispecific molecule.

[0213] The γδ TCR binding domain used by the multispecific molecule may originate from antibodies such as AS282152 (containing CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 66, 67, and 68, respectively), AS281876 (containing CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively), AS282067 (containing CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 50, 51, and 52, respectively), or AS288170 (containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with amino acid sequences of SEQ ID NO: 171, 172 or 206, 173, 175, 118, and 176, respectively), which recognize epitopes located in Vγ9 and the constant region of the γδ TCR. Since Vγ9 is part of the γδ TCR involved in non-peptide phosphate antigen recognition, blocking the interaction between the γδ TCR and its phosphate antigen can inhibit tumor cell killing. Therefore, if the tumor does not express the tumor antigen targeted by the multispecific molecule, the multispecific molecule using these antibodies as γδ T cell junction arms will have an inhibitory effect on tumor cell killing.

[0214] The γδ TCR binding domain used by the multispecific molecule may be derived from an antibody such as AS282116 (containing CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 58, 59, and 60, respectively) or AS287963 (containing CDR1, CDR2, and CDR3 with amino acid sequences of SEQ ID NO: 162, 59, and 163, respectively), which recognizes an epitope located in the constant region of the γδ TCR that is not involved in non-peptide phosphate antigen recognition. Therefore, if the tumor does not express the tumor antigen targeted by the multispecific molecule, the multispecific molecule using these antibodies as γδ T cell binding arms has a very small (in some cases) inhibitory effect on killing tumor cells.

[0215] In one embodiment, V as described herein HThis invention provides for the use of H, heavy chain antibodies, polypeptide constructs, general antibodies or their antigen-binding fragments, or multispecific molecules in the manufacture of drugs used to treat a disease of interest. Such diseases may include any disease that benefits from increased T cell (e.g., γδ T cell) proliferation and activation. As described herein, the disease may be cancer.

[0216] The cancers to which the treatment methods or uses described herein relate may include, but are not limited to, solid tumors and hematological cancers. The cancers may include hematological cancers such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma. The cancers may also include solid tumors such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (such as glioblastoma or other gliomas).

[0217] The cancers related to the treatment methods or uses described herein may be characterized by cancer antigens selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, Claudin 6, Claudin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, Mesothelin, HER2, PSMA, or PSCA.

[0218] The treatment methods or subjects of use described herein may be mammals, particularly humans.

[0219] In any one of the treatment methods or uses described herein, V disclosed herein H H, heavy chain antibodies, or polypeptide constructs can activate γδ T cells and / or increase γδ T cell proliferation, potentially producing an effective antitumor response.

[0220] In any of the therapeutic methods or uses described herein, the antibodies or antigen-binding fragments disclosed herein can activate γδ T cells and / or increase the proliferation of γδ T cells, potentially producing an effective antitumor response.

[0221] In any of the therapeutic methods or uses described herein, the multispecific molecules disclosed herein may also act as T cell engagers, which constitutively bind to γδ TCRs on T cells and cancer antigens on cancer cells. By producing a physical link between T cells and cancer cells, T cell engagers redirect T cells to cancer cells, activate T cells against cancer cells, and potentially produce an effective antitumor response.

[0222] In any one of the treatment methods or uses described herein, V disclosed herein H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions may be used alone.

[0223] Alternatively, V disclosed herein H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules or pharmaceutical compositions may be administered in combination with additional therapeutic agents or additional therapies. The additional therapeutic agents may be anticancer agents, such as immune checkpoint inhibitors, or cytotoxic agents, or cytokines. The additional therapies may be standard cancer treatments, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0224] In any one of the treatment methods or uses described herein, V disclosed herein HH, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions may be prepared in any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the desired route of administration and therapeutic use. One preferred dosage form is an injection. Such an injection may also be a sterile injectable solution. Alternatively, for ease of storage and use, the sterile injectable solution may be prepared as a sterile lyophilized powder (e.g., by vacuum drying or lyophilization).

[0225] In any one of the treatment methods or uses described herein, V disclosed herein H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, intraocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, local (e.g., powders, ointments, or infusions) or nasal administration. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous, intraperitoneal, or intramuscular). Those skilled in the art will understand that the route and / or mode of administration varies depending on the desired purpose. H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules or pharmaceutical compositions may be administered by intravenous injection or bolus.

[0226] In another embodiment, a method is provided for activating γδ T cells and / or increasing γδ T cell proliferation, wherein the method involves T cells (e.g., a T cell population) as disclosed herein. Hcontacting H, a heavy chain antibody, a polypeptide construct, an antibody or an antigen-binding fragment thereof, or a multispecific molecule, or a pharmaceutical composition. The T cells may be located in the body. The T cells may be located ex vivo.

[0227] Activation of γδ T cells may be determined by any method in the art, for example, the method according to Example 3 of the present specification, for example, by measuring CD25 and / or CD107a expression. An increase in the expression of CD25 and / or CD107a indicates activation. Examples

[0228] Unless otherwise specified, the following examples are for illustrative purposes only and are not intended to be limiting. Therefore, the present disclosure should not be construed as being limited to the following examples, but should be construed as covering any and all variations that become apparent from the teachings herein.

[0229] Example 1 Production and Characterization of Anti-γδ TCR Antibodies Internally, soluble γδ TCR protein was produced and used for animal immunization. To promote TCR heterodimerization, a pair of charge-complementary leucine zipper (LZ) sequences were linked to the C-termini of TCRγ and TCRδ chains via a flexible (G4S)3 linker. For the purpose of purification and detection, a 6xHis tag was added to the γ chain, and a flag tag was added to the C-terminus of the δ chain. TCRγ chain and TCRδ chain plasmids were prepared and used for the production of soluble γδ TCR. Soluble TCR (sTCR) was produced by FreeStyle 293-F cells and purified by anti-DYKDDDDK G1 affinity resin (Genscript, Catalog No. L00432). The constructs of human and cynomolgus monkey γ9δ2 TCR proteins are described in Figure 1.

[0230] According to all current animal welfare regulations, one camel was immunized with γ9δ2 TCR protein. The γ9δ2 TCR protein was prepared as an emulsion containing either a complete Freund's adjuvant (CFA) (for primary immunization) or an incomplete Freund's adjuvant (IFA) (for booster immunization). The antigen emulsion was administered by intramuscular two-point injection into the neck. The animal received three injections of 100-200 μg of human γ9δ2 TCR protein at two-week intervals, followed by two injections of 100 μg of cynomolgus monkey γ9δ2 TCR protein at weekly intervals. The animal was administered 50 μg of human γ9δ2 TCR protein as a final booster immunization. Five days later, a 150 mL blood sample was collected, and approximately 3 × 10¹⁶ ions were extracted from the blood. 8 Individual peripheral blood lymphocytes (PBLs) were isolated and used as the genetic source for general and heavy chain immunoglobulins.

[0231] All RNA was extracted from lymphocytes of immunized camels using TRIZOL® reagent (Invitrogen®, catalog number 15596026). cDNA was synthesized based on the RNA template using the PRIMESCRIPT® 1st Strand cDNA synthesis kit and oligo(dT)20 primer (Takara, catalog number 6110A). From camel cDNA, V H DNA encoding H (variable region of heavy-chain-only antibodies, also called single-domain antibodies or sdAb), VH, and VL was amplified, purified, and ligated into an internal phagemide vector (see Figure 1 of patent US20170089914A1). The ligated product was used to transform SS320 electrocompetent cells (Lucigen, catalog number 60512-1). The resulting sdAb and scFv libraries were supplemented with 20% glycerol and stored at -80°C.

[0232] The sdAb and scFv phage libraries were simultaneously rescued and stored at 4°C after sterilization by filtration for subsequent use. Binding proteins were isolated from the phage libraries using protein-based and cell-based panning. One round of panning was performed using both protein-based and cell-based panning methods. The proportion of γ9δ2 TCR-positive clones identified by ELISA reached at least 50%, and the sequence diversity of γ9δ2 TCR-specific clones was very high for all output phages. These outputs were used for subsequent high-throughput screening.

[0233] Selected output phages were used to infect exponentially growing E. coli cells. The output double-stranded DNA was extracted. The sdAb / scFv insertion fragment was excised from the phagemide vector and inserted into an antibody fragment expression vector for high-throughput screening. The resulting plasmid was used to transform exponentially growing E. coli cells, which were then inoculated and grown overnight at 37°C. Several thousand colonies were individually picked and grown in 96-deep-well plates containing 1 mL of 2YT medium. Antibody fragment expression was induced by adding 1.0 mM IPTG. The binding ability of the sdAb / scFv protein to the human γ9δ2 TCR protein in the supernatant was analyzed by ELISA, and the binding ability of these proteins to human γ9δ2 T cells was analyzed by flow cytometry. A total of 33 V phages with unique sequences were identified. H H and nine scFv-binding proteins were selected and used for further characterization. Full-length V H The IDs for the H / VH, VL, and CDR sequences are listed in Table 1.

[0234] [Table 1]

[0235] To evaluate the interspecies reactivity and specificity of the selected clones, ELISA was performed on internally produced cynomolgus monkey γ9δ2 TCR and human αβ TCR proteins (SEQ ID NO: 5-6) (Table 2). To classify binding proteins based on the recognized γδ TCR domain / region, five artificial TCR proteins were designed by replacing the γ9δ2 TCR domain with a control sequence derived from αβ TCR or another γδ TCR, and these proteins were produced internally (Figure 2). ELISA was performed using all recombinant TCR proteins as antigens to discover antigen domains that are crucial for antigen-antibody interactions. For example, the fact that AS281795 binds to human γ9δ2 TCR but not to human γ9δ1 TCR indicates that the epitope of AS281795 is located in the Vδ2 domain. Based on these binding epitopes, the selected binding proteins were classified into eight groups (Table 2). The results show that most binding proteins recognize human and cynomolgus monkey γ9δ2 TCRs, and the epitopes of these antibodies exhibit extremely high diversity.

[0236] [Table 2] TIFF2026529710000003.tif199160

[0237] Example 2: Determination of the affinity of heavy chain antibodies Camelidae V H Heavy chain-only antibodies (HCAb) or general IgG heavy and light chains were constructed using H / VH and VL sequences. Heavy and light chain plasmids were prepared and used to produce HCAb and IgG from HEK293 cells. Antibodies were purified using a Protein A Agarose column (Genscript, catalog number L00464), followed by size exclusion chromatography (Citiva, catalog number GE28-9893-35). Anti-γ9δ2 TCR HCAb, 6H1 (from patent US20190263908A1, SEQ ID NO:49) was prepared as a positive control.

[0238] The binding affinity between selected molecules and human and cynomolgus monkey γ9δ2 TCR proteins was determined by surface plasmon resonance (SPR) using a BIAcore T200 instrument (GE Healthcare). The experiment was performed as follows: Antibodies were captured on a sensor chip pre-coated with goat anti-human pAb (Jackson ImmunoResearch, catalog no. 109-005-098) by interaction between the polyclonal antibody and human Fc. Human or cynomolgus monkey γ9δ2 TCR at increasing concentrations (ranging from 40 nM to 2.56 μM) was injected onto the sensor chip surface to enable binding to the captured antibody, lasting 100 s, followed by injection of running buffer to enable dissociation of the complex, lasting 300 s. After each cycle, the surface was regenerated by injection of 10 mM glycine-HCl buffer (pH 2.0). The Langmuir model (which describes a simple 1:1 interaction between one ligand molecule and one analyte) was used to fit the experimental data and determine the association rate (k a ) and dissociation rate (k d The dynamical values ​​of ) are obtained, and these dynamical values ​​are used to obtain the equilibrium dissociation constant (K D The affinity (K) was calculated. According to the results summarized in Table 3, the antibodies had a wide range of affinity (K) of approximately 200 nM to 1 nM (with human γ9δ2 TCR) and approximately 3 μM to 5 nM (with cynomolgus monkey γ9δ2 TCR). D It bound to the γ9δ2 TCR.

[0239] [Table 3]

[0240] Example 3: Activation of γ9δ2 T cells induced by HCAb A flat-bottomed 96-well cell culture plate was coated overnight with 100 μL of 2 μg / mL goat anti-human IgG Fc (Thermo Scientific, catalog number 31125) at 4°C. The wells were washed with PBS and blocked with 200 μL of 4% BSA / PBS at room temperature for 30 minutes. The blocking solution was discarded, and the wells were incubated with 100 μL of HCAb (0.5 nM, 5 nM, and 50 nM) in PBS at 37°C for 2 hours. As described above (Makoto Kondo et al., September 9, 2011, (55):3182), human peripheral blood mononuclear cells (PBMCs) were used for the proliferation of γ9δ2 T cells. The obtained γ9δ2 T cells were resuspended using AIM-V medium (Gibco, catalog number A3830801) and added to the 96-well cell culture plate (1 × 10⁶ 5 γ9δ2 T cells (1 / 200 μL AIM-V / well) were incubated at 37°C. Flow cytometry was then used to evaluate γδ T cell degranulation after 4 hours of incubation and γδ T cell activation after 24 hours of incubation. As shown in Table 4, all HCAb tested showed the ability to drive γ9δ2 T cell activation and degranulation equivalent to or better than 6H1 HCAb, which was reflected by increased CD25 and CD107a expression, respectively.

[0241] [Table 4]

[0242] Example 4: Killing of AML-193 cells by TCE-KIH redirection To test the functional activity of anti-γδ TCR antibodies, 29 anti-γδ TCR V HH and six anti-γδ TCR scFv were incorporated into a CD33-targeting TCE (T cell binding)-KIH (knob-into-hole) bispecific antibody (BsAb) molecule (Figure 3). The BsAb contains three chains: one light chain and two heavy chains. Precise pairing of the heavy chains was achieved using the knob-into-hole technique. Chain 1 is the light chain of the CD33 arm and has a VL-CL structure. Chain 2 is the heavy chain of the CD33 arm and has a VH-CH1-hinge-CH2-CH3 structure. Chain 3 is the γδ T cell binding arm and contains anti-γδ TCR scFv. H It has an H / scFv-hinge-CH2-CH3 structure. The heavy chain constant regions of chains 2 and 3 contain mutations that eliminate arm exchange, reduce effector function, and support heavy chain heterodimerization. These BsAbs were produced by FreeStyle 293-F cells and purified using CH1-XL Affinity Matrix (Thermoscientific, catalog no. 1094462010). As a positive control, 5E7-KIH BsAb was further produced using 5E7 (from patent US20190263908A1, SEQ ID NO: 60).

[0243] Flow cytometry was used to evaluate the cytotoxicity of T cell redirection by using human γ9δ2 T cells as effector cells and the CD33+ human AML cell line AML-193 (ATCC, catalog number CRL-9589(trademark)) as target cells. In short, AML-193 cells were labeled with CellTrace(trademark) Violet (Thermo Fisher Scientific, catalog number C34557) and inoculated with γ9δ2 T cells in a 1:1 or 2:1 effector-to-target cell ratio (E:T) in a round-bottom 96-well plate (5 × 10⁻¹⁴). 4(100 tumor cells / well). A series of 5x dilutions of BsAb were added to the wells and incubated at 37°C for 48 hours. Cell lysis was assessed by loss of target cell membrane integrity by flow cytometry, which was reflected by nuclear uptake of propidium iodide (Sigma, catalog no. P4170-10MG). Samples were analyzed by flow cytometry using a BD FACSCelesta® flow cytometer, and data were processed using BD FACS Diva software version 8.0.1.1. Specific lysis was calculated using the following formula to quantify BsAb-induced killing. TIFF2026529710000006.tif10161

[0244] Killing was measured in two experiments, where the E:T ratio was 2:1 in the first experiment and 1:1 in the second. As shown in Table 5, there was clear baseline killing of AML-193 cells by γ9δ2 T cells, with approximately 45% of AML-193 cells being killed. The EC50 value of the positive control BsAb 5E7-KIH differed between the two experiments, which may be due to the difference in E:T ratios. Of the 35 BsAbs, most showed equivalent or better killing efficacy and effectiveness than 5E7-KIH, with the exception of AS281940-KIH, AS281949-KIH, AS281991-KIH, and AS287895-KIH, which had low EC50 values ​​(Figure 4 and Table 5).

[0245] [Table 5]

[0246] Example 5: Killing of various cancer cell lines by TCE-KIH redirection Five cancer cell lines with different CD33 expression levels were used as target cells, and cytotoxicity measurements were performed on the six BsAbs (i.e., AS281876-KIH, AS282067-KIH, AS282116-KIH, AS282152-KIH, AS287963-KIH, and AS288170-KIH) that showed the highest AML-193 killing effect and efficacy. Of these target cell lines, AML-193 and MOLM-13 (DSMZ, catalog number ACC 554) were high-expression AML lines, KG-1A (ATCC, catalog number CCL-246.1) and NALM6 (ATCC, catalog number CRL-3273) were low-expression cell lines, and CCRF-CEM (ATCC, catalog number CCL-119) was a CD33-acute lymphoblastic leukemia cell line. Killing measurements were performed as described in Example 4. In addition to the positive control 5E7-KIH, TCE-KIH BsAb controls were produced using three more anti-Vγ9Vδ2 sdAbs (i.e., 5C8, 6H1, 6H4) (from patent US20190263908A1, SEQ ID NO: 59 and 49, and patent US20220098301A1, SEQ ID NO: 54) and one anti-Vγ9Vδ2 scFv 7A5 (from patent WO2020227457A1, VH SEQ ID NO: 65, VL SEQ ID NO: 66), as described in Example 4.

[0247] Of all six BsAbs, AS281876-KIH and AS282152-KIH performed better than all five positive control BsAbs and showed the most favorable tumor cell-killing activity against all CD33+ cancer cell lines, as reflected, for example, high maximum killing capacity and the lowest EC50 values ​​(Figures 5a, 6a, 7a, and 8a, Table 6). AS288170-KIH and AS282067-KIH performed as well as or slightly better than all five positive control BsAbs (Figures 5b, 6b, 7b, and 8b, Table 6). AS282116-KIH and AS287963-KIH were comparable to the positive control BsAbs 5C8-KIH, 5E7-KIH, 6H1-KIH, and 6H4-KIH in killing CD33-high-expression cell lines, such as AML-193 and MOLM-13. However, they showed lower efficacy than 5C8-KIH, 5E7-KIH, 6H1-KIH, and 6H4-KIH in killing CD33-low-expression cell lines, such as KG-1A and NALM6, which was reflected, for example, by higher EC50 values. Nevertheless, AS282116-KIH and AS287963-KIH showed the highest efficacy in killing KG-1A and NALM6, which was reflected, for example, by the highest maximum killing of all 11 BsAbs (Figures 5c, 6c, 7c, and 8c, Table 6).

[0248] Because CCRF-CEM does not express the CD33 molecule on its surface, BsAb-dependent cancer cell killing was not observed (Figure 9). Only cancer cell killing was observed, which depended on the recognition of non-peptide phosphate antigens by the γ9δ2 TCR. However, for the majority of BsAbs, tumor cell killing was inhibited to a certain extent by high concentrations of BsAb, which may be due to these antibodies binding to the γδ TCR and blocking the recognition of non-peptide phosphate antigens. In particular, AS282152-KIH showed the highest killing inhibition. 5C8-KIH, 5E7-KIH, 6H1-KIH, 6H4-KIH, AS281876-KIH, AS288170-KIH, and AS282067-KIH showed similar killing inhibition. 7A5-KIH showed significantly lower killing inhibition, which may be due to the low affinity binding of 7A5 to the γ9δ2 TCR (although data are not shown, there is also submicron molar affinity to human and cynomolgus monkey γ9δ2 TCR). Only two antibodies, AS282116-KIH and AS287963-KIH, showed very little or no inhibition against cancer cell killing. The differences in inhibitory activity of these antibodies may be interpreted by differences in the epitopes they recognize. The epitopes of AS282152, AS281876, AS288170, and AS282067 are located in the Vγ9 and constant region of the γδ TCR. Since Vγ9 is part of the γδ TCR involved in non-peptide phosphate antigen recognition, they inhibited tumor cell killing by blocking the interaction between the γδ TCR and its homologous antigen. In another embodiment, AS282116 and AS287963 bind to the constant domain of the γδ TCR, which is not involved in antigen recognition. Therefore, BsAbs using these antibodies as γδ T cell junctioning arms have very little (in some cases) inhibitory effect on tumor cell killing.

[0249] [Table 6]

[0250] Example 6 Humanization of anti-γδ TCR antibody fragment Six types of camel antibodies (five sdAb and one scFv) were humanized using the CDR transplantation method (Winter G, Harris WJ 1993). H The H / VH and VL sequences were BLASTed against the NCBI Human Germline V Gene Databank, and therefore the human VH and VL germline sequences with the highest identity to the camel antibody (i.e., the human receptor) were identified. In the CDR transplantation method, the CDR of the human receptor was replaced with the CDR of the camel antibody to produce a straight-graft sequence. Straight-graft antibodies typically lose their binding activity, and this binding activity needs to be restored by replacing framework residues important for antibody activity with camel residues.

[0251] Camels and humanized V H Chain 3 of TCE-KIH was constructed using H / VH and VL sequences and expressed using the method described in Example 4. After humanization, all antibodies had at least one humanized variant that retained a corresponding binding affinity (Table 7). The sequence IDs of the humanized antibodies are listed in Table 8.

[0252] [Table 7]

[0253] [Table 8]

[0254] Example 7: Production of polyclonal γδ T cells by anti-γδ TCR antibody and its in vitro effect. Four antibodies, including AS281850, AS287435, AS288180, and AS287963, which specifically bind to the γδTCR, were tested to activate and proliferate polyclonal γδT cells from PBMCs. Cell culture plates were coated with these antibodies, and CAR-polyclonal γδT cells were produced in the media shown in Table 9. Both medium I and medium II contained basal media and were supplemented with human serum substitutes for different cytokines (e.g., human platelet lysates) and the Akt inhibitor MK-2206. Cell performance was then evaluated.

[0255] [Table 9]

[0256] In short, polyclonal γδ T cells were produced from PBMCs according to the following procedure: PBMCs were activated in medium I using the aforementioned antibody and kept active for 3–9 days. The activated cells were then transformed with a lentivirus or retrovirus expressing anti-BCMA CAR (SEQ ID NO: 62, WO2023020558A1) at an appropriate MOI. After 1–5 days of transformation, the cells were cultured in medium II for a further 6–12 days, and then the CAR-polyclonal γδ T cells were harvested. Cell number and viability were analyzed using a cellometer (Nexcelom, K2). For phenotypic and CAR positivity detection, the collected cells were stained with fluorescently labeled antibodies (CD3-BV785 [BioLegend, catalog number 344842], TCRVδ1-APC [Invitrogen, catalog number 344842], TCRVδ2-BV421 [Biolegend, catalog number 331428], and Alexa Fluor 488-labeled anti-mouse sdAb antibody [GenScript]) and analyzed by flow cytometry.

[0257] To further evaluate the correlation between different stimulants and their killing effects, proliferating CAR-γδ T cells were assessed in a repeated tumor challenge assay. In short, 2 × 10⁻⁶ 5 2 × 10¹ CAR+ γδ T cells5 NCI-H929 cells (ATCC, catalog number CRL-3580) were co-cultured with CAR+ γδ T cells in 24-well plates. After two days, the cells were harvested and the relative ratio of live T cells to tumor cells was determined. CAR+ γδ T cells were counted and re-inoculated with fresh NCI-H929 cells in a 1:1 ratio for use in the next round of tumor cell challenge.

[0258] In all four antibody types, γδ T cell proliferation was good, increasing at least 900-fold, and cell viability was >90% (Figures 10a to 10b). The purity of γδ T cells produced using all four antibody types was high, >98% (Figure 10c), and the composition of different γδ T cell subtypes was similar, with the highest percentage of Vδ2 T cells (approximately 65%) and Vδ1 T cells. - Vδ2 - The percentage of T cells was the lowest (approximately 10%) (Figure 10d). The percentage of CAR-positive cells was also similar across the four groups (Figure 10e). As shown in Figure 11a, polyclonal γδ T cells activated with different antibodies mediated the effective elimination of H929 cells up to the 7th round, and over the long-term killing period, the total γδ T proliferation multipliers and CAR γδ T proliferation multipliers were similar across all groups (Figures 11b to 11c).

[0259] array TIFF2026529710000012.tif242160TIFF2026529710000013.tif246160TIFF202 6529710000014.tif246160TIFF2026529710000015.tif247160TIFF20265297100 00016.tif246160TIFF2026529710000017.tif247160TIFF2026529710000018.t if247160TIFF2026529710000019.tif247160TIFF2026529710000020.tif176160

Claims

1. V that binds to γδ TCR Ｈ CDR1 contains the amino acid sequence of SEQ ID NO: 15, 19, 23, 27, 31, 35, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 85, 89, 93, 121, 125, 140, 150, 154, 158, 162, 178, 182 or 186, and CDR2 contains the amino acid sequence of SEQ ID NO: 16, 20, 24, 28, 32, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 82, 86, 90, 94, 122, 126, 137, 141, 151, 155, 159, 179, 183 or 187, and SEQ ID NO: CDR3 containing amino acid sequences of 17, 21, 25, 29, 33, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 83, 87, 91, 95, 123, 127, 138, 142, 152, 156, 160, 163, 180, 184 or 188, V Ｈ H.

2. (1) SEQ ID NO: 15, 16 and 17, or (2) SEQ ID NO: 19, 20 and 21, or (3) SEQ ID NO: 23, 24 and 25, or (4) SEQ ID NO: 27, 28 and 29, or (5) SEQ ID NO: 31, 32 and 33, or (6) SEQ ID NO: 35, 24 and 36, or (7) SEQ ID NO: 38, 39 and 40, or (8) SEQ ID NO: 42, 43 and 44, or (9) SEQ ID NO: 46, 47 and 48, or (10) SEQ ID NO: 50, 51 and 52, or (11) SEQ ID NO: 54, 55 and 56, or (12) SEQ ID NO: 58, 59 and 60, or (13) SEQ ID NO: 62, 63 and 64, or (14) SEQ ID NO: 66, 67 and 68, or (15) SEQ ID NO: 70, 71 and 72, or (16) SEQ ID NO: 74, 75 and 76, or (17) SEQ ID NO: 78, 79 and 80, or (18) SEQ ID NO: 46, 82 and 83, or (19) SEQ ID NO: 85, 86 and 87, or (20) SEQ ID NO: 89, 90 and 91, or (21) SEQ ID NO: 93, 94 and 95, or (22) SEQ ID NO: 121, 122 and 123, or (23) SEQ ID NO: 125, 126 and 127, or (24) SEQ ID NO: 54, 137 and 138, or (25) SEQ ID NO: 140, 141 and 142, or (26) SEQ ID NO: 150, 151 and 152, or (27) SEQ ID NO: 154, 155 and 156, or (28) SEQ ID NO: 158, 159 and 160, or (29) SEQ ID NO: 162, 59 and 163, or (30) SEQ ID NO: 178, 179 and 180, or (31) SEQ ID NO: 182, 183 and 184, or (32) SEQ ID NO: 186, 187 and 188 CDR1, CDR2, and CDR3 each contain the following amino acid sequences. V according to claim 1, including Ｈ H.

3. V according to claim 1 or 2, comprising an amino acid sequence of SEQ ID NO: 14, 18, 22, 26, 30, 34, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 84, 88, 92, 120, 124, 136, 139, 149, 153, 157, 161, 177, 181 or 185, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Ｈ H.

4. V according to any one of claims 1 to 3, which is humanized. Ｈ H.

5. V according to claim 4, comprising an amino acid sequence of SEQ ID NO: 199, 200, 201, 202, 203 or 204, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Ｈ H.

6. V according to any one of claims 1 to 5 Ｈ A polypeptide construct that contains H and an immunoglobulin Fc region and binds to the γδ TCR.

7. The polypeptide construct according to claim 6, wherein the immunoglobulin Fc region is an IgG Fc region, for example, an IgG1, IgG2, IgG3, or IgG4 Fc region.

8. An antibody or antigen-binding fragment thereof that binds to γδ TCR, a) i) HCDR1 containing the sequence SEQ ID NO: 97, 105, 113, 129, 144, 165, 171, 190 or 209, ii) HCDR2 containing the sequence SEQ ID NO: 98, 106, 114, 130, 145, 166, 172, 206, 191 or 210, iii) HCDR3 containing the sequence SEQ ID NO: 99, 107, 115, 131, 146, 167, 173, 192 or 211 Heavy chain variable region (VH) including, and / or, b) i) LCDR1 containing the sequence SEQ ID NO: 101, 109, 117, 133, 175, 194 or 213, ii) LCDR2 containing the sequence SEQ ID NO: 102, 110, 118, 134 or 214, iii) LCDR3 containing the sequence SEQ ID NO: 103, 111, 119, 135, 148, 169, 176, 195 or 215 Light chain variable region (VL) including An antibody or its antigen-binding fragment, including the above.

9. (1) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 97, 98, and 99, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 101, 102, and 103, respectively. (2) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 105, 106, and 107, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 111, respectively. (3) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 113, 114, and 115, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 117, 118, and 119, respectively. (4) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 129, 130, and 131, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively. (5) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 144, 145, and 146, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 148, respectively. (6) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 165, 166, and 167, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 109, 110, and 169, respectively. (7) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 171, 172, and 173, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. (8) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 171, 206, and 173, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. (9) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 190, 191, and 192, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 194, 110, and 195, respectively, (10) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 209, 210, and 211, respectively, and / or LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 213, 214, and 215, respectively. The antibody or antigen-binding fragment thereof according to claim 8, comprising:

10. VH containing an amino acid sequence of 96, 104, 112, 128, 143, 164, 170, 189, 205 or 208, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and / or VL containing an amino acid sequence of 100, 108, 116, 132, 147, 168, 174, 193, 207, or 212, or an amino acid sequence having at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto The antibody or antigen-binding fragment thereof according to claim 8 or 9, comprising:

11. (1) VH containing the amino acid sequence of SEQ ID NO: 96 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 100 or an amino acid sequence having at least 80% sequence identity thereto, (2) VH containing the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 108 or an amino acid sequence having at least 80% sequence identity thereto, (3) VH containing the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 116 or an amino acid sequence having at least 80% sequence identity thereto, (4) VH containing the amino acid sequence of SEQ ID NO: 128 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 132 or an amino acid sequence having at least 80% sequence identity thereto, (5) VH containing the amino acid sequence of SEQ ID NO: 143 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 147 or an amino acid sequence having at least 80% sequence identity thereto, (6) VH containing the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 168 or an amino acid sequence having at least 80% sequence identity thereto, (7) VH containing the amino acid sequence of SEQ ID NO: 170 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 174 or an amino acid sequence having at least 80% sequence identity thereto, (8) VH containing the amino acid sequence of SEQ ID NO: 189 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 193 or an amino acid sequence having at least 80% sequence identity thereto, (9) VH containing the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 212 or an amino acid sequence having at least 80% sequence identity thereto, (10) VH containing the sequence of SEQ ID NO: 205 or an amino acid sequence having at least 80% sequence identity thereto, and / or VL containing the sequence of SEQ ID NO: 207 or an amino acid sequence having at least 80% sequence identity thereto. An antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, comprising:

12. The antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) containing an amino acid sequence derived from the human immunoglobulin heavy chain constant region, Preferably, the heavy chain steady region is an IgG heavy chain steady region such as the IgG1, IgG2, IgG3, or IgG4 heavy chain steady region. Preferably, the antibody or its antigen-binding fragment further comprises a light chain constant region (CL) containing an amino acid sequence derived from a human immunoglobulin light chain constant region, such as a κ light chain constant region. An antibody or antigen-binding fragment thereof according to any one of claims 8 to 11.

13. scFv, Fab, Fab', (Fab') ２ An antibody or antigen-binding fragment thereof according to any one of claims 8 to 12, selected from Fv fragment, diabody, bispecific antibody, multispecific antibody, chimeric antibody, or humanized antibody.

14. The V according to any one of claims 1 to 5 Ｈ A multispecific molecule comprising a γδ TCR binding domain comprising H and an additional binding domain that binds to a target other than γδ TCR, and the multispecific molecule is preferably a bispecific antibody.

15. A multispecific molecule comprising a γδ TCR binding domain including the VH and VL regions of an antibody or antigen-binding fragment according to any one of claims 8 to 13, and an additional binding domain that binds to a target other than γδ TCR, preferably a bispecific antibody.

16. The multispecific molecule according to claim 15, wherein the γδ TCR binding domain is an scFv, for example, an scFv having the structure of VL-linker-VH or VH-linker-VL.

17. The multispecific molecule according to any one of claims 14 to 16, wherein the target other than the γδ TCR is a cancer antigen, for example, a tumor-specific antigen or a tumor-associated antigen.

18. The multispecific molecule according to claim 17, wherein the cancer antigen is an antigen of blood cancer, for example, an antigen of acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

19. The multispecific molecule according to claim 17, wherein the cancer antigen is an antigen of a solid tumor, for example, an antigen of ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, or brain tumors (for example, gliomas such as glioblastoma).

20. The multispecific molecule according to any one of claims 17 to 19, wherein the cancer antigen is selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, claudin 6, claudin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

21. The aforementioned additional binding domain is a monovalent antibody fragment, such as Fab, Fv, scFv, or V Ｈ A multispecific molecule according to any one of claims 14 to 20, wherein H.

22. The multispecific molecule according to any one of claims 14 to 21, wherein the multispecific molecule further comprises an immunoglobulin Fc domain composed of a first subunit and a second subunit, and preferably the Fc domain is an IgG Fc domain, for example, an IgG1 Fc domain or an IgG4 Fc domain.

23. The multispecific molecule according to claim 22, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding affinity to the Fc receptor and / or effector function, improve stability (e.g., prevent Fab arm exchange), and / or promote heterodimerization (e.g., form a knob into a hole).

24. The aforementioned Fc domain, For example, a knob-into-hole structure having substitution T366W in the first subunit of the Fc domain and substitution T366S and L368A (numbered based on the Kabat EU index) in the second subunit of the Fc domain. A multispecific molecule according to claim 23, comprising:

25. The multispecific molecule according to claim 23 or 24, wherein the Fc domain is a human IgG4 isotype Fc domain having amino acid substitutions S228P, L234A, and L235A (numbered based on the Kabat EU index).

26. The γδ TCR binding domain is V Ｈ H or scFv, wherein the additional binding domain is Fab, which consists of a light chain (VL-CL) and a heavy chain (VH-CH1), and the heavy chain of Fab is fused with one of the Fc domain subunits, and the V Ｈ The multispecific molecule according to any one of claims 22 to 25, wherein H or scFv is fused with the other of the Fc domain subunits.

27. The aforementioned multispecific molecule A first polypeptide chain comprising the aforementioned additional binding domain VL and light chain constant domain (CL), A second polypeptide chain comprising the VH of the additional binding domain, a first heavy chain constant domain 1 (CH1), and a first Fc subunit, A third polypeptide chain comprising the γδ TCR binding domain and a second Fc subunit Includes, Preferably, the first Fc subunit and the second Fc subunit include a knob-into-hole structure. The multispecific molecule according to claim 26.

28. The multispecific molecule according to claim 27, wherein the amino acid sequences of the first CH1 and the first Fc subunit are shown in SEQ ID NO: 197, and the amino acid sequence of the second Fc subunit is shown in SEQ ID NO:

198.

29. The multispecific molecule according to claim 27 or 28, wherein the amino acid sequence of CL is shown in SEQ ID NO:

196.

30. The multispecific molecule according to any one of claims 14 to 29, wherein the additional binding domain is a Fab that binds to CD33.

31. The multispecific molecule according to claim 30, wherein Fab comprises VH and VL, where VH comprises the sequence of SEQ ID NO: 216 or an amino acid sequence having at least 80% sequence identity thereto, and VL comprises the sequence of SEQ ID NO: 217 or an amino acid sequence having at least 80% sequence identity thereto.

32. V according to any one of claims 1 to 5 Ｈ H, an isolated nucleic acid molecule comprising a polypeptide construct according to claim 6 or 7, an antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, or a nucleotide sequence encoding a multispecific molecule according to any one of claims 14 to 31.

33. A vector comprising the nucleic acid molecule described in claim 32.

34. A cell comprising the nucleic acid molecule described in claim 32 or the vector described in claim 33.

35. V according to any one of claims 1 to 5 Ｈ H, a method for producing a polypeptide construct according to claim 6 or 7, an antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, or a multispecific molecule according to any one of claims 14 to 31, Culturing a host cell containing the isolated nucleic acid molecule described in claim 32 or the vector described in claim 33, or the cell described in claim 34, under conditions that enable protein expression, From the cultured cells, the V Ｈ H, recovering the polypeptide construct, the antibody or its antigen-binding fragment, or the multispecific molecule. Methods that include...

36. V according to any one of claims 1 to 5 Ｈ H, a polypeptide construct according to claim 6 or 7, an antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, or a multispecific molecule according to any one of claims 14 to 31, pharmaceutically acceptable carriers and / or excipients A pharmaceutical composition containing the following:

37. The pharmaceutical composition according to claim 36, further comprising an additional therapeutic agent.

38. The pharmaceutical composition according to claim 37, wherein the additional therapeutic agent is an anticancer agent, for example, an immune checkpoint inhibitor (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, or anti-CTLA-4 antibody), or a cytotoxic agent (e.g., alkylating agent, anti-mitotic agent, antitumor antibiotic, antimetabolites, topoisomerase inhibitor, tyrosine kinase inhibitor, or radionuclide), or a cytokine (e.g., immune cell activating cytokines such as IL-2, IL-15, IL-7, IL-6, IL-12, IL-18, IFNα, IFNβ, or IFNγ).

39. A method for treating a disease (e.g., cancer) in a subject, wherein an effective amount of the method described in any one of claims 1 to 5 is given to the subject in need. Ｈ A method comprising administering H, a polypeptide construct according to claim 6 or 7, an antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, a multispecific molecule according to any one of claims 14 to 31, or a pharmaceutical composition according to any one of claims 36 to 38.

40. The method according to claim 39, wherein the cancer is a blood cancer, for example, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

41. The method according to claim 39, wherein the cancer is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumor, mesothelioma, melanoma, sarcoma, or brain tumor (for example, a glioma such as glioblastoma).

42. The method according to claim 39, comprising administering the multispecific molecule to treat a cancer characterized by a cancer antigen targeted by the multispecific molecule.

43. The method according to any one of claims 39 to 42, wherein the cancer is characterized by a cancer antigen selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, Claudin 6, Claudin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, Mesothelin, HER2, PSMA, or PSCA.

44. The method according to any one of claims 39 to 43, wherein the subject is a mammal such as a human.

45. The aforementioned V Ｈ The method according to any one of claims 39 to 44, wherein H, the polypeptide construct, the antibody or its antigen-binding fragment, or the multispecific molecule, or the pharmaceutical composition is to be administered in combination with an additional therapeutic agent or additional therapy.

46. The aforementioned additional therapeutic agent or additional therapy is an anticancer drug or anticancer therapy. Preferably, the additional therapeutic agent is an immune checkpoint inhibitor, a cytotoxic agent, or a cytokine. Preferably, the additional therapy is a standard cancer treatment such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care. The method according to claim 45.

47. A method for activating γδ T cells, increasing γδ T cell proliferation, or producing polyclonal γδ T cells, wherein the T cells are V according to any one of claims 1 to 5. Ｈ A method comprising contacting H with a polypeptide construct according to claim 6 or 7, an antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, a multispecific molecule according to any one of claims 14 to 31, or a pharmaceutical composition according to any one of claims 36 to 38.

48. (i) A medicine for treating a disease (e.g., cancer) in the subject, (ii) Polyclonal γδ T cells In the manufacture of the V according to any one of claims 1 to 5 Ｈ H, use of a polypeptide construct according to claim 6 or 7, an antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, a multispecific molecule according to any one of claims 14 to 31, or a pharmaceutical composition according to any one of claims 36 to 38.

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