Anti-gamma delta TCR antibodies and uses thereof

Abstract

The present application provides molecules comprising antibodies that bind to T cell receptors (TCRs). In particular, the present application provides antibodies that bind to human [gamma] [delta] TCR wherein these human [gamma] [delta] T cells can be activated and modulated. The present application also provides bispecific tumor-targeting immunomodulators that bind to both tumor-associated antigens and gamma delta T cell receptors and enhance cell-mediated immune responses in the treatment of cancer. Also provided are methods of making these antibodies and methods of using these antibodies to kill cancer cells.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to international application No. PCT / CN2023 / 116291, filed on August 31, 2023, and international application No. PCT / CN2023 / 123953, filed on October 11, 2023, the full text of which is incorporated herein by reference.

[0003] sequence declaration

[0004] The following content, submitted as an XML file, is incorporated herein by reference in its entirety: a sequence list in computer-readable form (CRF) (filename: IEC240451PCT_SEQUENCE LISTING.xml, record date: August 26, 2024, size: 239,106 bytes). Technical Field

[0005] This disclosure relates to molecules including antibodies that bind to T-cell receptors (TCRs). In particular, this disclosure relates to antibodies that bind to human γδ TCRs, wherein human γδ T cells can be activated and modulated. This disclosure also relates to bispecific tumor-targeting immunomodulators that 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 using these antibodies to kill cancer cells are also provided. Background Technology

[0006] Human γδ T cells are a unique subset of immune cells, comprising 0.5%–5% of lymphocytes in peripheral blood. Unlike αβ T cells, γδ T cells express T cell receptors (TCRs) composed of γ and δ chains and recognize MHC-independent antigens. Generally, based on the TCR δ chain, human γδ T cells can be divided into four major groups: Vδ1, Vδ2, Vδ3, and Vδ5 γδ T cells. Vδ1 can be co-expressed with various Vγ chains (Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, and Vγ10) to form distinct Vδ1 γδ T cell subsets, which are primarily distributed in the skin, small intestine, and other mucosal tissues. Vδ1 γδ T cells have also been found in small numbers in the liver and spleen. Vδ2 is almost exclusively co-expressed with Vγ9 to form Vγ9Vδ2 T cells, which are the main γδ T cells in circulation. Vδ3 γδ T cells are primarily 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).

[0007] γδ T cells recognize tumor cells via γδ TCR and natural killer cell receptor (NKR). Vγ9Vδ2 TCR recognition depends on non-peptide phosphate antigens (such as isopentenyl pyrophosphate, IPP) of lactolipin 3A1 (BTN3A1) and lactolipin 2A1 (BTN2A1). Furthermore, Vγ9Vδ2 T cells also recognize tumor cells via NKG2D and DNAM-1 (which binds to their ligands (MICA / B, ULBP, Nectin-2, and PVR)) (Liu and Zhang, Cells (2020) 9(5):1206). Vδ1 T cells recognize lipid antigens presented by CD1d via Vδ1 TCR. NKG2D and natural cytotoxic receptors (NCR, NKp30, NKp44, NKp46) with their ligands are involved in Vδ1 T cell tumor cell recognition. Under continuous stimulation by TCR agonists, and in the presence of IL-15 or IL-2, Vδ1 T cells can express NCR (Mikulak et al., JCI Insight (2019) 19; 4(24):e125884, Almeida et al., Clin. Cancer Res (2016) 22:5795-5804). These cells exhibit strong antitumor activity against tumor cells and a high capacity for secreting IFNγ. Research on Vδ3 and Vδ5 TCR ligands is limited, and the antitumor mechanisms of these cells are poorly understood.

[0008] Currently, most clinical trials focus on autologous γδ T cells due to their unique characteristics (such as being unrestricted by MHC and lacking the risk of graft-versus-host disease (GVHD)). Adoptive transfer of autologous Vγ9Vδ2 T cells has been well-tolerated and can trigger anti-tumor immunity. Most strategies currently under evaluation incorporate tumor-targeting mechanisms, such as chimeric antigen receptors (CARs) or bispecific T cell conjugates (bsTCEs), which may be key to achieving more robust and consistent clinical responses. Preliminary results from these targeting approaches (both cell- and antibody-based) have shown great potential and confirmed 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 complexity, the need for specialized facilities, and the need for preparatory lymph node depletion chemotherapy regimens. Therefore, this disclosure relates to the development of antibodies that can effectively activate and reactivate γδ T cells. Summary of the Invention

[0009] This article presents a V-type TCR that combines γδ TCR. H H, the V HH comprises 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; 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 CDR2 containing the amino acid sequence of SEQ ID NO: CDR3 of 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.

[0010] In some embodiments, V HH contains CDR1, CDR2, and CDR3, which contain the following amino acid sequences: (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.

[0011] In some embodiments, V HH contains 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 with it.

[0012] In some embodiments, V H H is humanized. In some embodiments, V H H contains 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 with it.

[0013] This article also provides a peptide construct that binds to γδ TCR, the peptide construct containing the V disclosed above. H H and the Fc region of immunoglobulins.

[0014] In some embodiments, the immunoglobulin Fc region is an IgG Fc region, such as the IgG1, IgG2, IgG3, or IgG4 Fc region.

[0015] This article also provides an antibody or antigen-binding fragment thereof that binds to γδ TCR, the antibody or antigen-binding fragment thereof comprising:

[0016] a) Contains the following heavy chain variable regions (VH):

[0017] i) HCDR1 containing a sequence of SEQ ID NO: 97, 105, 113, 129, 144, 165, 171, 190 or 209;

[0018] ii) HCDR2 containing a sequence of SEQ ID NO: 98, 106, 114, 130, 145, 166, 172, 206, 191, or 210; and

[0019] iii) HCDR3 containing a sequence of SEQ ID NO: 99, 107, 115, 131, 146, 167, 173, 192, or 211; and / or

[0020] b) Contains the following light chain variable regions (VL):

[0021] i) LCDR1 containing a sequence of SEQ ID NO: 101, 109, 117, 133, 175, 194 or 213;

[0022] ii) LCDR2 containing a sequence of SEQ ID NO: 102, 110, 118, 134 or 214; and

[0023] iii) LCDR3 containing a sequence of SEQ ID NO: 103, 111, 119, 135, 148, 169, 176, 195 or 215.

[0024] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0025] (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;

[0026] (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;

[0027] (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;

[0028] (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;

[0029] (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;

[0030] (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;

[0031] (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;

[0032] (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;

[0033] (9) HCDR1, HCDR2, and HCDR3 respectively containing the amino acid sequences of SEQ ID NO: 190, 191, and 192; and / or LCDR1, LCDR2, and LCDR3 respectively containing the amino acid sequences of SEQ ID NO: 194, 110, and 195; or

[0034] (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.

[0035] In some embodiments, the antibody or its antigen-binding fragment comprises: VH, which comprises an amino acid sequence of 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 therewith; and / or VL, which comprises an amino acid sequence of 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 therewith.

[0036] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0037] (1) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0038] (2) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0039] (3) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0040] (4) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0041] (5) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0042] (6) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0043] (7) VH, which contains 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 with it; and / or VL, which contains 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 with it;

[0044] (8) VH, comprising 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 with it; and / or VL, comprising 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 with it; or

[0045] (9) VH, which contains 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 with it; and / or VL, which contains 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 with it.

[0046] In some embodiments, the antibody or its antigen-binding fragment is humanized. In some embodiments, the antibody or its antigen-binding fragment comprises: VH, which comprises 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 therewith; and / or VL, which comprises 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 therewith.

[0047] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region (CH) comprising an amino acid sequence derived from the heavy chain constant region of human immunoglobulins. Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the 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) comprising an amino acid sequence derived from the light chain constant region of human immunoglobulins, such as the κ light chain constant region.

[0048] In some embodiments, the antibody or its antigen-binding fragment is selected from scFv, Fab, Fab', (Fab')2, Fv fragment, biantibody, bispecific antibody, multispecific antibody, chimeric antibody, or humanized antibody.

[0049] A multispecific molecule is also provided, comprising a γδ TCR binding domain and an additional binding domain, wherein the γδ TCR binding domain comprises the V disclosed above. H H, the additional binding domain binds to targets other than γδTCR; preferably, the multispecific molecule is a bispecific antibody.

[0050] A multispecific molecule is also provided, comprising a γδ TCR-binding domain and an additional binding domain, the γδ TCR-binding domain comprising the VH and VL regions of the antibody or its antigen-binding fragment disclosed above, and the additional binding domain binding to a target other than the γδ TCR; preferably, the multispecific molecule is a bispecific antibody. In some embodiments, the γδ TCR-binding domain is an scFv, for example, an scFv comprising a VL-connector-VH or VH-connector-VL structure.

[0051] In some embodiments, targets other than γδ TCR are cancer antigens, such as tumor-specific antigens or tumor-associated antigens.

[0052] In some embodiments, the cancer antigen is an antigen of a blood cancer, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or multiple myeloma.

[0053] In some embodiments, the cancer antigen is an antigen of a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), 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).

[0054] In some embodiments, the cancer antigen is selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0055] In some embodiments, the additional binding domain is a monovalent antibody fragment, such as Fab, Fv, scFv, or V. H H.

[0056] In some embodiments, the multispecific molecule further comprises an immunoglobulin Fc domain composed of a first subunit and a second subunit; preferably, the Fc domain is an IgG Fc domain, such as an IgG1 Fc domain or an IgG4 Fc domain.

[0057] In some embodiments, the Fc domain contains 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).

[0058] In some embodiments, the Fc domain includes a pestle-and-mortar structure, for example, having a T366W substitution in a first subunit of the Fc domain and T366S and L368A substitutions (numbered according to the Kabat EU index) in a second subunit of the Fc domain.

[0059] In some embodiments, the Fc domain is the Fc domain of a human IgG4 isotype having amino acid substitutions of S228P, L234A, and L235A (numbered according to the Kabat EU index).

[0060] In some embodiments, the γδ TCR binding domain is V H H or scFv, the other binding domain is a Fab consisting of a light chain (VL-CL) and a heavy chain (VH-CH1), wherein the heavy chain of the Fab is fused with one of the subunits of the Fc domain, and V H Another fusion of H or scFv with the Fc structural domain subunit.

[0061] In some embodiments, the multispecific molecule comprises:

[0062] A first polypeptide chain comprising a VL binding domain and a light chain constant domain (CL).

[0063] A second polypeptide chain comprising an additional binding domain VH, a first heavy chain constant domain 1 (CH1), and a first Fc subunit;

[0064] A third polypeptide chain comprising a γδ TCR-binding domain and a second Fc subunit; preferably, the first Fc subunit and the second Fc subunit comprise a pestle-and-mortar structure.

[0065] In some embodiments, 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.

[0066] In some embodiments, the amino acid sequence of CL is shown in SEQ ID NO: 196.

[0067] In some embodiments, the additional binding domain is a CD33-binding Fab comprising 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 with it; and VL comprises the sequence of SEQ ID NO: 217 or an amino acid sequence having at least 80% sequence identity with it.

[0068] An isolated nucleic acid molecule is also provided, which contains encoding V as disclosed above. H H, the nucleotide sequence of a polypeptide construct, an antibody or its antigen-binding fragment, or a multispecific molecule.

[0069] A carrier containing the nucleic acid molecules disclosed above is also provided.

[0070] A cell containing nucleic acid molecules or vectors as disclosed above is also provided.

[0071] It also provides a method for generating V as disclosed above. H H, a method for manufacturing a polypeptide construct, an antibody or its antigen-binding fragment, or a multispecific molecule, the method comprising: culturing a host cell containing an isolated nucleic acid molecule or vector as disclosed above, or a host cell disclosed above, under conditions allowing protein expression, and recovering V from the culture of the cultured host cells. H H, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules.

[0072] A pharmaceutical composition is also provided, comprising V as disclosed above. H H, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, and pharmaceutically acceptable carriers and / or excipients.

[0073] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0074] In some embodiments, additional therapeutic agents are anticancer agents, such as immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, or anti-CTLA-4 antibodies) or 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).

[0075] A method for treating cancer in a subject is also provided, comprising administering to the subject in need an effective amount of V as disclosed above. H H, a polypeptide construct, an antibody or its antigen-binding fragment, or a multispecific molecule, or a pharmaceutical composition.

[0076] In some embodiments, the cancer is a blood cancer, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0077] In some embodiments, the cancer is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, stomach 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).

[0078] In some embodiments, the method includes administering a multispecific molecule for treating cancer, wherein the cancer is characterized by a cancer antigen targeted by the multispecific molecule.

[0079] In some embodiments, the cancer is characterized by cancer antigens selected from the following: CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, micin 6, micin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0080] In some embodiments, the subjects are mammals, such as humans.

[0081] In some embodiments, V H H, a polypeptide construct, antibody or its antigen-binding fragment, or a multispecific molecule, or a pharmaceutical composition, administered in combination with an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent or therapy is an anticancer agent or therapy; preferably, the additional therapeutic agent is an immune checkpoint inhibitor, or a cytotoxic agent, or a cytokine; preferably, the additional therapy is a standard cancer treatment, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0082] A method for activating or increasing the proliferation of γδ T cells is also provided, the method comprising reacting T cells with V as disclosed above. H H, contact with polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions.

[0083] It also provides the V disclosed above. H H. Use of a polypeptide construct, antibody or its antigen-binding fragment, or multispecific molecule, or pharmaceutical composition in the manufacture of a medicament for treating a subject’s cancer.

[0084] Further features and advantages of certain embodiments disclosed herein will become more fully apparent from the following description of embodiments thereof and the accompanying drawings, as well as from the claims. Attached Figure Description

[0085] Figure 1 . Design and sequence ID of soluble human and cynomolgus monkey γ9δ2 TCR.

[0086] Figure 2 Construct design and sequence IDs of 5 artificial TCR proteins.

[0087] Figure 3 Construct design and sequence ID of TCE-KIH targeting CD33. Anti-γδ TCR V H H( Figure 3 a) or anti-γδ TCR scFv ( Figure 3 b) Assembled into the CD33-targeting TCE-KIH BsAb molecule. The linker between VL and VH of scFv is (G4S)3.

[0088] Figure 4 CD33×γδ TCR TCE-KIH BsAb-redirected AML-193 cell killing. γ9δ2 T cells were used as effector cells, with an effector cell to target cell ratio of 2:1. Figure 4 a, 4b, 4c and 4d) or 1:1 ( Figure 4 (e, 4f, and 4g). A total of 35 BsAbs were tested in the two experiments, with 5E7-KIH used as a positive control.

[0089] Figure 5 CD33×γδ TCR TCE-KIH BsAb redirected CD33+ AML-193 cell killing. AS281876-KIH and AS282152-KIH showed Figure 5 In a, AS288170-KIH and AS282067-KIH are shown in Figure 5 b. AS282116-KIH and AS287963-KIH are shown in Figure 5 In c, 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls.

[0090] Figure 6 CD33×γδ TCR TCE-KIH BsAb redirected CD33+ MOLM-13 cell killing. AS281876-KIH and AS282152-KIH showed Figure 6 In a, AS288170-KIH and AS282067-KIH are shown in Figure 6 b. AS282116-KIH and AS287963-KIH are shown in Figure 6 In c, 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls.

[0091] Figure 7 CD33×γδ TCR TCE-KIH BsAb redirected CD33+KG-1A cell killing. AS281876-KIH and AS282152-KIH showed Figure 7 In a, AS288170-KIH and AS282067-KIH are shown in Figure 7 b. AS282116-KIH and AS287963-KIH are shown in Figure 7 In c, 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls.

[0092] Figure 8 CD33×γδ TCR TCE-KIH BsAb redirected CD33+ NALM6 cell killing. AS281876-KIH and AS282152-KIH showed Figure 8 In a, AS288170-KIH and AS282067-KIH are shown in Figure 8 b. AS282116-KIH and AS287963-KIH are shown in Figure 8 In c, 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls.

[0093] Figure 9 CD33×γδ TCR TCE-KIH BsAb redirected CD33-CCRF-CEM kill. AS281876-KIH and AS282152-KIH are shown in Figure 9 In a, AS288170-KIH and AS282067-KIH are shown in Figure 9 b. AS282116-KIH and AS287963-KIH are shown in Figure 9In c, 5E7-KIH, 5C8-KIH, 6H1-KIH, 6H4-KIH, and 7A5-KIH were used as positive controls.

[0094] Figure 10 Figure ae shows the fold increase in γδ T cells produced using different antibodies ( Figure 10 a) Vitality Figure 10 b) Purity ( Figure 10 c) Percentage of cell subtypes ( Figure 10 d) and CAR positivity rate ( Figure 10 e).

[0095] Figure 11 Figure ac shows the cytotoxicity of γδ T cells generated using different antibodies. Figure 11 a) Total T cell expansion ( Figure 11 b) and CAR+ T amplification after tumor cell killing ( Figure 11 c). Detailed Implementation

[0096] definition

[0097] In this disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions and explanations of the terms are provided below for a better understanding of this disclosure.

[0098] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Unless otherwise stated or clear from the context, the term "antibody" as used herein can include complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains. The term "antibody" can refer to a conventional antibody that typically comprises at least one heavy chain and at least one light chain. Antibody light chains can be classified as κ light chains and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V... H ) and heavy chain constant region (C H It consists of three structural domains (C). The heavy chain constant region is composed of three structural domains (C). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C.L Composition. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with relatively conservative regions called framing regions (FRs). V H and V L Each of these consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L These form antibody binding sites. The term "antibody" is not limited to any particular method used to produce antibodies.

[0099] As used in this article, the term "V" H H" or "V" H "H domain" refers to the antigen-binding portion of a single-domain antibody (such as camel heavy chain antibodies or shark heavy chain antibodies). V H H can contain three CDRs and four frame regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. V H H can be truncated at the N-terminus or C-terminus, so that it contains only a portion of FR1 and / or FR4, or lacks one or both of those frame regions, as long as V H H essentially maintains antigen binding and specificity. As used in this article, "humanized V" H "H" refers to V where one or more of the frame regions have been largely replaced by human frame regions. H H. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Furthermore, humanized V... H H can contain residues that are not present in the original V. H These residues were found in H and not in human framework sequences, but were included to further refine and optimize V. H The performance of H. As will be understood, humanized sequences can be identified by their primary sequence and do not necessarily represent the process of antibody production.

[0100] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the following definitions: 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, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art can readily identify the CDR defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). The CDR of the antibodies disclosed herein can be defined according to the Kabat, AbM, IMGT, or Chothia numbering systems, or any combination thereof. Unless otherwise specified or clear from the context, the CDR of the antibodies disclosed herein is preferably defined according to the AbM numbering system.

[0101] As used herein, the term “frame region” or “FR” residues refer to those amino acid residues in the variable region of an antibody, other than the CDR residues as defined above.

[0102] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing a full-length antibody fragment that retains the ability to specifically bind the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific antigen binding, and this polypeptide is also referred to as an "antigen-binding moiety." Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments can contain fragments of Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining regions (CDRs), single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and polypeptides containing at least a portion of an antibody (that portion is sufficient to confer specific antigen-binding ability upon the polypeptide).

[0103] As used in this article, the term "Fd fragment" refers to a fragment composed of V H and C H 1. Antibody fragments composed of structural domains; the term "dAb fragment" refers to antibody fragments composed of V... H Antibody fragments composed of structural domains; the term "Fab fragment" refers to an antibody fragment composed of V... L V H C L and C H An antibody fragment consisting of a 1-domain structure; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by disulfide bridges in the hinge region.

[0104] As used herein, the term "Fv fragment" refers to the V fragment formed by the antibody single arm. L and V H Antibody fragments are composed of structural domains. Fv fragments are generally considered to be the smallest antibody fragments capable of forming a complete antigen-binding site. It is believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (e.g., an Fd fragment containing only three CDRs specific to the antigen) can recognize and bind to the antigen, but with less affinity than the entire binding site.

[0105] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, having a generic structure of NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeating G4S amino acid sequences or variants thereof. In some cases, a disulfide bond may also exist between the VH and VL domains of scFv.

[0106] As used herein, the term "biantibody" refers to a dimer of scFv consisting of VH and VL domains linked by short peptide linkers. Linkers that are too short to form intra-chain pairing of the VH and VL domains are not possible. Instead, two such scFv fragments are co-expressed to form a multimer through inter-chain pairing (cross-pairing) of the VH and VL domains.

[0107] Each antigen-binding fragment maintains the ability to specifically bind the same antigen bound by the full-length antibody, 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., the complete antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as for the complete antibody.

[0108] 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 contain the hinge, CH2, and CH3. The Fc region can be any antibody heavy chain constant region isotype discussed herein. The Fc region can be IgG1, IgG2, IgG3, or IgG4.

[0109] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody type or subtype), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody type or subtype), provided that the antibody retains its activity in binding to the antigen of interest. For example, the term "chimeric antibody" can include antibodies (e.g., human-cameloid chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a cameloid antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0110] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its sequence homology with that of human antibodies. 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 intended 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.

[0111] As used herein, the term "specifically bind" or "specifically binding" refers to the non-random binding of two molecules, such as the reaction between an antibody and its target antigen. An antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) can refer to a binding with a specific binding rate of less than about 10... -5 M (for example, less than about 10) -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 Affinity (K) of M or smaller D Antibodies that bind to antigens.

[0112] As used in this article, the term "K" D "" refers to the dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity of an antibody to an antigen. The smaller the dissociation constant, the tighter the antibody binding and the higher the affinity between the antibody and the antigen. Typically, antibodies (e.g., the antibodies disclosed herein) have a dissociation constant of less than about 10. -5 M (for example, less than about 10) -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K (M or smaller) D The K binds to antigens (e.g., human γδ TCR). D Determined by, for example, surface plasmon resonance (SPR) in a BIACORE device.

[0113] As used herein, “isolated nucleic acid” is a nucleic acid (e.g., RNA, DNA, or a mixture of nucleic acids) that is substantially separate from other genomic DNA sequences and proteins or naturally occurring complexes such as ribosomes and polymerases. An “isolated” nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. Furthermore, “isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. One or more nucleic acid molecules encoding chimeric constructs as described herein may be isolated or purified. The term includes nucleic acid sequences that have been removed from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized from heterologous systems. A substantially pure molecule may include the isolated form of the molecule.

[0114] As used herein, the term "vector" refers to a nucleic acid medium into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can express its carried genetic material elements in a host cell through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, kinases, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)); bacteriophages (such as 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 (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papova viruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0115] As used herein, the term "host cell" refers to a cell that can be introduced into or transformed with a vector, including but not limited to prokaryotic cells (such as Escherichia coli or Bacillus subtilis) and eukaryotic cells (such as mammalian cells (e.g., mouse or human cells), insect cells, or yeast cells). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, or MDCKII cells.

[0116] As used herein, the culture medium may contain a basal medium. Any suitable mammalian cell culture medium (such as AIM-V™, X-VIVO, TexMACS, RPMI 1640, OPTMIZER CTS™ (Gibco, Life Technologies), XVIVO-10, XVIVO-15, or XVIVO-20 (Lonza)) can be used as a basal medium. The cell culture medium may contain L-glutamine, streptomycin sulfate, and gentamicin sulfate. The cell culture medium may contain L-glutamine, 50 µg / mL streptomycin sulfate, and 10 µg / mL gentamicin sulfate. Mammalian cell culture medium may contain serum or plasma. The cell culture medium may contain approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the basal medium by volume. Cell culture media can contain basal medium at concentrations greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by volume. Cell culture media can also contain basal medium at concentrations less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% by volume.

[0117] As used herein, the term "identity" refers to the degree of matching between two polypeptides or two nucleic acids. Two sequences used for comparison are identical at a site when they share the same base or amino acid monomer subunit (e.g., each of two DNA molecules has an adenine at a site, or each of two polypeptides has a lysine at a site). The percentage of identity between two sequences is calculated by dividing the number of shared identical sites by the total number of sites used for comparison and then multiplying by 100. For example, if six out of ten sites in two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six sites match). Typically, comparisons of two sequences are performed in a manner that yields the greatest possible identity. This alignment can be performed using computer programs such as the Align program (DNAstar, Inc.) based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) in the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) in the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0118] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. Such carriers and / or excipients include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate-buffered saline. 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, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, etc. Absorption delaying agents 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), etc. Stabilizers have the meaning commonly understood by those skilled in the art, meaning they stabilize the desired activity of an active ingredient in a pharmaceutical product (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 hydrolysates)). 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), antimicrobial 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.

[0119] As used herein, the term "treatment" refers to a method undertaken to achieve 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, disease reduction, stabilization (i.e., non-aggravation) of the disease state, delay or slowing disease progression, and symptom relief (partial or complete), whether detectable or undetectable. Furthermore, "treatment" also refers to prolonged survival compared to expected survival (without treatment). Beneficial or desired clinical outcomes described herein may include, but are not limited to: slowing tumor progression, cancer regression, enhanced anti-tumor immune response, reduction of tumor growth or size, tumor necrosis, reduction of the severity of at least one disease symptom, increased frequency and duration of asymptomatic periods of disease, prevention of damage or disability due to disease suffering, or other ways of improving a patient's disease symptoms.

[0120] 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 mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0121] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the intended effect. For example, an effective amount for treating a disease means an amount that effectively cures or at least partially blocks the disease and its complications in a patient suffering from the disease. The determination of such an effective amount is within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use depends on the severity of the disease to be treated, the general state of the patient's immune system, the patient's general condition (such as age, weight, and sex), the route of administration, and any concurrent therapies used.

[0122] As used herein, the terms “cancer” and “tumor” are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0123] V combined with γδ TCR H H antibody

[0124] On the one hand, it provides V that combines γδ TCR H H, heavy chain antibodies or those containing V H H is a polypeptide construct. The term "V" is also used. H H" or "V" HThe term "H domain" can be used interchangeably in this article to refer to the antigen-binding portion of a single-domain antibody (such as a camel heavy chain antibody or a shark heavy chain antibody). H H typically contains three CDRs and four frame regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some cases, V H H can be truncated at the N-terminus or C-terminus, so that it contains only a portion of FR1 and / or FR4, or lacks one or both of those frame regions, as long as V H H essentially maintains antigen binding and specificity.

[0125] 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 4-chain antibodies. HCAbs are known to be produced by camel-like animals (such as camels, llamas, or alpacas). HCAbs may contain sdAbs fused to the Fc region. HCAbs may also contain sdAbs fused to both the hinge and Fc region of human IgG1.

[0126] The V provided in this article H H, heavy chain antibodies or peptide constructs can specifically bind to γ9δ2 TCR (e.g., human γ9δ2 TCR containing the γ chain of SEQ ID NO: 1 and the δ chain of SEQ ID NO: 2).

[0127] On the one hand, this paper provides a V that combines γδ TCR (e.g., γ9δ2 TCR). H H, the V H H comprises 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; 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 CDR2 containing the amino acid sequence of SEQ ID NO: CDR3 is the amino acid sequence 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 CDRs described above can be defined according to the AbM numbering system.

[0128] On the one hand, this paper provides a V that combines γδ TCR (e.g., γ9δ2 TCR). H H, the V H H contains V as shown in any one 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, 185, 199, 200, 201, 202, 203, or 204. H The three CDRs contained in H. CDRs can be defined according to the Kabat, AbM, IMGT, or Chothia numbering systems or any combination thereof.

[0129] V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 15, 16, and 17. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 19, 20, and 21. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 23, 24, and 25. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 27, 28, and 29. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 31, 32, and 33. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 35, 24, and 36. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 38, 39, and 40. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 42, 43, and 44. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 46, 47, and 48. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 50, 51, and 52. V HH may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 54, 55, and 56. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 58, 59, and 60. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 62, 63, and 64. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 66, 67, and 68. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 70, 71, and 72. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 74, 75, and 76. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 78, 79, and 80. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 46, 82, and 83. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 85, 86, and 87. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 89, 90, and 91. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 93, 94, and 95. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 121, 122, and 123. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 125, 126, and 127. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 54, 137, and 138. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 140, 141, and 142. V HH may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 150, 151, and 152. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 154, 155, and 156. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 158, 159, and 160. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 162, 59, and 163. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences SEQ ID NO: 178, 179, and 180. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO: 182, 183, and 184. V H H may contain CDR1, CDR2, and CDR3, which respectively contain the amino acid sequences of SEQ ID NO:186, 187, and 188.

[0130] The V disclosed in this article H H can further include a frame region (FR).

[0131] V H H may contain a framework region (FR) derived from camel heavy chain antibodies.

[0132] V H H may contain 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% (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 with it.

[0133] Alternatively, V H H can be humanized V H H, where one or more frame regions have been largely replaced by human frame regions. V HH may contain a framework region (FR) derived from the variable region of the human immunoglobulin heavy chain, one or more of which can be mutated back to its cameloid control (i.e., a reversion mutation).

[0134] V H H may contain an amino acid sequence of SEQ ID NO: 199, 200, 201, 202, 203 or 204, 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 with it.

[0135] V H H can be selected from AS282152 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 66, 67, and 68, respectively), AS281876 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively), and AS282067 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 50, 51, and 52, respectively), which recognizes epitopes located at Vγ9 and constant regions of γδ TCR.

[0136] V H H can be selected from AS282116 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 58, 59, and 60, respectively) or AS287963 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 162, 59, and 163, respectively), which recognizes an epitope located in the constant region of γδ TCR.

[0137] In one aspect, this article also provides a heavy chain antibody that binds to γδ TCR (e.g., γ9δ2 TCR), which contains V as described herein. H H. Heavy chain antibodies can be dimers of two polypeptides, each containing at least one V. H H domain and Fc region. Heavy chain antibodies can cover multiple V domains. H Peptides with H domains.

[0138] In one aspect, this paper also provides a polypeptide construct that combines γδ TCR (e.g., γ9δ2 TCR) and contains V as described herein. H H and the Fc region of immunoglobulins.

[0139] The immunoglobulin Fc region can be an IgG Fc region, such as the IgG1, IgG2, IgG3, or IgG4 Fc region. The immunoglobulin Fc region can further be a human IgG Fc region, such as the human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0140] The Fc region of an immunoglobulin can be a natural Fc region containing the same amino acid sequence as that found in naturally occurring Fc regions.

[0141] Alternatively, the immunoglobulin Fc region can be a variant Fc region containing an amino acid sequence that differs from the native Fc region due to at least one amino acid mutation. One or more mutations (e.g., one or more amino acid substitutions) can be introduced into the constant region's Fc 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 such mutations. The immunoglobulin Fc region can have one or more effector functions that are reduced or eliminated.

[0142] V H H can fuse with the N-terminus of the Fc region of immunoglobulin (e.g., at its C-terminus) without a linker.

[0143] Alternatively, V H H can fuse with the N-terminus of the Fc region of an immunoglobulin (e.g., at its C-terminus) in the presence of a linker. The linker can be selected from cleavable linkers, non-cleavable linkers, peptide linkers, flexible linkers, rigid linkers, helical linkers, and non-helical linkers. The linker can be a peptide linker. The linker can contain a sequence of (GmS)n, where m is selected from an integer of 1-6 (e.g., 1, 2, 3, or 4), and n is selected from an integer of 1-6 (e.g., 1, 2, or 3).

[0144] In all the foregoing aspects, the V disclosed herein H H, heavy chain antibodies or peptide constructs can activate γδ T cells and / or increase γδ T cell proliferation, potentially generating an effective antitumor response.

[0145] Conventional antibodies that bind to γδ TCR

[0146] On one hand, conventional antibodies that bind to γδ TCRs are provided. The term "conventional antibody" typically refers to an antibody that comprises at least one heavy chain and at least one light chain. In some cases, it refers to a conventional Y-shaped antibody consisting of two heavy chains and two light chains. In some cases, the conventional antibodies provided herein specifically bind to γ9δ2 TCRs (e.g., human γ9δ2 TCRs comprising the γ chain of SEQ ID NO: 1 and the δ chain of SEQ ID NO: 2).

[0147] CDR and variable area

[0148] In one respect, this article provides an antibody or antigen-binding fragment thereof that binds to γδ TCR (e.g., γ9δ2 TCR), the antibody or antigen-binding fragment thereof comprising:

[0149] a) Contains the following heavy chain variable regions (VH):

[0150] i) HCDR1 containing a sequence of SEQ ID NO: 97, 105, 113, 129, 144, 165, 171, 190 or 209;

[0151] ii) HCDR2 containing a sequence of SEQ ID NO: 98, 106, 114, 130, 145, 166, 172, 206, 191, or 210; and

[0152] iii) HCDR3 containing a sequence of SEQ ID NO: 99, 107, 115, 131, 146, 167, 173, 192, or 211; and / or

[0153] b) Contains the following light chain variable regions (VL):

[0154] i) LCDR1 containing a sequence of SEQ ID NO: 101, 109, 117, 133, 175, 194 or 213;

[0155] ii) LCDR2 containing a sequence of SEQ ID NO: 102, 110, 118, 134 or 214; and

[0156] iii) LCDR3 containing a sequence of SEQ ID NO: 103, 111, 119, 135, 148, 169, 176, 195, or 215. CDRs can be defined according to the AbM numbering system.

[0157] In one aspect, this document 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 the three heavy chain CDRs contained in a VH shown in any one of SEQ ID NO: 96, 104, 112, 128, 143, 164, 170, 205, 189 or 208; and / or the three light chain CDRs contained in a VL shown in any one of SEQ ID NO: 100, 108, 116, 132, 147, 168, 174, 207, 193 or 212. CDRs may be defined according to the Kabat, AbM, IMGT or Chothia numbering system or any combination thereof.

[0158] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 97, 98, and 99, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 101, 102, and 103, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 97, 98, 99, 101, 102, and 103, respectively.

[0159] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 105, 106, and 107, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 109, 110, and 111, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 105, 106, 107, 109, 110, and 111, respectively.

[0160] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 113, 114, and 115, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 117, 118, and 119, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 113, 114, 115, 117, 118, and 119, respectively.

[0161] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 129, 130, and 131, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 129, 130, 131, 133, 134, and 135, respectively.

[0162] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 144, 145, and 146, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 109, 110, and 148, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 144, 145, 146, 109, 110, and 148, respectively.

[0163] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 165, 166, and 167, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 109, 110, and 169, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 165, 166, 167, 109, 110, and 169, respectively.

[0164] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 171, 172, and 173, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 171, 172, 173, 175, 118, and 176, respectively.

[0165] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 171, 206, and 173, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 175, 118, and 176, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 171, 206, 173, 175, 118, and 176, respectively.

[0166] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 190, 191, and 192, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 194, 110, and 195, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 190, 191, 192, 194, 110, and 195, respectively.

[0167] The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 209, 210, and 211, respectively; and / or LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 213, 214, and 215, respectively. The antibody or its antigen-binding fragment may comprise: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 209, 210, 211, 213, 214, and 215, respectively.

[0168] The conventional antibody may be AS288170 (containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 containing amino acid sequences of SEQ ID NO: 171, 172 or 206, 173, 175, 118 and 176 respectively), which recognizes epitopes located at Vγ9 and constant regions of γδ TCR.

[0169] The antibodies or antigen-binding fragments thereof disclosed herein may further include a frame region (FR). Antibodies or antigen-binding fragments thereof may include a frame region (FR) derived from mammalian immunoglobulins (e.g., camelids or humans).

[0170] Antibodies or their antigen-binding fragments may contain a frame region (FR) derived from conventional antibodies from camels.

[0171] The antibody or its antigen-binding fragment may contain a VH containing 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 with it.

[0172] The antibody or its antigen-binding fragment may contain a VL containing 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 with it.

[0173] An antibody or its antigen-binding fragment may comprise: a 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 with it; and / or a 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 96; and a VL containing the sequence of SEQ ID NO: 100.

[0174] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 104; and a VL containing the sequence of SEQ ID NO: 108.

[0175] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 112; and a VL containing the sequence of SEQ ID NO: 116.

[0176] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 128; and a VL containing the sequence of SEQ ID NO: 132.

[0177] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 143; and a VL containing the sequence of SEQ ID NO: 147.

[0178] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 164; and a VL containing the sequence of SEQ ID NO: 168.

[0179] An antibody or its antigen-binding fragment may comprise: 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 therewith; 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 therewith. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 170; and a VL containing the sequence of SEQ ID NO: 174.

[0180] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 189; and a VL containing the sequence of SEQ ID NO: 193.

[0181] An antibody or its antigen-binding fragment may comprise: 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 with it; 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 with it. An antibody or its antigen-binding fragment may comprise: a VH containing the amino acid sequence of SEQ ID NO: 208; and a VL containing the sequence of SEQ ID NO: 212.

[0182] Alternatively, the antibody or its antigen-binding fragment can be humanized, wherein the amino acid sequence has been modified to increase its homology with human antibody sequences. Typically, camel CDR regions can be transplanted onto human frame sequences to generate humanized antibodies. In cases of loss of affinity in these straight-graft antibodies, several frame residues can be mutated back to their camel control (i.e., reversion mutations) to restore the antibody's binding affinity.

[0183] Antibodies or their antigen-binding fragments may include a framework region (FR) derived from human immunoglobulins, and optionally include one or more reversion mutations from human residues to cameloid residues. Antibodies or their antigen-binding fragments may include a heavy chain FR derived from the variable region sequence of the human immunoglobulin heavy chain. Antibodies or their antigen-binding fragments may include a light chain FR derived from the variable region sequence of the human immunoglobulin light chain.

[0184] An antibody or its antigen-binding fragment may comprise a VH containing 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 with it; and / or a VL containing 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 with it. An antibody or its antigen-binding fragment may comprise a VH containing the sequence of SEQ ID NO: 205; and a VL containing the sequence of SEQ ID NO: 207.

[0185] constant region

[0186] The antibodies or antigen-binding fragments disclosed herein may further include constant region sequences derived from mammalian (e.g., camel or human) immunoglobulins. The antibodies or antigen-binding fragments disclosed herein may include constant region sequences derived from human immunoglobulins.

[0187] Any immunoglobulin constant region can be used in the antibodies disclosed herein. The heavy chain constant region can be the heavy chain constant region of human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules. The light chain constant region can be λ or κ. The heavy chain constant region can further be an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. The light chain constant region can further be κ.

[0188] The selection of the constant region depends in part on whether antibody-dependent complement and / or cell-mediated cytotoxicity is desired. Heavy chain constant regions with fewer or reduced effector functions may be preferred. Alternatively, heavy chain constant regions with effector functions (e.g., ADCC) or enhanced effector functions may be preferred.

[0189] The heavy chain of the antibody or its antigen-binding fragment disclosed herein may contain a heavy chain constant region (CH) containing an amino acid sequence derived from the heavy chain constant region of human immunoglobulins.

[0190] The antibodies or antigen-binding fragments disclosed in this article may contain a heavy chain constant region, which is a wild-type heavy chain constant region.

[0191] 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) may 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 antibodies without such mutations.

[0192] The light chain of the antibody or its antigen-binding fragment disclosed herein may contain a light chain constant region (CL) containing an amino acid sequence derived from the light chain constant region of human immunoglobulins.

[0193] antigen-binding fragments

[0194] The antigen-binding fragments disclosed in this article can be any type of antibody fragment with a conventional "Y"-shaped structure, which essentially retains the ability to specifically bind to γδ TCRs and promote the activation or proliferation of γδ T cells.

[0195] The antibodies or antigen-binding fragments disclosed in this article may be selected from the following groups: scFv, Fab, Fab', (Fab')2, Fv fragment, biantibody, bispecific antibody, multispecific antibody, chimeric antibody, or humanized antibody.

[0196] In all of the foregoing aspects, the antibodies or antigen-binding fragments disclosed herein can activate γδ T cells and / or increase γδ T cell proliferation, potentially generating an effective antitumor response.

[0197] It also provides antibody sequences (including V) provided in this article. H "Conserved sequence modifications" (H, heavy chain antibodies, and conventional antibodies) refer to nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody to the antigen encoded by the nucleotide sequence or containing the amino acid sequence. For example, modifications can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. The resulting modified antibodies can be screened for their binding activity.

[0198] Conserved sequence modifications include conserved amino acid substitutions, wherein 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 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 uncharged polar 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 can preferably be replaced by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art. 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).

[0199] Multispecific molecules that bind γδ TCR

[0200] On the other hand, multispecific molecules that bind to γδ TCRs are provided. As used herein, a “multispecific” molecule is a molecule that has at least two binding specificities, such as a polypeptide.

[0201] Multispecific molecules can be bispecific antibodies with two binding specificities.

[0202] Multispecific molecules can contain V as described herein. H H's γδ TCR binding domain.

[0203] Multispecific molecules may contain a γδ TCR-binding domain comprising the VH and VL regions of an antibody or its antigen-binding fragment as described herein. The γδ TCR-binding domain may be an 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 contain a (GmS)n sequence, where m is an integer selected from 1 to 6 (e.g., 1, 2, 3, or 4), and n is an integer selected from 1 to 6 (e.g., 1, 2, or 3). The linker may contain a (G4S)3 sequence.

[0204] Multispecific molecules may contain additional binding domains that bind to targets other than γδ TCR.

[0205] Cancer antigens

[0206] Targets other than γδ TCR can be cancer antigens, such as tumor-specific antigens or tumor-associated antigens.

[0207] Cancer antigens can be antigens of blood cancers. Blood cancers can be selected from, for example, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or multiple myeloma.

[0208] Cancer antigens can be antigens of solid tumors. Solid tumors can be selected from, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, stomach 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).

[0209] Cancer antigens can be selected from CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0210] Multispecific molecules may contain CD33-binding domains comprising VH and VL. The CD33-binding domains may contain VH and VL, wherein VH contains the sequence of SEQ ID NO: 216 and VL contains the sequence of SEQ ID NO: 217.

[0211] Structural form

[0212] The multispecific molecules presented in this article can have several different structural forms known in the art.

[0213] Additional binding domains that bind to targets other than γδ TCR may contain monovalent antibody fragments. These monovalent antibody fragments may be selected from Fab, Fv, scFv, or V. H H.

[0214] Multispecific molecules as described herein may further include immunoglobulin Fc domains. Exemplary Fc domains may be selected from the heavy chain constant region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4); more particularly, the heavy chain constant region of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0215] The Fc domain can be composed of a first subunit and a second subunit. The γδ TCR-binding domain and an additional binding domain can each fuse to the first and second Fc subunits. The γδ TCR-binding domain can fuse to the N-terminus of one of the Fc subunits with or without a linker (e.g., a peptide linker, at its C-terminus). The additional binding domain can fuse to the N-terminus of another Fc subunit with or without a linker (e.g., a peptide linker, at its C-terminus).

[0216] The first and second Fc subunits can be engineered for heterodimerization.

[0217] Heterodimerization of dissimilar polypeptides in multispecific molecules can be facilitated by methods known in the art, including but not limited to heterodimerization via the pestle-and-mortar technique. This technique is developed by introducing a "knob" (or protuberance) by substituting a large amino acid residue for a small amino acid residue in the CH3 domain of one Fc, and by introducing a "hole" (or cavity) by substituting a smaller amino acid residue for one or more large amino acid residues in the CH3 domain of another Fc. One chain of the Fc fragment in the fusion protein may contain a pestle, and a second chain of the Fc fragment may contain a hole.

[0218] Preferred residues for forming the pestle 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). Tryptophan (W) and tyrosine (Y) are most preferred. Exemplary amino acid substitutions in the CH3 domain for forming the pestle include, but are not limited to, substitutions with T366W, T366Y, or F405W.

[0219] Preferred residues for forming the mortar 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 generating the mortar include, but are not limited to, substitutions of T366S, L368A, F405A, Y407A, Y407T, and Y407V.

[0220] In some cases, the pestle contains T366W substitutions, and the mortar contains T366S / L368A substitutions.

[0221] It should be understood that this disclosure also considers and covers other modifications to the Fc region known in the art that promote heterodimerization.

[0222] In some cases, multispecific molecules contain an Fc domain that includes a pestle-and-mortar structure, for example, with a T366W substitution in the first subunit of the Fc domain and T366S and L368A substitutions in the second subunit of the Fc domain.

[0223] Typically, the residues in the immunoglobulin heavy chain are numbered using EU index numbers, such as those in Kabat.

[0224] In other cases, the Fc domain is altered, for example by mutation, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the 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 the 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 exchange). In some cases, the Fc domain is the Fc domain of the human IgG4 isotype with amino acid substitutions of S228P, L234A, and L235A (numbered according to the Kabat EU index).

[0225] Furthermore, multifunctional molecules may include half-life extenders, such as human serum albumin or antibodies against human serum albumin.

[0226] The Fc subunit containing a pestle may contain the sequence of SEQ ID NO: 197.

[0227] The Fc subunit containing mortar may contain the sequence of SEQ ID NO: 198.

[0228] γδ TCR binding domains can be V HH or scFv, and the additional binding domain can be a Fab consisting of a light chain (VL-CL) and a heavy chain (VH-CH1). The heavy chain of the Fab can be fused with one of the Fc domain subunits (e.g., its N-terminus), and V H H or scFv can be fused with another (e.g., its N-terminus) in the Fc domain subunit.

[0229] As described in this article, multispecific molecules can include:

[0230] A first polypeptide chain comprising a VL binding domain and a light chain constant domain (CL).

[0231] A second polypeptide chain comprising an additional binding domain VH, a first heavy chain constant domain 1 (CH1), and a first Fc subunit;

[0232] The third polypeptide chain contains a γδ TCR-binding domain and a second Fc subunit.

[0233] 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 contain a club-and-mortar structure.

[0234] The Fc domain can contain CH2 and CH3. Alternatively, the Fc domain can contain hinge, CH2, and CH3.

[0235] The VL of another binding domain can (e.g., at its C-terminus) fuse with the N-terminus of the CL.

[0236] The additional binding domain VH can (e.g., at its C-terminus) fuse with the N-terminus of CH1, and subsequently with the N-terminus of the first Fc subunit. Alternatively, the additional binding domain VH can (e.g., at its C-terminus) fuse with the N-terminus of a full-length heavy chain constant domain (CH) comprising CH1, a hinge, CH2, and CH3.

[0237] γδ TCR binding domains (e.g., V H H or scFv) can (e.g., at its C-terminus) fuse with the N-terminus of the second Fc subunit.

[0238] Multispecific molecules may further include: a linker between CL and a VL with an additional binding domain, a linker between CH1 and a VH with an additional binding domain, a linker between CH1 and a first Fc subunit, a linker between the γδ TCR binding domain and a second Fc subunit, or combinations thereof. 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 contain a (GmS)n sequence, where m is selected from an integer of 1-6 (e.g., 1, 2, 3, or 4), and n is selected from an integer of 1-6 (e.g., 1, 2, or 3).

[0239] Multispecific molecules may include a first binding arm targeting γδ TCR and a second binding arm targeting targets other than γδ TCR (e.g., cancer antigens), comprising chains 1, 2, and 3, wherein:

[0240] Chain 1 is a light chain of the second arm, with a VL-CL structure;

[0241] Chain 2 is the heavy chain of the second arm, with a structure of VH-CH1-hinge-CH2-CH3; where VH and VL form binding sites for targets other than γδ TCR;

[0242] Chain 3 is a γδ T cell binding arm with anti-γδ TCR V. H The structure of H / scFv-hinge-CH2-CH3.

[0243] Chain 1 may contain a VL of the second binding arm and a CL containing the sequence of SEQ ID NO: 196. Chain 2 may contain a VH of the second binding arm and a CH containing the sequence of SEQ ID NO: 197. Chain 3 may contain a V of the first binding arm. H H or scFv and Fc fragments containing the sequence of SEQ ID NO: 198.

[0244] In all the foregoing aspects, the multispecific molecules disclosed herein can be T-cell conjugates that bind concurrently to γδ TCRs on T cells and cancer antigens on cancer cells. By establishing a physical connection between T cells and cancer cells, T-cell conjugates redirect T cells to cancer cells, activating T cells targeting cancer cells and potentially generating an effective anti-tumor response.

[0245] Antibody production

[0246] On the other hand, V is provided for generating the disclosure herein. H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or multispecific molecules, nucleic acid molecules, vectors and host cells.

[0247] An isolated nucleic acid molecule is provided, which contains encoding V as described herein. H The nucleotide sequence of H.

[0248] An isolated nucleic acid molecule is also provided, which contains a nucleotide sequence encoding a heavy chain antibody as described herein.

[0249] An isolated nucleic acid molecule is also provided, which contains a nucleotide sequence encoding a polypeptide construct as described herein.

[0250] Also provided is an isolated nucleic acid molecule comprising nucleotide sequences encoding conventional antibodies or antigen-binding fragments thereof as described herein, or their heavy chain variable regions and / or light chain variable regions. The isolated nucleic acid molecule may 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 antigen-binding fragment disclosed herein, respectively.

[0251] An isolated nucleic acid molecule is also provided, which contains a nucleotide sequence encoding a multispecific molecule as described herein. The isolated nucleic acid molecule may contain different nucleotide sequences encoding different polypeptide chains, respectively, that encode multispecific molecules.

[0252] A vector (e.g., a cloning vector or an expression vector) is also provided that contains the isolated nucleic acid molecules disclosed herein.

[0253] The vectors disclosed in this article may be, for example, plasmids, granules, or bacteriophages.

[0254] The carrier may contain encoding the V disclosed herein. H The nucleotide sequence of H.

[0255] The vector may contain a nucleotide sequence encoding a heavy chain antibody as described herein.

[0256] The vector may contain a nucleotide sequence encoding the polypeptide construct disclosed herein.

[0257] The vector may contain 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, respectively, of the conventional antibody or its antigen-binding fragment disclosed herein. The first nucleotide sequence and the second nucleotide sequence may be located on the same or different vectors.

[0258] Vectors can contain different nucleotide sequences of different polypeptide chains that encode multispecific molecules. Different nucleotide sequences can be located on the same or different vectors.

[0259] A host cell is also provided, which contains or is transformed with the following: the isolated nucleic acid molecules or vectors disclosed herein. Such host cells include, but are not limited to, prokaryotic cells (such as Escherichia coli cells) and eukaryotic cells (such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells and human cells)).

[0260] A method for generating the V disclosed herein is also provided. H Methods involving H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules, comprising culturing host cells containing the isolated nucleic acid molecules or vectors disclosed herein, or the host cells disclosed herein, under conditions allowing protein expression, and recovering V from the culture of the cultured host cells. H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules.

[0261] Pharmaceutical Composition

[0262] In another aspect, a pharmaceutical composition is provided comprising the V disclosed herein. H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules.

[0263] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0264] Provides V as described in this article H The pharmaceutical composition of H. The pharmaceutical composition may contain an effective amount of V. H H. V H H may be the only active ingredient contained in a pharmaceutical composition.

[0265] Pharmaceutical compositions comprising heavy chain antibodies as described herein are also provided. The pharmaceutical composition may contain an effective amount of heavy chain antibody. The heavy chain antibody may be the sole active ingredient contained in the pharmaceutical composition.

[0266] Pharmaceutical compositions comprising a polypeptide construct as described herein are also provided. The pharmaceutical composition may contain an effective amount of the polypeptide construct. The polypeptide construct may be the sole active ingredient contained in the pharmaceutical composition.

[0267] Pharmaceutical compositions comprising conventional antibodies or antigen-binding fragments thereof as described herein are also provided. The pharmaceutical composition may comprise an effective amount of a conventional antibody or antigen-binding fragment thereof. The conventional antibody or antigen-binding fragment thereof may be the sole active ingredient contained in the pharmaceutical composition.

[0268] Pharmaceutical compositions comprising multispecific molecules as described herein are also provided. The pharmaceutical composition may contain an effective amount of the multispecific molecule. The multispecific molecule may be the only active ingredient contained in the pharmaceutical composition.

[0269] The pharmaceutical compositions disclosed herein may further comprise additional therapeutic agents. These additional therapeutic agents may be anticancer agents. Alternatively, 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, additional therapeutic agents may be cytotoxic agents, such as alkylating agents, antimitotic agents, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, or radionuclides. Alternatively, 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).

[0270] The V disclosed in this article H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules, and other therapeutic agents may be provided as separate components or as multiple components of a single composition. The V disclosed herein may be... H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules are used in combination with other agents, either simultaneously, separately or sequentially.

[0271] The pharmaceutical composition may be provided in unit dosage form (i.e., the dose for a single administration).

[0272] Pharmaceutical compositions can be formulated using one or more pharmaceutically acceptable carriers and / or excipients. The formulation depends on the chosen route of administration. For parenteral administration, the pharmaceutical composition is preferably sterile and substantially isotonic and manufactured under GMP conditions. For example, the V disclosed herein can be... H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules are formulated in an aqueous solution for injection, preferably in a physiologically compatible buffer such as water for injection (WFI), antibacterial 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), or Ringer's solution. The solution may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, V disclosed herein... HH, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules may be in lyophilized form so that they can be prepared with a suitable medium (e.g., sterile pyrogen-free water) before use.

[0273] The pharmaceutical compositions described herein can be used to promote the activation and / or proliferation of γδ T cells and / or to treat diseases such as cancer.

[0274] Methods or uses for treating diseases

[0275] On the other hand, it provides the use of V as described in this article. H H. Methods or uses for treating diseases, including heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules.

[0276] On one hand, a method for treating a subject's disease is provided, which includes administering an effective amount of V, as described herein, to the subject in need. H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules. Such diseases include any disease that would benefit from increased proliferation and activation of T cells (such as γδ T cells).

[0277] As discussed in this article, a disease can be cancer.

[0278] A method for treating cancer is provided, which includes administering V, a drug that targets γδ TCR, as disclosed herein. H H, heavy chain antibodies, peptide constructs, or conventional antibodies or their antigen-binding fragments can be used to increase the proliferation and / or activation of γδ T cells.

[0279] A method for treating cancer is also provided, which involves increasing the proliferation and / or activation of γδ T cells by administering a multispecific molecule disclosed herein that binds γδ TCRs and cancer antigens. Cancer may be characterized by cancer antigens targeted by the multispecific molecule.

[0280] The γδ TCR binding domain employed by the multispecific molecule can be derived from the following antibodies: AS282152 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 66, 67, and 68, respectively), AS281876 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively), AS282067 (containing CDR1, CDR2, and CDR3 containing the amino acid sequences of SEQ ID NO: 50, 51, and 52, respectively), or AS288170 (containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 171, 172, or 206, 173, 175, 118, and 176, respectively), which recognize epitopes located at Vγ9 and constant regions of the γδ TCR. Vγ9 is part of the γδ TCR involved in the recognition of non-peptide phosphate antigens; therefore, by blocking the interaction between the γδ TCR and its phosphate antigen, tumor cell killing can be inhibited. Thus, in cases where tumors do not express tumor antigens targeted by multispecific molecules, using these antibodies as multispecific molecules of the γδ T cell conjugating arm can inhibit tumor cell killing.

[0281] The γδ TCR-binding domain employed by multispecific molecules can be derived from antibodies such as AS282116 (containing CDR1, CDR2, and CDR3, respectively, with amino acid sequences of SEQ ID NO: 58, 59, and 60) or AS287963 (containing CDR1, CDR2, and CDR3, respectively, with amino acid sequences of SEQ ID NO: 162, 59, and 163), which recognize epitopes located in constant regions of the γδ TCR that are not involved in the recognition of non-peptide phosphate antigens. Therefore, in cases where the tumor does not express tumor antigens targeted by multispecific molecules, the use of these antibodies as multispecific molecules acting as γδ T cell binding arms exhibits minimal (if any) inhibitory activity against tumor cell killing.

[0282] On the one hand, it provides V as described in this article. H H, heavy chain antibodies, polypeptide constructs, conventional antibodies or their antigen-binding fragments, or multispecific molecules, in the manufacture of a medicament for use in treating a subject's disease. Such a disease may include any condition that would benefit from increased proliferation and activation of T cells, such as γδ T cells. As described herein, a disease may be cancer.

[0283] The cancers involved in the treatments or uses described herein may include, but are not limited to, solid tumors and hematologic malignancies. Cancers may be hematologic malignancies, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or multiple myeloma. Cancers may be solid tumors, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), 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).

[0284] The cancers involved in the treatment methods or uses described herein may be characterized by cancer antigens selected from the following: CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, mesothelin 6, mesothelin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

[0285] The subjects involved in the treatment methods or uses described in this article may be mammals, especially humans.

[0286] In any of the treatment methods or uses described herein, the V disclosed herein H H, heavy chain antibodies or peptide constructs can activate γδ T cells and / or increase γδ T cell proliferation, potentially generating an effective antitumor response.

[0287] In any of the treatment methods or uses described herein, the antibodies or antigen-binding fragments disclosed herein can activate γδ T cells and / or increase γδ T cell proliferation, potentially generating an effective antitumor response.

[0288] In any of the therapeutic methods or uses described herein, the multispecific molecules disclosed herein can act as T-cell conjugates, which concomitantly bind to γδ TCRs on T cells and cancer antigens on cancer cells. By establishing a physical connection between T cells and cancer cells, the T-cell conjugate redirects T cells to cancer cells, activating T cells targeting cancer cells and potentially generating an effective anti-tumor response.

[0289] In any of the treatment methods or uses described herein, the 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.

[0290] Alternatively, the V disclosed in this article can be used as a reference.H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions, administered in combination with other therapeutic agents or therapies. Other therapeutic agents may be anticancer agents, such as immune checkpoint inhibitors, or cytotoxic agents, or cytokines. Other therapies may be standard cancer treatments, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0291] In any of the treatment methods or uses described herein, the V disclosed herein H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions can be formulated into 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), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. Alternatively, for ease of storage and use, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying).

[0292] In any of the treatment methods or uses described herein, the 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, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, groin, bladder, local (e.g., powder, ointment, or drops), or nasal administration. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose. The 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 intravenous injection or bolus.

[0293] On the other hand, a method for activating γδ T cells and / or increasing γδ T cell proliferation is also provided, the method comprising causing T cells (e.g., T cell populations) to react with the V cells disclosed herein. H H, heavy chain antibodies, polypeptide constructs, antibodies or their antigen-binding fragments, or multispecific molecules, or pharmaceutical compositions may come into contact. T cells can be present in vivo. T cells can be present in vitro.

[0294] Activation of γδ T cells can be determined by any method in the art, such as that provided in Example 3 of this document, for example by measuring CD25 and / or CD107a expression. Increased expression of CD25 and / or CD107a indicates activation.

[0295] Example

[0296] Unless otherwise stated, the examples provided below are for illustrative purposes only and are not intended to be limiting. Therefore, this disclosure should in no way be construed as limited to the examples below, but rather as covering any and all variations that become apparent as a result of the teachings provided herein.

[0297] Example 1: Generation and Characterization of Anti-γδ TCR Antibodies

[0298] Soluble γδ TCR protein was generated internally for animal immunization. To promote TCR heterodimerization, a pair of charge-complementary leucine zipper (LZ) sequences were attached to the C-terminus of the TCRγ and TCRδ chains via a flexible (G4S)3 linker. Additionally, a 6xHis tag was added to the γ chain, and a flag tag was added to the C-terminus of the δ chain for simultaneous purification and detection. TCRγ and TCRδ chain plasmids were prepared and used for the production of soluble γδ TCR. Soluble TCR (sTCR) was produced from FreeStyle 293-F cells and purified using anti-DYKDDDDK G1 affinity resin (Genscript, catalog number L00432). Constructs of human and cynomolgus monkey γ9δ2 TCR protein were depicted on [the image / image / etc.]. Figure 1 middle.

[0299] In accordance with all current animal welfare regulations, a camel was immunized with γ9δ2 TCR protein. The γ9δ2 TCR protein was formulated as an emulsion with either complete Freund's adjuvant (CFA) (for primary immunization) or incomplete Freund's adjuvant (IFA) (for booster immunization). The antigen emulsion was administered via a two-point intramuscular injection in the neck. The animal received three injections of 100–200 μg human γ9δ2 TCR protein at 2-week intervals, followed by two injections of 100 μg cynomolgus monkey γ9δ2 TCR protein at weekly intervals. A final booster was given to the animal with 50 μg human γ9δ2 TCR protein. Five days later, 150 mL of blood was collected, and approximately 3 × 10⁻⁶ TCR proteins were isolated from the blood. 8 Peripheral blood lymphocytes (PBLs) serve as the genetic source of conventional and heavy chain immunoglobulins.

[0300] Whole RNA was extracted from lymphocytes of immunized camels using TRIZOL® reagents (Invitrogen™, catalog number 15596026). cDNA was synthesized based on the RNA template using the PRIMESCRIPT™ 1st Strand cDNA Synthesis Kit and oligo(dT)20 primers (Takara, catalog number 6110A). The camel cDNA encoding V was amplified. H DNA from H (the variable region of heavy chain antibodies only, also known as single-domain antibodies, sdAb), VH, and VL was purified and ligated into an internal phage vector (see patent number US 20170089914 A1). Figure 1 The ligation 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.

[0301] Simultaneously, sdAb and scFv phage libraries were rescued and stored at 4°C after filtration sterilization for further use. Binding proteins were isolated from the aforementioned phage libraries using both protein-based and cell-based panning methods. A first round of panning was performed using both protein-based and cell-based methods. At least 50% of the γ9δ2TCR-positive clones were identified by ELISA, and high sequence diversity was observed for all output phages. These outputs were then used for subsequent high-throughput screening.

[0302] E. coli cells were grown using the selected output phage infection index. The output double-stranded DNA was extracted. The sdAb / scFv insert was cut from the phage vector and inserted into an antibody fragment expression vector for high-throughput screening. E. coli cells were transformed using the resulting plasmid, and these cells were then seeded and grown overnight at 37°C. Thousands of colonies were individually picked and grown in 96-well plates containing 1 mL of 2YT medium. Antibody fragment expression was induced by adding 1.0 mM IPTG. The ability of the sdAb / scFv protein in the supernatant to bind to human γ9δ2 TCR protein was analyzed by ELISA, and the ability of these proteins to bind to human γ9δ2 T cells was analyzed by flow cytometry. A total of 33 V cells with unique sequences were selected. H H and 9 scFv-binding proteins were used for further characterization. Full-length V H The IDs of the H / VH, VL, and CDR sequences are listed in Table 1.

[0303] Table 1. Anti-γδTCR antibody sequence IDs in camels

[0304]

[0305] To assess the cross-species reactivity and specificity of the selected clones, ELISA was performed on internally generated cynomolgus monkey γ9δ2 TCR and human αβ TCR proteins (SEQ ID NO: 5-6) (Table 2). To classify binding proteins based on identified γδ TCR domains / regions, five artificial TCR proteins were designed by replacing the γ9δ2 TCR domain with a control derived from αβ TCR or a sequence from other γδ TCRs, and these proteins were generated internally. Figure 2 ELISA was performed using all recombinant TCR proteins as antigens to identify antigenic domains crucial for antigen-antibody interactions. For example, the fact that AS281795 binds to the human γ9δ2 TCR but not the human γ9δ1 TCR indicates that the epitope of AS281795 is located in the Vδ2 domain. Based on their binding epitopes, the selected binding proteins were classified into eight groups (Table 2). According to the results, almost all binding proteins recognized the human and cynomolgus monkey γ9δ2 TCRs, and the epitopes of these antibodies exhibited great diversity.

[0306] Table 2. Binding activity, specificity, and binding epitopes of anti-γδ TCR antibodies

[0307]

[0308] Example 2: Determination of Affinity for Heavy Chain Antibodies

[0309] Using camel-like animals V H Heavy chain-only antibodies (HCAbs) or conventional IgG heavy and light chains were constructed using H / VH and VL sequences. Heavy and light chain plasmids were prepared and used for the production of HCAbs 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 number US 20190263908 A1, SEQ ID NO: 49) was prepared as a positive control.

[0310] The binding affinity of the selected molecules to human and cynomolgus monkey γ9δ2 TCR proteins was determined by surface plasmon resonance (SPR) on a BIAcore T200 instrument (GE Healthcare). Experiments were performed as follows: antibodies were captured onto a sensor chip pre-coated with goat anti-human pAb (Jackson ImmunoResearch, catalog number 109-005-098) via the interaction between a polyclonal antibody and human Fc. Incremental concentrations (ranging from 40 nM to 2.56 µM) of human or cynomolgus monkey γ9δ2 TCR were injected onto the sensor chip surface, allowing binding of the captured antibody for 100 s, followed by injection of run buffer to allow dissociation of the complex for 300 s. After each cycle, the surface was regenerated by injection of 10 mM glycine-HCl buffer (pH 2.0). The experimental data were fitted using a Langmuir model (which describes a simple 1:1 interaction between a ligand molecule and an analyte) to obtain the association rate (kJ / kJ / kJ). a ) and dissociation rate (k d The kinetic values ​​of ) are used to calculate the equilibrium dissociation constant (K). D Based on the results summarized in Table 3, the antibodies exhibit a wide range of affinities (K0) from approximately 200 nM to 1 nM (with human γ9δ2 TCR) and from approximately 3 μM to 5 nM (with cynomolgus monkey γ9δ2 TCR). D () Combined with γ9δ2 TCR.

[0311] Table 3. Binding affinity of purified antibody to human and cynomolgus monkey γ9δ2 TCR protein

[0312]

[0313] Example 3: HCAb-induced activation of γ9δ2 T cells

[0314] Flat-bottomed 96-well cell culture plates were coated overnight at 4°C with 100 μL of 2 μg / mL goat anti-human IgG Fc (ThermoScientific, catalog 31125). The wells were washed with PBS and blocked for 30 min at room temperature with 200 μL of 4% BSA / PBS. The blocking solution was discarded, and the wells were incubated for 2 h at 37°C with 100 μL of HCAb (0.5 nM, 5 nM, and 50 nM) in PBS. Human peripheral blood mononuclear cells (PBMCs) were used for the expansion of γ9δ2 T cells as previously described (Makoto Kondo et al., Sep 9, 2011;(55):3182). The resulting γ9δ2 T cells were resuspended in AIM-V medium (Gibco, catalog A3830801) and added to 96-well cell culture plates (1 × 10⁻⁶). 5 γ9δ2 T cells / 200 μL LAIM-V / well were incubated at 37°C. γδ T cell degranulation after 4 hours of incubation and γδ T cell activation after 24 hours of incubation were then assessed by flow cytometry. As shown in Table 4, all HCAbs tested demonstrated comparable or better ability to drive γ9δ2 T cell activation and degranulation than 6H1 HCAbs, reflected by increased CD25 and CD107a expression, respectively.

[0315] Table 4. HCAb-induced activation of γ9δ2 T cells

[0316]

[0317] Example 4: TCE-KIH Redirected AML-193 Cell Killing

[0318] To test the functional activity of anti-γδ TCR antibodies, 29 anti-γδ TCR V antibodies were used. H H and 6 anti-γδ TCRscFv are assembled into a CD33-targeting TCE (T cell conjugation)-KIH (pestle) bispecific antibody (BsAb) molecule. Figure 3 BsAb consists of three chains: one light chain and two heavy chains. The heavy chains are paired correctly using a pestle-and-mortar technique. Chain 1 is the light chain of the CD33 arm, with a VL-CL structure. Chain 2 is the heavy chain of the CD33 arm, with a VH-CH1-hinge-CH2-CH3 structure. Chain 3 is the γδ T cell binding arm, possessing anti-γδ TCR V... HThe structure is H / scFv-hinge-CH2-CH3. 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 number 1094462010). As a positive control, 5E7-KIHBsAbs were also produced using 5E7 (from patent number US 20190263908 A1, SEQ ID NO: 60).

[0319] The cytotoxicity of T cell redirection was evaluated by flow cytometry using human γ9δ2 T cells as effector cells and CD33+ human AML cell line AML-193 (ATCC, catalog number CRL-9589™) as target cells. In short, AML-193 cells were labeled with CellTrace™ Violet (Thermo Fisher Scientific, catalog number C34557) and seeded together with γ9δ2 T cells at an effector cell to target cell ratio (E:T) of 1:1 or 2:1 in 96-well round-bottom plates (5 × 10⁶ cells per well). 4 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 flow cytometry as loss of target cell membrane integrity, reflected by nuclear uptake of propidium iodide (Sigma, catalog number P4170-10MG). Samples were analyzed by flow cytometry on a BD FACSCelesta™ flow cytometer, and data were processed using BD FACS Diva software version 8.0.1.1. To quantify BsAb-induced killing, specific lysis was calculated using the following formula:

[0320]

[0321] The killing assay was performed in two experiments, with an E:T ratio of 2:1 in the first experiment and 1:1 in the second. As shown in Table 5, there was significant baseline killing of AML-193 cells by γ9δ2 T cells, with approximately 45% of AML-193 cells killed. The EC50 value of the positive control BsAb 5E7-KIH differed between the two experiments, possibly due to the difference in E:T. Among the 35 BsAbs, almost all showed comparable or better killing efficacy and potency to 5E7-KIH, except for AS281940-KIH, AS281949-KIH, AS281991-KIH, and AS287895-KIH, which had lower EC50 values. Figure 4 (and Table 5).

[0322] Table 5. TCE-KIH targeting CD33-induced tumor cell killing

[0323]

[0324] Example 5: TCE-KIH-redirected killing of multiple cancer cell lines

[0325] Five cancer cell lines with different CD33 expression levels were used as target cells. Cytotoxicity assays were performed on six BsAbs (AS281876-KIH, AS282067-KIH, AS282116-KIH, AS282152-KIH, AS287963-KIH, and AS288170-KIH) that showed the highest killing efficacy against AML-193. Among these target cell lines, AML-193 and MOLM-13 (DSMZ, catalog number ACC 554) were high-expressing AML lines; KG-1A (ATCC, catalog number CCL-246.1) and NALM6 (ATCC, catalog number CRL-3273) were low-expressing cell lines; and CCRF-CEM (ATCC, catalog number CCL-119) was a CD33-acute lymphoblastic leukemia cell line. Killing assays were performed as described in Example 4. In addition to the positive control 5E7-KIH, three anti-Vγ9Vδ2sdAbs (i.e., 5C8, 6H1, and 6H4) (from patent numbers US 20190263908 A1, SEQ ID NO: 59 and 49, and US 20220098301 A1, SEQ ID NO: 54, respectively) and one anti-Vγ9Vδ2 scFv 7A5 (from patent number WO2020227457 A1, VH SEQ ID NO: 65, VL SEQ ID NO: 66) were used to generate the TCE-KIH BsAb control, as described in Example 4.

[0326] Of the six BsAbs, AS281876-KIH and AS282152-KIH were superior to all five positive control BsAbs and showed the best tumor cell killing activity against all CD33+ cancer cell lines, as reflected by high maximum killing and lowest EC50 values. Figure 5 a, Figure 6 a, Figure 7 a and Figure 8 a, Table 6). AS288170-KIH and AS282067-KIH were comparable to or slightly better than all five positive control BsAbs (a, Table 6). Figure 5 b、 Figure 6 b、 Figure 7 b and Figure 8(b, 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, but 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, as reflected by higher EC50 values. However, AS282116-KIH and AS287963-KIH showed the highest efficacy in killing KG-1A and NALM6, as reflected by the highest maximum kill of all 11 BsAbs. Figure 5 c. Figure 6 c. Figure 7 c and Figure 8 c, Table 6).

[0327] Since CCRF-CEM does not express CD33 molecules on its surface, no BsAb-dependent cancer cell killing was observed. Figure 9 Cancer cell killing could only be observed, depending on the recognition of non-peptide phosphate antigens by the γ9δ2 TCR. However, for most BsAbs, tumor cell killing was inhibited to some extent by high concentrations of BsAbs, possibly because these antibodies bind to the γδTCR and block 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 much lower killing inhibition, possibly due to the low affinity of 7A5 for the γ9δ2 TCR (data not shown, but also submicromolar affinity for the γ9δ2 TCR in humans and cynomolgus monkeys). Only two antibodies, AS282116-KIH and AS287963-KIH, showed little or no inhibition of cancer cell killing. The difference in inhibitory activity among these antibodies can be explained by the differences in the epitopes they recognize. The epitopes of AS282152, AS281876, AS288170, and AS282067 are located at Vγ9 and the constant domain of the γδ TCR. Vγ9 is part of the γδ TCR involved in non-peptide phosphate antigen recognition; therefore, tumor cell killing is inhibited by blocking the interaction between the γδ TCR and its homologous antigens. On the other hand, AS282116 and AS287963 bind to the constant domain of the γδ TCR, which is not involved in antigen recognition. Therefore, using these antibodies as BsAbs as γδ T cell conjugating arms has little (if any) inhibitory effect on tumor cell killing.

[0328] Table 6. TCE-KIH targeting CD33-induced tumor cell killing in AML cell lines with high and low CD33 expression levels.

[0329]

[0330] Example 6: Humanization of anti-γδ TCR antibody fragments

[0331] Humanization of six camel antibodies (five sdAbs and one scFv) was performed using the CDR transplantation method (Winter G, Harris WJ 1993). The V of the selected camel antibodies was then... H The H / VH and VL sequences were BLASTed against the NCBI human germline V gene database, thus identifying the human VH and VL germline sequences with the highest identity to the camel antibody (i.e., the human receptor). In the CDR transplantation approach, the human receptor's CDR is replaced by the camel antibody's CDR, resulting in a straight-graft sequence. Straight-grafted antibodies typically lose binding activity, which needs to be restored by replacing key framework residues for antibody activity with camel residues.

[0332] Using camels and humanized V H The H / VH and VL sequences were used to construct chain 3 of TCE-KIH and expressed using the method described in Example 4. After humanization, all antibodies possessed at least one humanized variant that retained considerable binding affinity (Table 7). The sequence IDs of the humanized antibodies are listed in Table 8.

[0333] Table 7. Affinity of humanized anti-γδ TCR antibodies

[0334]

[0335] Table 8. Sequence IDs of Humanized Antibodies

[0336]

[0337] Example 7: Production of polyclonal γδ T cells and their in vitro efficacy using anti-γδ TCR antibodies

[0338] To activate and expand polyclonal γδT cells from PBMCs, four antibodies specifically binding to γδTCRs were tested, including AS281850, AS287435, AS288180, and AS287963. These antibodies were coated onto cell culture plates, and CAR-polyclonal γδT cells were generated using the media shown in Table 9. Media I and Media II both contained basal medium, human serum substitutes supplemented with different cytokines (e.g., human platelet lysate), and the Akt inhibitor MK-2206. Cell performance was then assessed.

[0339] Table 9

[0340]

[0341] In summary, polyclonal γδT cells were generated from PBMCs as follows: PBMCs were activated in medium I using the aforementioned antibody for 3–9 days. Then, activated cells were transduced with a lentivirus or retrovirus expressing anti-BCMA CAR (SEQ ID NO: 62, WO2023020558 A1) at an appropriate multiple of infection (MOI). After 1–5 days of transduction, the cells were cultured in medium II for another 6–12 days, and then CAR-polyclonal γδT cells were harvested. Cell number and viability were analyzed using a Cellometer (Nexcelom, K2). For phenotypic and CAR positivity detection, harvested 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.

[0342] To further evaluate the correlation between different stimuli and cytotoxic efficacy, expanded CAR-γδT cells were assessed in repeated tumor challenge assays. In short, 2 × 10⁶ cells were used. 5 CAR+ γδT cells and 2 × 10 5 Two NCI-H929 cells (ATCC, catalog number CRL-3580) were co-cultured together in a 24-well plate. After two days, cells were harvested to determine the relative ratio of live T cells to tumor cells. CAR+ γδ T cells were counted and reseeded at a 1:1 ratio with fresh NCI-H929 cells for the next round of tumor cell attack.

[0343] In all four antibody cases, γδT cells expanded well, at least 900-fold, and cell viability was >90%.Figure 10 a to Figure 10 b). The purity of γδT cells generated using all four antibodies was high, >98% ( Figure 10 c), and the composition of different γδT cell subtypes is similar: Vδ2 T cells have the highest percentage (approximately 65%), and Vδ1... - Vδ2 - The percentage of T cells was the lowest (approximately 10%). Figure 10 d). The percentage of CAR-positive cells was also similar in the four groups ( Figure 10 e). For example Figure 11 As described in a, the effective elimination of H929 cells mediated by polyclonal γδT cells activated by different antibodies, up to round 7, showed that the total γδT amplification fold and CAR γδT amplification fold were similar across all groups during long-term killing. Figure 11 b to Figure 11 c).

[0344] sequence

[0345] .

Claims

1. A V that combines γδ TCR H H, the V H H comprises 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; 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 CDR2 containing the amino acid sequence of SEQ ID NO: CDR3 of 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.

2. The V as described in claim 1 H H, the V H H contains CDR1, CDR2, and CDR3, which respectively contain the following amino acid sequences: (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.

3. The V as described in claim 1 or 2 H H, the V H H contains 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 with it.

4. The V as described in any one of claims 1-3 H H, the V H H is humanized.

5. The V as described in claim 4 H H, the V H H contains 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 with it.

6. A polypeptide construct that binds to γδ TCR, said polypeptide construct comprising V as described in any one of claims 1 to 5 H H and the Fc region of immunoglobulins.

7. The polypeptide construct of claim 6, wherein the immunoglobulin Fc region is an IgG Fc region, such as an IgG1, IgG2, IgG3 or IgG4 Fc region.

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

9. The antibody or antigen-binding fragment thereof as claimed in claim 8, wherein the antibody or antigen-binding fragment thereof comprises: (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 respectively containing the amino acid sequences of SEQ ID NO: 190, 191, and 192; and / or LCDR1, LCDR2, and LCDR3 respectively containing the amino acid sequences of SEQ ID NO: 194, 110, and 195; or (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.

10. The antibody or antigen-binding fragment thereof as claimed in claim 8 or 9, wherein the antibody or antigen-binding fragment comprises a VH, the VH comprising an amino acid sequence of 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 therewith; and / or a VL, the VL comprising an amino acid sequence of 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 therewith.

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

12. The antibody or antigen-binding fragment thereof as claimed in any one of claims 8 to 11, wherein the antibody or antigen-binding fragment thereof further comprises: a heavy chain constant region (CH) comprising an amino acid sequence derived from the heavy chain constant region of human immunoglobulins. Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as the 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 light chain constant region of human immunoglobulins, such as the κ light chain constant region.

13. The antibody or antigen-binding fragment thereof as described in any one of claims 8 to 12, wherein the antibody or antigen-binding fragment thereof is selected from scFv, Fab, Fab', (Fab')2, Fv fragment, biantibody, bispecific antibody, multispecific antibody, chimeric antibody or humanized antibody.

14. A multispecific molecule comprising a γδ TCR binding domain and another binding domain, the γδ TCR binding domain comprising V as described in any one of claims 1 to 5. H H, the additional binding domain binds to targets other than γδ TCR; preferably, the multispecific molecule is a bispecific antibody.

15. A multispecific molecule comprising a γδ TCR binding domain and an additional binding domain, the γδ TCR binding domain comprising the VH and VL regions of an antibody or an antigen-binding fragment thereof as described in any one of claims 8 to 13, the additional binding domain binding to a target other than the γδ TCR; preferably, the multispecific molecule is a bispecific antibody.

16. The multispecific molecule of claim 15, wherein the γδ TCR binding domain is an scFv, for example, an scFv comprising a VL-connector-VH or a VH-connector-VL structure.

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

18. The multispecific molecule of claim 17, wherein the cancer antigen is an antigen of a blood cancer, such as acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or multiple myeloma.

19. The multispecific molecule of claim 17, wherein the cancer antigen is an antigen of a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), 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).

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

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

22. The multispecific molecule of 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; preferably, the Fc domain is an IgG Fc domain, such as an IgG1 Fc domain or an IgG4 Fc domain.

23. The multispecific molecule of 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 mordant).

24. The multispecific molecule of claim 23, wherein the Fc domain comprises a mortar and pestle structure, for example having a T366W substitution in the first subunit of the Fc domain and T366S and L368A substitutions (numbered according to the Kabat EU index) in the second subunit of the Fc domain.

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

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

27. The multispecific molecule of claim 26, wherein the multispecific molecule comprises: A first polypeptide chain, the first polypeptide chain comprising the VL of the additional binding domain and the light chain constant domain (CL). The second polypeptide chain includes the additional binding domain VH, the first heavy chain constant domain 1 (CH1), and the first Fc subunit. A third polypeptide chain, wherein the third polypeptide chain comprises the γδ TCR binding domain and the second Fc subunit; preferably, the first Fc subunit and the second Fc subunit comprise a pestle-and-mortar structure.

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

196.

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

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

32. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the following: V as described in any one of claims 1 to 5 H H. The polypeptide construct as described in claim 6 or 7, the antibody or its antigen-binding fragment as described in any one of claims 8 to 13, or the multispecific molecule as described in any one of claims 14 to 31.

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

34. A cell comprising a nucleic acid molecule as claimed in claim 32 or a carrier as claimed in claim 33.

35. A method for producing V as described in any one of claims 1 to 5 H H. A method comprising: a polypeptide construct as described in claim 6 or 7, an antibody or antigen-binding fragment thereof as described in any one of claims 8 to 13, or a multispecific molecule as described in any one of claims 14 to 31, said method comprising: Culture host cells containing the isolated nucleic acid molecule as described in claim 32 or the vector as described in claim 33, or the cells as described in claim 34, under conditions that allow protein expression, and recover the V from the culture of the cultured cells. H H, the polypeptide construct, the antibody or its antigen-binding fragment, or the multispecific molecule.

36. A pharmaceutical composition comprising V as claimed in any one of claims 1 to 5 H H. The polypeptide construct as described in claim 6 or 7, the antibody or its antigen-binding fragment as described in any one of claims 8 to 13, or the multispecific molecule as described in any one of claims 14 to 31, and a pharmaceutically acceptable carrier and / or excipient.

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

38. The pharmaceutical composition of claim 37, wherein the additional therapeutic agent is an anticancer agent, such as an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, or an anti-CTLA-4 antibody), or a cytotoxic agent (e.g., an alkylating agent, an antimitotic agent, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, or a 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 of treating a subject for a disease (e.g., cancer), the method comprising administering to a subject in need an effective amount of V as described in any one of claims 1 to 5. H H. The polypeptide construct as described in claim 6 or 7, the antibody or its antigen-binding fragment as described in any one of claims 8 to 13, or the multispecific molecule as described in any one of claims 14 to 31, or the pharmaceutical composition as described in any one of claims 36 to 38.

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

41. The method of claim 39, wherein the cancer is a solid tumor, such as ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell lung cancer or non-small cell lung cancer), 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 tumor (e.g., glioma, such as glioblastoma).

42. The method of claim 39, the method comprising administering the multispecific molecule for treating cancer, the cancer being characterized by a cancer antigen targeted by the multispecific molecule.

43. The method of any one of claims 39-42, wherein the cancer is characterized by cancer antigens selected from the group consisting of: CD33, CD19, CD20, CD22, CD30, CD70, CLL-1, BCMA, DLL-3, dentin 6, dentin 18.2, GPC3, GPC2, GPRC5D, CD229, FcRH5, GUCY2C, mesothelin, HER2, PSMA, or PSCA.

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

45. The method of any one of claims 39 to 44, wherein the V H H, the administration of the polypeptide construct, the antibody or its antigen-binding fragment, or the multispecific molecule, or the pharmaceutical composition in combination with another therapeutic agent or another therapy.

46. ​​The method of claim 45, wherein the additional therapeutic agent or additional therapy is an anticancer agent or 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, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

47. A method for activating γδ T cells, or increasing γδ T cell proliferation, or generating polyclonal γδ T cells, said method comprising reacting T cells with V as described in any one of claims 1 to 5 H H. Contact with a polypeptide construct as described in claim 6 or 7, an antibody or antigen-binding fragment thereof as described in any one of claims 8 to 13, a multispecific molecule as described in any one of claims 14 to 31, or a pharmaceutical composition as described in any one of claims 36 to 38.

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

Images