isolated GAMMA DELTA T cell population

Abstract

This invention relates to an in vitro method for regulating Vδ1T cells using anti-Vδ1 antibodies or fragments thereof.

Description

Technical Field

[0001] This invention relates to a population of γδT cells in contact with an anti-TCR delta variable 1 (anti-Vδ1) antibody. Background Technology

[0002] The growing interest in cancer T-cell immunotherapy focuses on the apparent ability of CD8+ and CD4+ alpha beta (αβ) T cell subsets to recognize cancer cells and mediate host protective functions, particularly when repressive effects are achieved through clinically mediated antagonism of inhibitory pathways imposed by PD-1, CTLA-4, and other receptors. However, αβ T cells are MHC-restricted and can contribute to graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) represent a subset of T cells that express a unique, defined γδ T cell receptor (TCR) on their surface. This TCR consists of a gamma (γ) and a delta (δ) chain, each undergoing chain rearrangement, but with a limited number of V genes compared to αβ T cells. The major TGRV gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV8, TRGV9, and TRGV11, as well as the non-functional genes TRGV10, TRGV11, TRGVA, and TRGVB. The most common TRDV gene segments encode Vδ1, Vδ2, and Vδ3, along with several V segments that are named with both Vδ and Vα (Adams et al., 296:30-40 (2015) Cell Immunol.). Human γδ T cells can be broadly classified according to their TCR chains, as certain γ and δ types are more prevalent, though not specific, in cells of one or more tissue types. For example, most blood-dwelling γδT cells express Vδ2TCR, typically Vγ9Vδ2, which is less common in tissue-dwelling γδT cells, such as those in the skin, which more often use Vδ1TCR paired with the gamma chain, such as Vγ4 in the gut.

[0004] To utilize γδT cells for immunotherapy, methods are needed for either in situ cell expansion or cell harvesting, ex vivo expansion, and subsequent re-infusion. The latter approach, using the addition of exogenous cytokines, has been previously described, for example, see WO2017 / 072367 and WO2018 / 212808. Methods for expanding a patient's own γδT cells have been described using pharmacologically modified forms of hydroxymethylbut-2-enyl pyrophosphate (HMBPP) or clinically approved aminobisphosphonates. These methods have been used to treat 250 cancer patients and appear to be safe, although complete remission is rare. Nevertheless, there remains a need for activators that have been shown to expand large numbers of γδT cells. Invention Overview

[0006] According to a first aspect of the invention, an in vitro method for regulating Vδ1T cells is provided, the method comprising administering to a cell population containing Vδ1T cells a human anti-TCR delta variable 1 (anti-Vδ1) antibody or a fragment thereof that binds to an epitope of a variable delta 1 (Vδ1) chain of the γδT cell receptor (TCR), said epitope comprising one or more amino acid residues in a region of the following amino acid domain:

[0007] (i) SEQ ID NO: 1, 3-20; and / or

[0008] (ii) 37-77 of SEQ ID NO: 1.

[0009] According to another aspect of the present invention, a method for in vitro regulation of Vδ1T cells is provided, the method comprising administering an anti-Vδ1 antibody or a fragment thereof to a cell population containing Vδ1T cells, the anti-Vδ1 antibody comprising one or more of the following:

[0010] CDR3, which contains a sequence having at least 80% sequence identity with any one of SEQ ID NO: 2-25;

[0011] CDR2, which comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 26-37 and SEQUENCES: A1-A12 (Table 2); and / or

[0012] CDR1 contains a sequence that has at least 80% sequence identity with any of SEQ ID NO: 38-61.

[0013] According to another aspect of the invention, a population of Vδ1T cells obtained by an in vitro method as defined herein is provided.

[0014] According to another aspect of the invention, a composition comprising a Vδ1T cell population as defined herein is provided.

[0015] According to another aspect of the invention, a pharmaceutical composition comprising a Vδ1T cell population as defined herein is provided.

[0016] According to another aspect of the invention, a method is provided for treating cancer, infectious disease, or inflammatory disease in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of a Vδ1T cell population or pharmaceutical composition as defined herein. Attached Figure Description

[0017] Figure 1 ELISA detection of directly coated antigens was performed using anti-Vδ1Ab (REA173, Miltenyi Biotec). Detection was only observed in antigens containing the Vδ1 domain. The leucine zipper (LZ) format appeared to be more efficient than the Fc format, consistent with cell-based flow cytometry competitive assays (data not shown).

[0018] Figure 2 : Polyclonal phage DELFIA data used for DV1 selection. A) Heterodimer selection: Heterodimer LZ TCR format in rounds 1 and 2, with heterodimeric LZ TCR deselected in both rounds. B) Homodimeric selection: Round 1 using homodimeric Fc fusion TCR, with human IgG1 Fc deselected, followed by round 2 using heterodimeric LZ TCR, with heterodimeric LZ TCR deselected. Each plot contains two bars for each target to represent selections from different libraries.

[0019] Figure 3 IgG capture: Left) Sensing plot of anti-L1 IgG interaction with L1, Right) Steady-state fit (if any). All experiments were performed at room temperature on a MASS-2 instrument. Steady-state fit was performed based on Langmuir 1:1 binding.

[0020] Figure 4 Results of TCR downregulation assays of clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07 and 1251_P02_C05(A), or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09, 1251_P02_G10 and 1252_P01_C08(B).

[0021] Figure 5Results of T-cell degranulation assays of clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07 and 1251_P02_C05(A), or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09 and 1251_P02_G10(B).

[0022] Figure 6 Results of lethality assays (based on THP-1 flow cytometry) of clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07 and 1251_P02_C05(A), or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09 and 1251_P02_G10(B).

[0023] Figure 7 Epitope mapping data for SEQ ID NO:1. Illustration of the epitope binding site of SEQ ID NO:1 for SEQ ID NO:1.

[0024] Figure 8 Epitope mapping data for SEQ ID NO:1 1252_P01_C08. Illustration of the epitope binding site of SEQ ID NO:1 1245_P01_C08.

[0025] Figure 9 Epitope mapping data for SEQ ID NO:1. Illustration of the epitope binding sites of SEQ ID NO:1 for 1245_P02_G04.

[0026] Figure 10 Epitope mapping data for SEQ ID NO:1. Illustration of the epitope binding sites of SEQ ID NO:1 for 1251_P02_C05.

[0027] Figure 11 Epitope mapping data for 1141_P01_E01. Illustration of the epitope binding sites of 1141_P01_E01 on SEQ ID NO:1.

[0028] Figure 12 Total cell counts during Experiment 1 of Example 10. Samples were cultured with different concentrations of anti-Vδ1 antibody as described herein and compared with samples cultured with comparative antibody or control. The figure shows total cell counts on (A) day 7, (B) day 14, and (C) day 18.

[0029] Figure 13 Vδ1T cell analysis during Experiment 1 of Example 10. The figure shows (A) the percentage of Vδ1T cells, (B) the Vδ1T cell count, and (C) the fold change of Vδ1 in the sample on day 18.

[0030] Figure 14 Total cell counts during Experiment 2 of Example 10. Samples were cultured with different concentrations of anti-Vδ1 antibody as described herein and compared with samples cultured with comparative antibody or control. The figure shows the total cell counts on (A) day 7, (B) day 11, (C) day 14, and (D) day 17.

[0031] Figure 15 Vδ1T cell analysis during Experiment 2 of Example 10. The figure shows (A) the percentage of Vδ1T cells, (B) the Vδ1T cell count, and (C) the fold change of Vδ1 in the sample on day 17.

[0032] Figure 16 Cell composition analysis. Cell types present in the samples (including non-Vδ1 cells) were measured on day 17 of Experiment 2. Cells were harvested and the surface expression of Vδ1, Vδ2, and αβTCR was analyzed by flow cytometry. Percentage values ​​are also provided in Table 6.

[0033] Figure 17 Results of SYTOX-flow cytometry killing assay. Cell function was tested using the SYTOX-flow cytometry killing assay. The results are shown as follows: (A) Day 14 of Experiment 1, using cells with an effector-to-target (E:T) ratio of 10:1, and (B) Day 17 of Experiment 2 (after freeze-thaw), using cells with E:T ratios of 1:1 and 10:1.

[0034] Figure 18 Total cell count after freeze-thaw. The figure shows the total cell count of cultures that were in contact with antibodies B07, C08, E07, G04, or OKT-3 before freezing, 7 days after freeze-thaw.

[0035] Figure 19 Monitor cell expansion. Monitor the total cell count of cells cultured after freeze-thaw cycles until day 42.

[0036] Figure 20 Anti-Vδ1 antibodies confer regulation and proliferation of tumor-infiltrating lymphocytes (TILs) in human tumors. A study of + / - antibodies in renal cell carcinoma (RCC). A) Increased fold in TIL Vδ1+ cells. B) Total number of TIL Vδ1+ cells. C) Example of gating strategies. D) Comparative cell surface phenotypic profiles of TIL Vδ1+ cells. E) Analysis of TIL Vδ1 negative gating fractions. Detailed Implementation

[0037] definition

[0038] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, the following terms have the meanings assigned to them as follows.

[0039] Gamma delta (γδ) T cells represent a small subset of T cells that express a unique, defined T cell receptor (TCR) on their cell surface. This TCR consists of a gamma (γ) chain and a delta (δ) chain. Each chain contains a variable (V) region, a constant (C) region, a transmembrane region, and a cytoplasmic tail region. The V region contains the antigen-binding site. Human γδ T cells have two main subtypes: one predominant in peripheral blood and the other in non-hematopoietic tissues. These subtypes can be defined by the type of δ and / or γ present on the cell. For example, γδ T cells predominant in peripheral blood primarily express the delta variable 2 chain (Vδ2). γδ T cells predominant in non-hematopoietic tissues (i.e., tissue-resident γδ T cells) primarily express the delta variable 1 (Vδ1) chain. The term "Vδ1 T cell" refers to a γδ T cell possessing the Vδ1 chain, i.e., Vδ1. + T cells.

[0040] The term "δ variable 1" can also refer to Vδ1 or Vd1, and the nucleotide encoding the TCR chain containing this region can be called "TRDV1". Antibodies or fragments thereof that interact with the Vδ1 chain of γδTCR are effective antibodies or fragments thereof that bind to Vδ1, and can be called "anti-TCRδ variable 1 antibody or fragment thereof" or "anti-Vδ1 antibody or fragment thereof".

[0041] This document also refers to other delta chains, such as “delta variable 2” chains. These chains can be referred to in a similar manner. For example, a delta variable 2 chain can be called Vδ2, and the nucleotide encoding the TCR chain containing this region can be called “TRDV2”. In a preferred embodiment, the antibody or fragment thereof interacting with the Vδ1 chain of the γδTCR does not interact with other delta chains such as Vδ2.

[0042] This article also refers to "gamma variable chains". These chains may be called γ-chains or Vγ, and the nucleotides encoding the TCR chain containing this region may be called TRGV. For example, TRGV4 refers to the Vγ4 chain. In a preferred embodiment, an antibody or fragment thereof that interacts with the Vδ1 chain of the γδTCR does not interact with gamma chains such as Vγ4.

[0043] The term "antibody" includes any antibody protein construct containing at least one antibody variable domain, which contains at least one antigen-binding site (ABS). Antibodies include, but are not limited to, immunoglobulins of IgA, IgG, IgE, IgD, and IgM types (and their subtypes). The overall structure of immunoglobulin G (IgG) antibodies is well established and highly conserved in mammals, consisting of two identical heavy (H) chains and two identical light (L) chain polypeptides (Padlan (1994) Mol. Immunol. 31:169-217).

[0044] Conventional antibodies or immunoglobulins (Ig) are proteins consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into constant regions and variable domains. The heavy (H) chain variable domain is abbreviated as VH in this paper, and the light (L) chain variable domain is abbreviated as VL in this paper. These domains, their associated domains, and domains derived from them are referred to in this paper as immunoglobulin chain variable domains. The VH and VL domains (also known as the VH and VL regions) can be further subdivided into regions called complementarity-determining regions ("CDRs"), in which more conserved regions called "frame regions" ("FRs"). The frame regions and complementarity-determining regions have been precisely defined (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, (1991) NIHP Publication Number 91-3242). For CDR sequences, there are also some alternative numbering rules, such as those listed in Chothia et al. (1989) Nature 342:877-883. In conventional antibodies, each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers of two immunoglobulin heavy chains and two immunoglobulin light chains are formed by interchain linkages of the immunoglobulin heavy and light chains, for example, disulfide bonds, with the heavy chains linked in a similar manner. The constant region of the heavy chain contains three domains, CH1, CH2, and CH3. The constant region of the light chain consists of one domain, CL. The variable domains of the heavy and light chains are binding domains that interact with the antigen. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0045] As used herein, an antibody fragment (which may also be referred to as an "antibody fragment," "immunoglobulin fragment," "antigen-binding fragment," or "antigen-binding polypeptide") refers to a portion of an antibody (or a construct containing said portion) that specifically binds to the delta variable 1 (Vδ1) chain of the γδT cell receptor (e.g., a molecule in which one or more immunoglobulin chains are not full-length but specifically bind to the target). Examples of binding fragments included in the term antibody fragment include:

[0046] (i) Fab fragment (a monovalent fragment composed of VL, VH, CL and CH1 domains);

[0047] (ii) F(ab')2 fragment (a divalent fragment consisting of two Fab fragments connected by disulfide bridges in the hinge region);

[0048] (iii) Fd fragment (composed of VH and CH1 domains);

[0049] (iv) Fv fragment (composed of the VL and VH domains of the antibody single arm);

[0050] (v) Single-chain variable fragments, scFv (composed of VL and VH domains linked by synthetic linkers using recombination methods, enabling them to be prepared as a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule);

[0051] (vi)VH (variable domains of the immunoglobulin chain composed of VH domains);

[0052] (vii)VL (variable domains of the immunoglobulin chain composed of VL domains);

[0053] (viii) Domain antibodies (dAbs, composed of VH or VL domains);

[0054] (ix) Minibody (consisting of a pair of scFv fragments linked by a CH3 domain); and

[0055] (x) Diabody (consisting of a non-covalent dimer of scFv fragments, which are composed of a VH domain from one antibody linked to a VL domain from another antibody via a small peptide linker).

[0056] "Human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human subject administered the human antibody does not develop a cross-species antibody response to the primary amino acids contained in the antibody (e.g., a HAMA reaction – human anti-mouse antibody). The human antibody may, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in the CDR, particularly in CDR3 (e.g., mutations introduced by random or site-specific mutagenesis or by somatic mutation). However, this term is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence. Human antibodies prepared, expressed, produced, or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combined human antibody library, antibodies isolated from transgenic animals (e.g., mice) containing human immunoglobulin genes, or antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences, may also be referred to as "recombinant human antibodies."

[0057] When at least one amino acid residue in the framework region of a non-human immunoglobulin variable domain is replaced by a corresponding residue from a human variable domain, it is called "humanization." Humanization of the variable domain can reduce immunogenicity in humans.

[0058] "Specificity" refers to the number of different types of antigens or antigenic determinants that a particular antibody or fragment can bind to. Antibody specificity is the ability of an antibody to recognize a specific antigen as a unique molecular entity and distinguish it from other antigens. The term "specifically binding" an antibody to an antigen or epitope is well-known in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with a stronger affinity for a specific target antigen or epitope compared to alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds to it with greater affinity, avidity, easier, and / or longer duration of action compared to other substances.

[0059] "Affinity" is expressed as the equilibrium constant for the dissociation of the antigen and the antigen-binding polypeptide (KD). It is a measure of the binding strength between the antigenic determinant and the antigen-binding site on the antibody (or its fragments): the smaller the KD value, the stronger the binding strength between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can also be expressed as the affinity constant (KA), i.e., 1 / KD. Affinity can be determined by known methods, depending on the specific target antigen.

[0060] Any less than 10 -6KD values ​​are considered to indicate binding. Specific binding of an antibody or fragment thereof to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (e.g., using fluorescence assays) and various variations thereof known in the art.

[0061] Affinity is a measure of the strength of binding between an antibody or fragment thereof and its associated antigen. Affinity is related to the affinity between the antigenic determinant and its antigen-binding site on the antibody, as well as the number of associated binding sites present on the antibody.

[0062] “Human tissue Vδ1+ cells,” “hematopoietic and blood Vδ1+ cells,” and “tumor-infiltrating lymphocytes (TIL) Vδ1+ cells” are defined as Vδ1+ cells contained in or derived from human tissues or the hematopoietic system or human tumors, respectively. All of the aforementioned cell types can be identified by their (i) location or place of origin and (ii) their Vδ1+ TCR expression.

[0063] "Regulatory antibodies" are antibodies that confer measurable changes when they come into contact with or bind to cells expressing an antibody target. These changes include, but are not limited to, measurable changes in cell cycle, and / or cell number, and / or cell viability, and / or one or more cell surface markers, and / or the secretion and / or function (cytotoxicity to target or diseased cells) of one or more secretory molecules (e.g., cytokines, chemokines, leukotrienes, etc.).

[0064] A method of “regulating” cells or populations thereof refers to a method in which at least one measurable change is triggered in the one or more cells or their secretions, resulting in one or more “regulated cells”.

[0065] "Immune response" refers to a measurable change (including but not limited to cell-mediated response, humoral response, cytokine response, and chemokine response) in at least one cell, cell type, endocrine pathway, or exocrine pathway of the immune system when a regulatory antibody is added.

[0066] "Immune cells" are defined as cells of the immune system, including but not limited to CD34+ cells, B cells, CD45+ (lymphocyte common antigen) cells, Alpha-Beta T cells, cytotoxic T cells, helper T cells, plasma cells, neutrophils, monocytes, macrophages, erythrocytes, platelets, dendritic cells, phagocytes, granulocytes, innate lymphocytes, natural killer (NK) cells, and Gamma Delta T cells. Typically, immune cells are classified using combined cell surface molecular analyses (e.g., flow cytometry) to identify, group, or cluster them into subsets. These subsets can then be further subdivided using additional analyses. For example, CD45+ lymphocytes can be further subdivided into vδ-positive and vδ-negative subsets.

[0067] A “model system” is a biological model or biological representation designed to help understand how drugs, such as antibodies or fragments thereof, function as drugs to improve signs or symptoms of disease. Such models typically involve the use of diseased cells, healthy cells, effector cells, and tissues in vitro, ex vivo, and in vivo, and the performance of the drug is studied and compared within the model.

[0068] "Disease-caused cells" exhibit phenotypes associated with disease progression, such as cancer, infection (e.g., viral infection), or inflammatory disorders or diseases. For example, disease-caused cells can be tumor cells, autoimmune tissue cells, or virus-infected cells. Therefore, disease-caused cells can be defined as tumor cells, virus-infected cells, or inflammatory cells.

[0069] "Healthy cells" refer to normal cells that are not diseased. They may also be referred to as "normal" or "disease-free" cells. Disease-free cells include non-cancerous, uninfected, or non-inflammatory cells. These cells are often used in conjunction with relevant diseased cells to determine the disease-specificity of a drug and / or to better understand the therapeutic index of the drug.

[0070] "Disease-cell specificity" is a measure of how effectively effector cells or populations (e.g., Vδ1+ cell populations) can distinguish and kill diseased cells (e.g., cancer cells) while preserving non-diseased or healthy cells. This potential can be measured in a model system and can include comparing the tendency of effector cells or effector cell populations to selectively kill or lyse diseased cells with the potential of said effector cells to kill or lyse non-diseased or healthy cells. The disease-cell specificity can inform the potential therapeutic index of a drug.

[0071] "Enhanced disease-cell specificity" describes the phenotype of effector cells, such as Vδ1+ cells or populations thereof, which have been modulated to further increase their ability to specifically kill disease-cells. This enhancement can be measured in a variety of ways, including fold changes or percentage increases in disease-cell killing specificity or selectivity.

[0072] Suitablely, the antibodies or fragments thereof (i.e., polypeptides) of the present invention are isolated. An “isolated” polypeptide is a polypeptide removed from its original environment. The term “isolated” can be used to refer to an antibody substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds Vδ1 substantially free of antibodies that specifically bind antigens other than Vδ1). The term “isolated” can also be used to refer to formulations, when formulated as the active ingredient of a pharmaceutical composition, wherein the isolated antibody is sufficiently pure for therapeutic administration, or at least 70-80% (w / w) pure, more preferably at least 80-90% (w / w) pure, even more preferably 90-95% pure; and most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure.

[0073] Suitablely, the polynucleotides used in this invention are isolated. An "isolated" polynucleotide is a polynucleotide removed from its original environment. For example, a naturally occurring polynucleotide is considered isolated if it is separated from some or all of the coexisting substances in a natural system. Similarly, a polynucleotide is considered isolated if it is cloned into a vector that is not part of its natural environment or if it is contained in cDNA.

[0074] Antibodies or fragments thereof can be “functionally active variants,” which also include naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As known in the art, allelic variants are alternative forms of (poly)peptides characterized by substitutions, deletions, or additions of one or more amino acids that substantially do not alter the biological function of the polypeptide. As a non-limiting example, the functionally active variant may remain functional when the framework containing the CDR is modified, when the CDR itself is modified, when the CDR is grafted onto an alternative framework, or when an N- or C-terminal extension is incorporated. Furthermore, the binding domain containing the CDR may pair with different mating chains, such as those shared with another antibody. The binding domain may remain functional after sharing with a so-called “shared” light chain or “shared” heavy chain. Furthermore, the binding domain may function upon polymerization. Additionally, “antibodies or fragments thereof” may also contain functional variants in which the VH or VL or constant domains have been modified away or toward a different canonical sequence (e.g., listed on IMGT.org) and remain functional.

[0075] To compare two closely related polypeptide sequences, you can use NCBI BLAST v2.0 with the standard settings for polypeptide sequences (BLASTP) to calculate the "sequence identity %" between the first and second polypeptide sequences. To compare two closely related polynucleotide sequences, you can use NCBI BLAST v2.0 with the standard settings for nucleotide sequences (BLASTN) to calculate the "sequence identity %" between the first and second nucleotide sequences.

[0076] A polypeptide or polynucleotide sequence is said to be identical or "identical" to other polypeptides or polynucleotide sequences if it shares 100% sequence identity across its entire length. Residues in the sequence are numbered from left to right: polypeptides are numbered from the N-terminus to the C-terminus; polynucleotides are numbered from the 5' to the 3' end.

[0077] A “difference” between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at a position in a second sequence compared to a first sequence. Two polypeptide sequences can contain one, two, or more such amino acid differences. An insertion, deletion, or substitution in a second sequence that is identical to the first sequence (100% sequence identity) results in a reduced percentage of sequence identity. For example, if the identical sequence is 9 amino acid residues long, a substitution in the second sequence results in 88.9% sequence identity. If the first and second polypeptide sequences are both 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share more than 66% identity (the first and second polypeptide sequences share 66.7% identity).

[0078] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence can be determined to produce the second sequence. "Addition" is the addition of one amino acid residue to the sequence of the first polypeptide (including addition to either end of the first polypeptide). "Substitution" is the substitution of one amino acid residue in the sequence of the first polypeptide with a different amino acid residue. The substitution can be conserved or non-conserved. "Deletion" is the removal of one amino acid residue from the sequence of the first polypeptide (including deletion at either end of the first polypeptide).

[0079] "Conservative" amino acid substitution is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a similar chemical structure and which is expected to have little effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitution is appropriately defined as substitution in which one amino acid from the following group is replaced by another amino acid residue from the same group:

[0080]

[0081] Suitablely, the hydrophobic amino acid residue is a nonpolar amino acid. More preferably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W, or C.

[0082] As used herein, peptide sequence numbering and the definitions of CDR and FR are based on the Kabat system (Kabat et al., 1991, incorporated herein by reference in their entirety). The “corresponding” amino acid residues between the first and second peptide sequences are amino acid residues in the first sequence that share the same position as amino acid residues in the second sequence, although the amino acid residues in the second sequence may differ from those in the first sequence. Suitablely, if the frame and CDR are of the same length according to the Kabat definition, the corresponding residues will share the same numbers (and letters). Alignment can be performed manually or using known computer algorithms for sequence alignment, such as NCBI BLAST v2.0 (BLASTP or BLASTN), using standard settings.

[0083] The term "epitope" as used in this article refers to the target region that is specifically bound by an antibody or its fragments. An epitope can also be called an "antigenic determinant." When an antibody and another antibody both recognize the same or spatially overlapping epitopes, then the antibody binds to "substantially the same epitope" with the other antibody. A common method for determining whether two antibodies bind to the same or overlapping epitopes is a competitive assay, which uses labeled antigens or antibodies and can be configured in various different formats (e.g., using radiolabeled or enzyme-labeled well plates, or flow cytometry on cells expressing the antigen).

[0084] Epitopes found on protein targets can be defined as "linear epitopes" or "conformational epitopes." Linear epitopes are formed from a continuous amino acid sequence in a protein antigen. Conformational epitopes are formed from amino acids that are not continuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0085] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which other DNA segments can be linked. Another type of vector is a viral vector, in which other DNA segments can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian and yeast vectors). After introduction into a host cell, other vectors (e.g., non-free-living mammalian vectors) can integrate into the host cell's genome and replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, this invention aims to include other forms of expression vectors with equivalent functionality, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as well as phage and phage particle systems. As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell in which a recombinant expression vector has been introduced. Such terms are intended to refer not only to a specific test cell but also to the progeny of such cells, for example, when said progeny are used to prepare a cell line or cell bank which is subsequently optionally stored, provided, sold, transferred, or used to manufacture antibodies or fragments thereof as described herein.

[0086] The terms "subject," "patient," or "individual" refer to a subject to treatment, particularly a mammalian subject. Mammal subjects include humans, non-human primates, farm animals (e.g., dairy cows), athletic animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the subject is a human. In alternative embodiments, the subject is a non-human mammal, such as a mouse.

[0087] The term "sufficient amount" refers to an amount sufficient to produce the desired effect. The term "therapeutic effective amount" is an amount that effectively improves the symptoms of a disease or ailment. Therapeutic effective amount can also be "preventive effective amount," since prevention can be considered treatment.

[0088] As used herein, the term “about” includes up to 10% and including 10% higher and down to 10% and including 10% higher than the specified value, appropriately up to 5% and including 5% higher and down to 5% and including 5% lower than the specified value, particularly the specified value. The term “between” includes values ​​that specify boundaries.

[0089] The disease or disorder is “improved” if the severity of the signs or symptoms of the disease or disorder, the frequency with which the subject experiences such signs or symptoms, or both are reduced.

[0090] As used herein, “treating a disease or ailment” means reducing the frequency and / or severity of at least one sign or symptom of a disease or ailment experienced by the subject.

[0091] As used in this article, “cancer” refers to the abnormal growth or division of cells. Typically, cancer cells grow and / or live beyond the normal cells and tissues around them, and are out of sync with them. Cancer can be benign, precancerous, or malignant. Cancer occurs in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile ducts, gallbladder, pancreas), respiratory system (e.g., larynx, lungs, bronchi), bones, joints, skin (e.g., basal cells, squamous cells, meningioma), breast, reproductive system (e.g., uterus, ovaries, prostate, testes), urinary system (e.g., bladder, kidneys, ureters), eyes, nervous system (e.g., brain), endocrine system (e.g., thyroid), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia).

[0092] Methods for regulating γδT cells

[0093] According to a first aspect of the invention, an in vitro method for modulating delta variable 1 chain (Vδ1) T cells is provided, comprising administering an anti-Vδ1 antibody or a fragment thereof, as defined herein, to a cell population containing Vδ1 T cells. It should be understood that "administering" the antibody or a fragment thereof includes "contacting" the Vδ1 T cells.

[0094] Regulation of Vδ1T cells may include:

[0095] -Vδ1T cell expansion, for example, by selectively increasing the number of Vδ1T cells or promoting the survival of Vδ1T cells;

[0096] - Stimulate Vδ1T cells, for example, by increasing the efficacy of Vδ1T cells, i.e., increasing the killing of target cells;

[0097] - Prevent Vδ1T cell depletion, for example, by increasing Vδ1T cell persistence;

[0098] Degranulation of -Vδ1T cells;

[0099] Immunosuppression of Vδ1T cells, for example, by downregulating Vδ1TCR expression on the cell surface, i.e., by inducing Vδ1TCR internalization or reducing Vδ1TCR protein expression, or by blocking Vδ1TCR binding.

[0100] - Reduce the number of Vδ1T cells, for example, by inhibiting Vδ1T cell proliferation or by inducing Vδ1T cell death (i.e., killing Vδ1T cells).

[0101] Such regulation of Vδ1T cells may include, for example, Vδ1T cell activation or Vδ1T cell suppression. In one embodiment, Vδ1T cells are activated by administration of an anti-Vδ1 antibody or a fragment thereof as defined herein. In an alternative embodiment, Vδ1T cells are suppressed by administration of an anti-Vδ1 antibody or a fragment thereof as defined herein. In an alternative embodiment, Vδ1T cells are not suppressed after administration of an anti-Vδ1 antibody or a fragment thereof as defined herein to a patient.

[0102] In one embodiment, the regulation of Vδ1T cells includes administering a variable antibody against TCRdelta 1 or a fragment thereof to Vδ1T cells in a culture (i.e., in vitro or ex vivo). Vδ1T cells may be present in mixed cell populations, for example, in cell populations containing other lymphocyte types (e.g., αβT cells or NK cells).

[0103] In one embodiment, a cell population containing Vδ1 T cells (i.e., from a sample as described herein) is isolated prior to administration of an anti-Vδ1 antibody or a fragment thereof. In another embodiment, the T cell population is enriched prior to administration of an anti-Vδ1 antibody or a fragment thereof. In yet another embodiment, the γδT cell population is enriched prior to administration of an anti-Vδ1 antibody or a fragment thereof.

[0104] This method can also be performed on a cell population containing a purified fraction of γδT cells. In such an embodiment, cell types other than γδT cells present in the sample, such as αβT cells and / or NK cells, are depleted from the cell population before administration of the anti-Vδ1 antibody or a fragment thereof. Alternatively or additionally, cell types that may contain Vδ1, such as T cells and / or γδ cells, can be enriched in the cell population before administration of the anti-Vδ1 antibody or a fragment thereof. For example, T cells of the sample can be enriched, or γδT cells enriched, or αβT cells or non-γδT cells depleted, before culturing the sample. In one embodiment, αβT cells of the sample are depleted first, followed by enrichment of CD3+ cells. Enrichment or depletion can be achieved using techniques known in the art, such as using magnetic beads coated with an antibody that binds to molecules on the cell surface associated with the phenotype to be enriched / removed.

[0105] The presence of cell types other than lymphocytes in the cell culture may inhibit the proliferation of Vδ1 cells. Such cells, such as stromal cells, epithelial cells, tumor cells, and / or feeder cells, can be removed prior to culture. Therefore, in one embodiment, the cell population does not come into direct contact with stromal cells during culture. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematologic tumor cells. Preferably, lymphocytes do not come into direct contact with fibroblasts during culture. In one embodiment, the cell population does not come into direct contact with epithelial cells during culture. In one embodiment, the cell population does not come into direct contact with tumor cells and / or feeder cells during culture.

[0106] In one embodiment, the method includes culturing Vδ1T cells without significant stromal cell contact. In another embodiment, the method includes culturing Vδ1T cells without substantial fibroblast contact.

[0107] In one embodiment, the method includes culturing Vδ1T cells in a substantially serum-free medium (e.g., a serum-free medium or a medium containing a serum substitute (SR)). Therefore, in one embodiment, the method includes culturing in a serum-free medium. Such a serum-free medium may also include a serum substitute medium, wherein the serum substitute is based on chemically determined components to avoid the use of human or animal-derived serum. In an alternative embodiment, the method includes culturing in a serum-containing medium (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the medium contains a serum substitute. In one embodiment, the medium does not contain animal-derived products.

[0108] It should be understood that samples cultured in serum-free media have the advantages of avoiding filtration, precipitation, contamination, and serum supply issues. Furthermore, animal-derived products are unsuitable for clinical-grade preparation of human therapeutic agents. Using serum-free media for cells (especially Vδ1T cells) significantly increases the number of cells obtained from the sample compared to using media containing AB serum.

[0109] In one embodiment, the anti-Vδ1 antibody or a fragment thereof is in a soluble or fixed form. For example, the antibody or a fragment thereof may be applied to Vδ1T cells in a soluble form. Alternatively, the antibody or a fragment thereof may be applied to Vδ1T cells when it is bound to or covalently attached to a surface, such as beads or a plate (i.e., in a fixed form). In one embodiment, the antibody is fixed to a surface, such as Fc-coated pores. Alternatively, the antibody or a fragment thereof binds to a cell surface (e.g., fixed to the surface of antigen-presenting cells (APCs)). In another embodiment, the antibody is not fixed to a surface when the cell population comes into contact with the antibody.

[0110] Cell populations contacted with anti-Vδ1 antibodies or fragments thereof can be obtained from a variety of sample types (separation methods are further described below). In one embodiment, the sample is a non-hematopoietic tissue sample. "Non-hematopoietic tissue" or "non-hematopoietic tissue sample" as used herein includes skin (e.g., human skin) and intestines (e.g., human intestines). Non-hematopoietic tissue is tissue other than blood, bone marrow, lymphoid tissue, lymph node tissue, or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is skin (e.g., human skin). In some embodiments, the cell population (e.g., γδT cells) is not obtained from a specific type of biological fluid sample (e.g., blood or synovial fluid). In some embodiments, the cell population (e.g., γδT cells) is obtained from skin (e.g., human skin), which can be obtained by methods known in the art. For example, a cell population can be obtained from a non-hematopoietic tissue sample by culturing the sample on a synthetic scaffold configured to facilitate cell egress from the non-hematopoietic tissue sample. Alternatively, these methods can be applied to cell populations (e.g., γδT cells) obtained from the gastrointestinal tract (e.g., colon or intestine), breast, lung, prostate, liver, spleen, pancreas, uterus, vagina, and other skin, mucous membranes, or serous membranes.

[0111] In an alternative embodiment, the sample is a hematopoietic sample or a fraction thereof (i.e., the cell population is obtained from a hematopoietic sample or a fraction thereof). "Hematopoietic sample" or "hematopoietic tissue sample" as referred to herein includes blood (e.g., peripheral blood or cord blood), bone marrow, lymphoid tissue, lymph node tissue, thymic tissue, and fractions or enriched portions thereof. The sample is preferably blood, including peripheral blood or cord blood or fractions thereof, including erythrocyte sedimentation rate (ESR) amber layer cells, products of leukocyte removal, peripheral blood mononuclear cells (PBMCs), and low-density mononuclear cells (LDMCs). In some embodiments, the sample is human blood or a fraction thereof. Cells can be obtained from blood samples using techniques known in the art, such as density gradient centrifugation. For example, whole blood can be layered on an equal volume of FICOLL-HYPAQUE and then centrifuged at 400 x g for 15–30 minutes at room temperature. The interfacial material will contain low-density mononuclear cells, which can be collected, washed in culture medium, and centrifuged at 200 x g for 10 minutes at room temperature.

[0112] Cell populations can be obtained from cancerous tissue samples (e.g., breast or prostate tumors); i.e., γδT cells can also reside in cancerous tissue samples. In some embodiments, the cell populations may be derived from human cancerous tissue samples (e.g., solid tumor tissue). In other embodiments, the cell populations may be derived from samples other than human cancerous tissue (e.g., tissue without a large number of tumor cells). For example, the cell populations may be derived from a skin region separated from nearby or adjacent cancerous tissue (e.g., healthy skin). Therefore, in some embodiments, the cell populations are not obtained from cancerous tissue (e.g., human cancerous tissue).

[0113] The cell population can be obtained from human or non-human animal tissues. Therefore, the method may additionally include the step of obtaining the cell population from human or non-human animal tissues. In one embodiment, the sample has been obtained from a human. In an alternative embodiment, the sample has been obtained from a non-human animal subject.

[0114] Expansion of γδT cells

[0115] In one embodiment, regulation includes the activation of Vδ1T cells, particularly the expansion of Vδ1T cells. Therefore, according to one aspect of the invention, an in vitro method for expanding Vδ1T cells is provided, comprising administering an anti-Vδ1 antibody or a fragment thereof, as defined herein, to a cell population containing Vδ1T cells. Such expansion of Vδ1T cells can be achieved by selectively increasing the number of Vδ1T cells and / or by promoting Vδ1T cell survival. In one embodiment, the expansion of Vδ1T cells comprises administering an anti-TCR delta 1 variable antibody or a fragment thereof (i.e., in vitro or ex vivo) to Vδ1T cells in a culture. Vδ1T cells may be present in mixed cell populations, for example, in cell populations containing other lymphocyte types (e.g., αβT cells or NK cells).

[0116] Therefore, the present invention provides a method for generating an enriched population of γδT cells (e.g., Vδ1T cells) in vitro. An enriched population can be generated from an isolated mixed cell population (e.g., obtained from a sample taken from a patient / donor) by contacting the mixed cell population or a purified fraction thereof with an antibody or a fragment thereof. The antibody (or a fragment thereof) selectively amplifies Vδ1T cells by binding to an epitope specific to the Vδ1 chain of the γδTCR.

[0117] An expanded population of Vδ1T cells obtained according to the methods defined herein is also provided. According to this aspect of the invention, it should be understood that such an expanded population of Vδ1T cells can be obtained and / or expanded in vitro or in vitro. In one aspect, an expanded population of Vδ1 cells obtained according to the methods defined herein is provided, wherein the Vδ1 population is isolated and expanded in vitro or in vitro.

[0118] The antibodies or fragments thereof described herein can be used in methods for amplifying γδT cells (e.g., Vδ1T cells). These methods can be performed in vitro. If the amplification method is performed in vitro, the antibody (or fragments thereof) can be applied to isolated γδT cells (e.g., Vδ1T cells) obtained as described above. In some embodiments, γδT cells are amplified from a cell population that has already been isolated from a non-hematopoietic tissue sample. In an alternative embodiment, γδT cells are amplified from a cell population that has already been isolated from a hematopoietic tissue sample (e.g., a blood sample).

[0119] Expansion of γδT cells (e.g., Vδ1T cells) may include culturing samples in the presence of antibodies or fragments thereof as described herein, as well as cytokines. Cytokines may include interleukins, lymphokines, interferons, colony-stimulating factors, and chemokines. In one embodiment, the cytokines are selected from interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-33 (IL-33), insulin-like growth factor 1 (IGF-1), interleukin-1β (IL-1β), interferon-γ (IFN-γ), and stromal cell-derived factor-1 (SDF-1). It should be understood that references to the cytokines described herein may include any compound that has the same activity as the cytokines in terms of its ability to promote similar physiological effects in cultured Vδ1T cells, and include, but are not limited to, its mimics or any functional equivalents.

[0120] In one embodiment, the cytokine is a typical cytokine receptor gamma chain (γ-ray) C ) Cytokine family. In another implementation, γ C -Cytokines selected from: IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-21 or mixtures thereof.

[0121] The cytokines used (e.g., interleukins) can be of human or animal origin, preferably human origin. They can be wild-type proteins or any bioactive fragments or variants, i.e., capable of binding to their receptors. This binding can induce the activation of γδT cells under the conditions of the method according to the invention. More preferably, the cytokines can be in a soluble form, fused to or complexed with another molecule, such as a peptide, polypeptide, or bioactive protein. Preferably, recombinant human cytokines are used. More preferably, the interleukin concentration range can vary between 1-10000 U / ml, and even more preferably between 100-1000 U / ml.

[0122] In a further implementation, the cytokines are chemokines. It should also be understood that the chemokines will vary and be selected based on the sample used to obtain γδT cells.

[0123] In one embodiment, the method includes culturing a cell population in the presence of IL-2, IL-9, and / or IL-15. In a further embodiment, the method includes culturing a cell population (i.e., IL-2, IL-15, or a combination thereof) in the presence of IL-2 and / or IL-15. In an alternative embodiment, the method includes culturing a cell population (i.e., IL-9, IL-15, or a combination thereof) in the presence of IL-9 and / or IL-15. In one embodiment, the method includes culturing a cell population in the presence of IL-2, IL-9, and / or IL-15, and additional growth factors (e.g., IL-21). In other embodiments, the method includes culturing a cell population in a culture medium that does not contain growth factors other than IL-2 and / or IL-15. In an alternative embodiment, the method includes culturing a cell population in a culture medium that does not contain growth factors other than IL-9 and / or IL-15. In yet another embodiment, the method includes culturing a cell population in a culture medium consisting of a basal medium supplemented with IL-2, IL-9, and / or IL-15. In another embodiment, the method includes culturing a cell population in a culture medium consisting of a basal medium supplemented with IL-2 and / or IL-15.

[0124] In one implementation, the method includes culturing a cell population in the presence of IL-21.

[0125] In one embodiment, the method includes culturing a cell population in the presence of IL-4. IL-4 promotes the physiological functions of Vδ1T cells (as described in WO2016 / 198480) by reducing the expression levels of NKG2D and NCR, inhibiting cytotoxic function, and improving selective survival. Furthermore, it has been previously shown that the absence of IL-4 a few days after culture can alter the physiological characteristics of cells to a phenotype more suitable for use as an antitumor or antiviral therapy. Therefore, in one embodiment, the amplification method includes further culturing the sample in the absence of a growth factor with IL-4-like activity (e.g., IL-4). In one embodiment, the amplification method includes culturing the sample in the absence of IL-4.

[0126] In one embodiment, the cytokine is a growth factor with interleukin-15-like activity, i.e., any compound having the same activity as IL-15 in promoting similar physiological effects on cultured Vδ1T cells, including but not limited to IL-15 and IL-15 mimics, or any functional equivalent of IL-15, including IL-2 and IL-7. IL-15, IL-2, and IL-7 promote substantially the same physiological effects on cultured Vδ1T cells (as described in WO2016 / 198480), namely, inducing cell differentiation into a more cytotoxic phenotype. Furthermore, it has been previously shown that the absence of IL-2, IL-7, and IL-15 in the first few days of culture leads to starvation and apoptosis in contaminating cells (including TCRαβ+T and Vδ2+T cells) that are highly dependent on these cytokines for survival. Therefore, in one embodiment, the amplification method includes first culturing the sample in the absence of growth factors with IL-15-like activity.

[0127] Therefore, in one embodiment, the method includes culturing a cell population in a first culture medium containing IL-4 and then culturing the cell population in a second culture medium containing IL-15.

[0128] In one embodiment, the first culture medium is free of IL-15, IL-2, and / or IL-7. In one embodiment, the second culture medium is free of IL-4.

[0129] Therefore, in one embodiment, the amplification method includes:

[0130] (1) Cells in the sample were cultured in a first culture medium containing the antibody or fragment thereof as described herein and IL-4; in the absence of IL-15, IL-2 and IL-7; and

[0131] (2) The cells obtained in step (1) are cultured in a second medium containing the antibody or a fragment thereof as described herein and IL-15; IL-4 is not present.

[0132] As described herein, the culture medium may also contain other growth factors, including cytokines that can further enhance Vδ1T cell expansion. Examples of such cytokines include, but are not limited to: (i) IFN-γ and any growth factor with IFN-γ-like activity, (ii) IL-21 and any growth factor with IL-21-like activity, and (iii) IL-1β and any growth factor with IL-1β-like activity. Examples of other growth factors that may be added include co-stimulatory molecules, such as human anti-SLAM antibodies, any soluble ligand of CD27, or any soluble ligand of CD7. Any combination of these growth factors may be included in the culture medium.

[0133] In one embodiment, the first or second culture medium, or both culture media, contain one or more additional cytokines. The first and / or second culture media may contain second, third, and / or fourth cytokines. In a further embodiment, the additional cytokines are selected from IL-21, IFN-γ, and IL-1β.

[0134] In one embodiment, the method includes culturing a cell population in the presence of IL-15 and one of the following factors: IL-2, IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, IFN-γ, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1).

[0135] Amplification of γδT cells may include culturing samples in the presence of at least one additional T cell mitogen. The term "T cell mitogen" (also referred to as "γδTCR agonist") refers to any agent that can stimulate T cells via TCR signaling, including but not limited to phytohemagglutinins such as phytohemagglutinin (PHA) and concanavalin A (ConA) and lectins of non-plant origin. In one embodiment, the T cell mitogen is an anti-CD3 monoclonal antibody (mAb). Other mitogens include phorbol 12-myristate-13-acetic acid (TPA) and related compounds such as mezerein, or bacterial compounds (e.g., staphylococcal enterotoxin A (SEA) and streptococcal protein A). T cell mitogens may be soluble or fixed, and more than one T cell mitogen may be used in the amplification method.

[0136] As used herein, references to “amplified” or “amplified γδT cell population” include cell populations that are larger or contain a greater number of cells than unamplified populations. Such populations can be large, small, or mixed populations in which a portion of the population or a particular cell type is amplified. It should be understood that the term “amplification method” refers to the method that results in amplification or an amplified population. Therefore, an amplified or expanded population may be larger in number or contain a greater number of cells compared to a population that has not undergone an amplification step or has not undergone any amplification step. It should also be understood that any numbers specified herein indicating amplification (e.g., fold increase or amplification factor) describe an increase in the number or size of the cell population, or an increase in the number of cells, and indicate the amount of amplification.

[0137] In one embodiment, the method includes culturing the cell population for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 18 days, at least 21 days, at least 28 days, or longer, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days). In a further embodiment, the method includes culturing the cell population for at least 7 days, e.g., at least 11 days or at least 14 days.

[0138] In one embodiment, the method includes culturing a cell population for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 18 days, at least 21 days, at least 28 days or longer, e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days or about 21 days), said period of time being sufficient to effectively generate an expanded population of γδT cells.

[0139] In one embodiment, the cell population is cultured for 5 to 60 days, for example, at least 7 to 45 days, 7 to 21 days, or 7 to 18 days. If the method includes a separation culture period (e.g., 1 to 40 days, such as 14 to 21 days), in some embodiments, the separation and amplification steps may last for 21 to 39 days.

[0140] This method may include periodically adding anti-Vδ1 antibodies or fragments thereof and / or growth factors during culture. For example, anti-Vδ1 antibodies or fragments thereof and / or growth factors may be added every 2 to 5 days, more preferably every 3 to 4 days. In one embodiment, anti-Vδ1 antibodies or fragments thereof and / or growth factors are added after 7 days of culture, and then every 3 to 4 days thereafter.

[0141] The amplification method provides an expanded population of γδT cells in greater numbers than a reference population. In some embodiments, the expanded population of γδT cells (e.g., Vδ1T cells) is larger than the isolated population of γδT cells prior to the amplification step (e.g., at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 1000-fold, or more). In one embodiment, the expanded population of γδT cells (e.g., Vδ1T cells) is larger than a population cultured for the same length of time in the absence of an antibody or fragment thereof. In one implementation, the expanded population of γδT cells (e.g., Vδ1T cells) is larger than that of a population cultured for the same duration in the presence of TS8.2 or TS-1.

[0142] The expansion method provides an expanded Vδ1T cell population with a higher percentage of Vδ1T cells than a reference population. In some embodiments, the expanded Vδ1T cell population contains more than about 50% Vδ1T cells, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, or 95% Vδ1T cells. In a further embodiment, the expanded Vδ1T cell population contains more than about 85% Vδ1T cells, such as more than about 90% Vδ1T cells.

[0143] In some embodiments, the expanded γδT cell population (e.g., Vδ1T cells) contains less than about 10% αβT cells, for example, less than about 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% αβT cells. In another embodiment, the expanded Vδ1T cell population contains less than about 1% αβT cells. T cells with αβ receptors are highly reactive, therefore, in the context of this invention, a suitable cell population for administration to a patient contains only low levels of αβT cells. The antibodies described herein can be used to selectively expand Vδ1T cell populations, thereby reducing the need for large-scale purification methods to remove αβT cells after expansion.

[0144] In some embodiments, the expanded γδT cell population (e.g., Vδ1T cells) contains less than about 10% Vδ2T cells, such as less than about 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% Vδ2T cells. In another embodiment, the expanded Vδ1T cell population contains less than about 0.5% Vδ2T cells.

[0145] In some embodiments, the expanded γδT cell population (e.g., Vδ1T cells) contains less than about 10% natural killer (NK) cells (also known as CD56+CD3- cells), for example, less than about 5%, 4%, 3%, 2.5%, 2%, 1.5%, or 1% NK cells. In a further embodiment, the expanded Vδ1T cell population contains less than about 2% NK cells.

[0146] Increased or decreased expression of cell surface markers, including CD27, CD69, TIGIT, PD-1, and TIM-3, can be used, either additionally or alternatively, to characterize one or more expanded Vδ1T cell populations. In some embodiments, the expanded Vδ1T cell population expresses high levels of CD27 (CD27...). 高 For example, greater than about 70%, such as greater than about 80%, 85%, or 90%, of the expanded Vδ1T cell population expresses CD27 (i.e., CD27+). In some embodiments, the expanded Vδ1T cell population has a higher average expression of CD27 compared to, for example, the isolated Vδ1T cell population prior to expansion. In some embodiments, the expanded Vδ1T cell population expresses low levels of CD69, TIGIT, PD-1, and / or TIM-3. For example, less than about 40%, such as less than about 30%, of the expanded Vδ1T cell population expresses CD69, TIGIT, PD-1, and / or TIM-3. In some embodiments, the expanded Vδ1T cell population has a lower average expression of one or more markers selected from CD69, TIGIT, PD-1, and TIM-3 compared to the isolated Vδ1T cell population.

[0147] There are many basal media suitable for γδT cell proliferation, particularly media such as AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies), EXVIVO-10, EXVIVO-15, or EXVIVO-20 (Lonza), in which serum or plasma is present. The media may be supplemented with other culture factors as defined herein, such as serum, serum proteins, and selection reagents, such as antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1 X MEM non-essential amino acids (Life Technologies)), and 10 μl / L β-mercaptoethanol. In an alternative embodiment, AIM-V medium may be supplemented with a CTS immune serum substitute and amphotericin B. In some embodiments, the culture medium may be further supplemented with IL-2, IL-4, IL-9, and / or IL-15, as described herein. Conveniently, during isolation and / or expansion, cells are cultured in a suitable culture medium at 37°C in a humid environment containing 5% CO2.

[0148] Other factors may also be added to the expansion culture of γδT cells. In one embodiment, these factors are used to selectively promote the expansion of γδT cells. For example, expansion may additionally include the addition of exogenous cytokines, such as interleukins, to the expansion culture. Such expansion may include culturing γδT cells in the presence of IL-2 and IL-15. Alternatively, expansion may include culturing γδT cells in the presence of IL-9 and IL-15. It should be understood that any expansion step is performed for a period of time to effectively generate an expanded population of γδT cells.

[0149] Methods for expanding γδT cells may include population doubling times of less than 5 days (e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.4 days, less than 3.3 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

[0150] Methods for isolating γδT cells

[0151] As described herein, antibodies (or fragments thereof) can be applied to cultured γδT cells, i.e., γδT cells obtained from a sample. In one embodiment, a cell population is isolated from the sample prior to the administration of an anti-Vδ1 antibody or a fragment thereof. Therefore, a method for regulating (particularly expanding) Vδ1T cells is provided, the method comprising administering an anti-Vδ1 antibody or a fragment thereof, as defined herein, to a γδT cell population isolated from a sample (e.g., a cell population containing Vδ1T cells).

[0152] γδT cells, which predominate in non-hematopoietic tissues (i.e., tissue-resident cells), primarily comprise the delta variable 1 chain; therefore, the anti-Vδ1 antibodies described herein are particularly suitable for γδT cells isolated from non-hematopoietic tissues. Thus, in one embodiment, the sample is a non-hematopoietic tissue sample, such as skin. Alternatively, the method of the present invention can be used to amplify the Vδ1T cell population in samples that do not primarily comprise the Vδ1 chain, such as blood samples. Therefore, the method can be used to increase the number of Vδ1T cells in a sample.

[0153] The term "isolation" or "separation" of cells, particularly γδT cells, as used herein refers to a method or process for removing, separating, purifying, enriching, or otherwise extracting cells from a tissue or cell pool. It should be understood that such references include terms such as "separated," "removed," "purified," "enriched," etc. Isolation of γδT cells includes separating or separating cells from intact non-hematopoietic tissue samples or from stromal cells (e.g., fibroblasts or epithelial cells) of non-hematopoietic tissue. Such isolation may alternatively or additionally include separating or separating γδT cells from other hematopoietic cells (e.g., αβT cells or other lymphocytes). Isolation can continue for a defined period of time, for example, from the placement of tissue explants or biopsies in an isolation culture to the collection of cells from the culture, such as by centrifugation or other means to transfer the isolated cell population to an expansion culture, or for other purposes, or to remove the original tissue explants or biopsies from the culture. The isolation step can continue for at least about 3 days to about 45 days. In one embodiment, the isolation step continues for at least about 10 days to at least 28 days. In a further embodiment, the separation step lasts for at least 14 to at least 21 days. Therefore, the separation step can last for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 days, approximately 35, approximately 40, or approximately 45 days. It is understood that although cell proliferation may not be significant during this separation step, it is not necessarily absent. In fact, it is recognized by those skilled in the art that the separated cells may also begin to divide to generate numerous such cells within the separation container containing the sample.

[0154] Therefore, the terms "isolated γδT cells," "isolated γδT cell population," or "isolated γδT cell population" in this document will be understood to refer to γδ cells that have been isolated, separated, removed, purified, or enriched from a sample (e.g., a raw non-hematopoietic tissue sample) such that these cells are substantially not in contact with cells contained in a intact (non-hematopoietic tissue) sample. Similarly, the terms "isolated Vδ1T cells," "isolated Vδ1T cell population," "isolated Vδ1T cell population," "separated Vδ1T cells," "separated Vδ1T cell population," or "separated Vδ1T cell population" in this document will be understood to refer to Vδ1T cells that have been isolated, separated, removed, purified, or enriched from a sample (e.g., a raw non-hematopoietic tissue sample) such that these cells are substantially not in contact with cells contained in a intact (non-hematopoietic tissue) sample.

[0155] Cell populations can be obtained by any suitable method that allows the isolation of lymphocytes, particularly Vδ1T cells, from human or non-human animal samples (e.g., non-hematopoietic tissue samples). One such method is described in Clark et al. (2006) J. Invest. Dermatol. 126(5):1059-70, which describes a three-dimensional skin explant protocol for isolating lymphocytes from human skin. The explants can be attached to a synthetic scaffold to facilitate the release of lymphocytes from the explants onto the scaffold. A synthetic scaffold is a non-natural three-dimensional structure suitable for supporting cell growth. Synthetic scaffolds can be made of materials such as polymers (e.g., natural or synthetic polymers such as polyvinylpyrrolidone, polymethyl methacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (tantalum, titanium, platinum, and combinations of metals and alloys in the same element group as platinum, niobium, hafnium, and tungsten). According to methods known in the art, biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitamins, antibodies and fragments thereof), and cytokines (e.g., IL-2 or IL-15 and combinations thereof) can be coated onto the surface of a scaffold or encapsulated within a scaffold material to enhance cell adhesion, migration, survival, or proliferation. This and other methods can be used to isolate cell populations from many other non-hematopoietic tissue types, such as the intestine, prostate, and breast. Other examples of suitable isolation methods utilize a “crawl-out” approach, which may include culturing cell populations and / or samples in the presence of cytokines and / or chemokines sufficient to induce the isolation or separation of γδT cells (particularly Vδ1T cells). Isolation of γδT cells from a sample (e.g., a non-hematopoietic tissue sample) may include culturing the sample in the presence of IL-2 and IL-15.

[0156] Lymphocytes residing in non-hematopoietic tissues can be harvested and separated from stromal cells (e.g., dermal fibroblasts), for example, by vigorous pipetting. The lymphocyte harvest can be further washed through a 40 μm nylon mesh to retain any fibroblast aggregates that may have become loose in the process. Lymphocytes can also be separated using fluorescent or magnetically correlated cell sorting, for example, with CD45 antibodies.

[0157] Alternatively, isolating γδT cells from a sample (e.g., a hematopoietic tissue sample) may include the presence of T cell mitogens (e.g., γδTCR agonists) and cytokines (especially common cytokine receptors gamma chains (γδTCRs)). cSamples can be cultured in the presence of T cell mitogens and cytokines, as described in WO2012 / 156958. Alternatively, isolating γδT cells from a sample (e.g., a hematopoietic tissue sample) may include culturing the sample in the presence of T cell mitogens and cytokines, as described in WO2016 / 198480.

[0158] Isolation of γδT cells may include culturing a sample in the presence of at least one cytokine. For example, the method may include culturing the sample in the presence of at least one reagent, such as a chemokine. It will be further understood that the chemokine will be selected based on the isolated γδT cells. Furthermore, the chemokine will be modified and selected based on the sample used to isolate the γδT cells.

[0159] Isolation of γδT cells may include further culturing the sample in the presence of at least one cytokine. The cytokine may be different from the cytokine used in the initial culture.

[0160] Isolation methods may include culturing samples. As used herein, "culturing" includes adding a sample (including cells isolated, separated, removed, purified, or enriched from the sample) to a culture medium containing the cells and / or preferred growth factors and / or essential nutrients required by the sample. It should be understood that such culture conditions may be adjusted based on the cells or cell populations to be isolated from the sample, or based on the cells or cell populations to be isolated and expanded from the sample.

[0161] In some embodiments, the sample is cultured for a period sufficient to isolate γδT cells from the sample. In some embodiments, the culture duration is at least 14 days. In some embodiments, the culture duration is less than 45 days, for example less than 30 days, or even less than 25 days. In a further embodiment, the culture duration is between 14 and 35 days, for example between 14 and 21 days. In yet another embodiment, the culture duration is approximately 21 days.

[0162] In certain embodiments, γδT cells are collected from the culture after the sample has been cultured. The collection of γδT cells may include physically collecting γδT cells from the culture, isolating γδT cells from other lymphocytes (e.g., αβT cells and / or NK cells), or isolating and / or separating γδT cells from other cells present in the sample (e.g., stromal cells, such as fibroblasts). In one embodiment, γδT cells are collected by mechanical methods (e.g., pipetting). In another embodiment, γδT cells are collected by magnetic separation and / or labeling methods. In yet another embodiment, γδT cells are collected by flow cytometry techniques such as FACS. Therefore, in some embodiments, γδT cells are collected by specifically labeling them. It should be understood that such collection of γδT cells may include physical removal from the culture of the sample, transfer to a separate culture container, or transfer to separate or different culture conditions.

[0163] It should be understood that the collection of γδT cells is performed after a sufficient period of time to achieve isolation of the γδT cell population from the sample. In some embodiments, γδT cells are collected after culturing the sample for at least one week, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. Suitablely, γδT cells are collected after 40 days or less, such as 38 days or less, 36 days or less, 34 days or less, 32 days or less, 30 days or less, 28 days or less, 26 days or less, or 24 days or less. In one embodiment, γδT cells are collected after culturing the sample for at least 14 days. In another embodiment, γδT cells are collected after culturing the sample for 14 to 21 days.

[0164] In one embodiment, the sample is cultured in a medium that is substantially serum-free (e.g., a serum-free medium or a medium containing a serum substitute (SR)). Therefore, in one embodiment, the sample is cultured in a serum-free medium. Such a serum-free medium may also include a serum substitute medium, wherein the serum substitute is based on chemically determined components to avoid the use of serum of human or animal origin. In one embodiment, the medium does not contain animal-derived products. In an alternative embodiment, the sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)).

[0165] The culture medium may additionally contain other components that can assist in the growth and expansion of γδT cells. Examples of other components that may be added include, but are not limited to, plasma or serum, purified proteins such as albumin, lipid sources such as low-density lipoprotein (LDL), vitamins, amino acids, steroids, and any other supplements that support or promote cell growth and / or survival.

[0166] The dominant γδT cells in the blood are mainly Vδ2T cells, while the dominant γδT cells in non-hematopoietic tissues are mainly Vδ1T cells. Therefore, Vδ1T cells account for approximately 70-80% of the γδT cell population residing in non-hematopoietic tissues. In a preferred embodiment, the isolated γδT cells comprise a Vδ1T cell population.

[0167] Antibody or fragment thereof

[0168] This article provides an antibody or fragment thereof that can specifically bind to the delta variable 1 chain (Vδ1) of the γδT cell receptor (TCR).

[0169] In one embodiment, the antibody or a fragment thereof is scFv, Fab, Fab', F(ab')2, Fv, a variable domain (e.g., VH or VL), a biantibody, a microantibody, or a monoclonal antibody. In a further embodiment, the antibody or a fragment thereof is scFv.

[0170] The antibodies described herein can be of any type, such as IgG, IgA, IgM, IgE, IgD, or their isotypes, and may contain a kappa or lambda light chain. In one embodiment, the antibody is an IgG antibody, such as at least one isotype IgG1, IgG2, IgG3, or IgG4. In a further embodiment, the antibody may be a format that has been modified to impart desired properties, such as an IgG format, for example, having an Fc mutation to reduce effector function, prolong half-life, alter ADCC, or improve hinge stability. Such modifications are well known in the art.

[0171] In one embodiment, the antibody or a fragment thereof is human. Therefore, the antibody or a fragment thereof may be derived from a human immunoglobulin (Ig) sequence. The CDR, framework, and / or constant regions of the antibody (or its fragment) may be derived from a human Ig sequence, particularly a human IgG sequence. The CDR, framework, and / or constant regions of a human Ig sequence, particularly a human IgG sequence, may be substantially identical. One advantage of using human antibodies is that they have low or no immunogenicity in humans.

[0172] Antibodies or fragments thereof can also be chimeric, such as mouse-human chimeric antibodies.

[0173] Alternatively, the antibody or a fragment thereof may be derived from a non-human species, such as a mouse. Such non-human antibodies can be modified to increase their similarity to naturally occurring human antibody variants, thus allowing the antibody or a fragment thereof to be partially or fully humanized. Therefore, in one embodiment, the antibody or a fragment thereof is humanized.

[0174] Antibodies targeting epitopes

[0175] This article provides antibodies (or fragments thereof) that bind to the Vδ1 epitope of γδTCR. This binding may optionally have an effect on γδTCR activity, such as activation or inhibition.

[0176] In one embodiment, the epitope may be an activating epitope of γδT cells. An “activating” epitope may include, for example, stimulating TCR function, such as degranulation, TCR downregulation, cytotoxicity, proliferation, mobilization, increased survival or resistance to exhaustion, intracellular signaling, cytokine or growth factor secretion, phenotypic alterations, or alterations in gene expression. For example, binding to an activating epitope can stimulate the expansion (i.e., proliferation) of a γδT cell population, preferably a Vδ1+ T cell population. Therefore, these antibodies can be used to modulate γδT cell activation, thereby modulating the immune response. Thus, in one embodiment, binding to an activating epitope downregulates γδTCR. In another or alternative embodiment, binding to an activating epitope activates γδT cell degranulation. In a further, alternative embodiment, binding to an activating epitope activates γδT cell killing.

[0177] Alternatively, an antibody (or a fragment thereof) may have a blocking effect by preventing the binding or interaction of another antibody or molecule. In one embodiment, the present invention provides a dissociative antibody or a fragment thereof that blocks Vδ1 and prevents TCR binding (e.g., by steric hindrance). By blocking Vδ1, the antibody can prevent TCR activation and / or signaling. The epitope may be an inhibitory epitope of γδT cells. An "inhibitory" epitope may include, for example, blocking TCR function, thereby inhibiting TCR activation.

[0178] The epitope preferably consists of at least one extracellular portion, soluble portion, hydrophilic portion, external portion, or cytoplasmic portion of the Vδ1 chain of γδTCR.

[0179] Specifically, the epitope is not contained in the hypervariable region of the Vδ1 chain of the γδTCR, particularly epitopes present in CDR3 of the Vδ1 chain. In a preferred embodiment, the epitope is located within the nonvariable region of the Vδ1 chain of the γδTCR. It should be understood that this binding allows for unique recognition of the Vδ1 chain, unrestricted by the highly variable TCR sequence (particularly CDR3). Various γδTCR complexes that recognize MHC-like peptides or antigens can be recognized solely by the presence of the Vδ1 chain. Therefore, it should be understood that antibodies or fragments thereof, as defined herein, can be used to recognize any γδTCR containing the Vδ1 chain, regardless of the specificity of the γδTCR. In one embodiment, the epitope comprises one or more amino acid residues within amino acid regions 1-24 and / or 35-90 of SEQ ID NO:1, e.g., portions of the Vδ1 chain that are not part of the CDR1 and / or CDR3 sequences. In one embodiment, the epitope does not comprise amino acid residues within amino acid regions 91-105 (CDR3) of SEQ ID NO:1.

[0180] Similar to well-characterized αβT cells, γδT cells utilize a diverse set of somatic rearrangements of variable (V), diverse (D), connected (J), and constant (C) genes, although γδT cells contain fewer V, D, and J segments compared to αβT cells. In one embodiment, the epitope bound by the antibody (or a fragment thereof) is not an epitope present in the J region of the Vδ1 chain (e.g., one of the four J regions encoded in the human delta1 chain germline: SEQ ID NO: 131 (J1*0) or 132 (J2*0) or 133 (J3*0) or 134 (J4*0)). In one embodiment, the epitope bound by the antibody (or a fragment thereof) is not an epitope present in the C region of the Vδ1 chain (e.g., SEQ ID NO: 135 (C1*0), which contains a C-terminal juxtamembrane / transmembrane region). In one embodiment, the epitope bound by the antibody (or a fragment thereof) is not an epitope present in the N-terminal leader sequence of the Vδ1 chain (e.g., SEQ ID NO: 129). Therefore, the antibody or fragment may bind only to the V region of the Vδ1 chain (e.g., SEQ ID NO: 130). Thus, in one embodiment, the epitope consists of an epitope in the V region of the γδTCR (e.g., amino acid residues 1-90 of SEQ ID NO: 1).

[0181] The epitope is derived from the Vδ1 sequence described in Luoma et al. (2013) Immunity 39:1032-1042 and RCSB Protein Data Bankentries: 4MNH and 3OMZ, and is shown in SEQ ID NO: 1: AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGESLTRADKLIFGKGTRVTVEPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 1)

[0182] SEQ ID NO:1 represents a soluble TCR containing the V region (also known as the variable domain), D region, J region, and TCR constant region. The V region contains amino acid residues 1-90, the D region contains amino acid residues 91-104, the J region contains amino acid residues 105-115, and the constant region contains amino acid residues 116-209. Within the V region, CDR1 is defined as amino acid residues 25-34 of SEQ ID NO:1, CDR2 is defined as amino acid residues 50-54 of SEQ ID NO:1, and CDR3 is defined as amino acid residues 93-104 of SEQ ID NO:1 (Xu et al., PNAS USA 108(6):2414-2419(2011)).

[0183] Therefore, in one embodiment, the isolated antibody or a fragment thereof binds to an epitope of the variable delta1 (Vδ1) chain of the γδT cell receptor (TCR), said epitope comprising one or more amino acid residues within the following amino acid region:

[0184] (i) SEQ ID NO: 1, 3-20; and / or

[0185] (ii) 37-77 of SEQ ID NO: 1.

[0186] In another embodiment, the antibody or a fragment thereof additionally recognizes the polymorphic V region containing epitopes 1-90 of amino acid residues SEQ ID NO: 128. Therefore,

[0187] When defining the epitopes described herein, amino acids 1-90 of SEQ ID NO: 1 and the polymorphic germline variant sequence (amino acids 1-90 of SEQ ID NO: 128) can be considered interchangeable. The antibodies of the present invention can recognize both variants of this germline sequence. For example, when indicating that an antibody or fragment thereof, as defined herein, recognizes an epitope containing one or more amino acid residues within amino acid regions 1-24 and / or 35-90 of SEQ ID NO: 1, this also indicates the same region of SEQ ID NO: 128; specifically amino acid regions 1-24 and / or 35-90 of SEQ ID NO: 128.

[0188] In one embodiment, the antibody or a fragment thereof recognizes one or more amino acid residues within amino acid regions 1-90 of SEQ ID NO: 1 and within amino acid regions 1-90 of the equivalent location of SEQ ID NO: 128. More specifically, in one embodiment, the antibody or a fragment thereof, as defined herein, recognizes a human germline epitope, wherein the germline encodes alanine (A) or valine (V) at position 71 of SEQ ID NO: 1.

[0189] In one embodiment, the epitope comprises one or more, such as two, three, four, five, six, seven, eight, nine, ten or more amino acid residues within the region.

[0190] In a further embodiment, the epitope comprises one or more (e.g., 5 or more, e.g., 10 or more) amino acid residues within amino acid regions 3-20 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one or more (e.g., 5 or more, e.g., 10 or more) amino acid residues within amino acid regions 37-77 (e.g., amino acid regions 50-54) of SEQ ID NO: 1. In yet another embodiment, the epitope comprises one or more (e.g., 5 or more, e.g., 10 or more) amino acid residues within amino acid regions 3-20 (e.g., 5-20 or 3-17) of SEQ ID NO: 1, and one or more (e.g., 5 or more, e.g., 10 or more) amino acid residues within amino acid regions 37-77 (e.g., 62-77 or 62-69) of SEQ ID NO: 1.

[0191] It will be further understood that the antibody (or a fragment thereof) does not need to bind to all amino acids within the defined range. Such an epitope may be referred to as a linear epitope. For example, an antibody that binds to an epitope containing amino acid residues within amino acid region 5-20 of SEQ ID NO: 1 may bind only to one or more amino acid residues within the range, for example, to amino acid residues at both ends of the range (i.e., amino acids 5 and 20), and optionally to amino acids included in the range (i.e., amino acids 5, 9, 16, and 20).

[0192] In one embodiment, the epitope comprises at least one of amino acid residues 3, 5, 9, 10, 12, 16, 17, 20, 37, 42, 50, 53, 59, 62, 64, 68, 69, 72, or 77 of SEQ ID NO:1. In a further embodiment, the epitope comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acids selected from amino acid residues 3, 5, 9, 10, 12, 16, 17, 20, 37, 42, 50, 53, 59, 62, 64, 68, 69, 72, or 77 of SEQ ID NO:1.

[0193] In one embodiment, the epitope comprises one or more amino acid residues within the following amino acid region of SEQ ID NO:1 (or SEQ ID NO:128, as described above):

[0194] (i)3-17;

[0195] (ii)5-20;

[0196] (iii)37-53;

[0197] (iv)50-64;

[0198] (v)59-72;

[0199] (vi)59-77;

[0200] (vii)62-69; and / or

[0201] (viii)62-77.

[0202] In a further embodiment, the epitope comprises one or more amino acid residues within the amino acid region of SEQ ID NO:1: 5-20 and 62-77; 50-64; 37-53 and 59-72; 59-77; or 3-17 and 62-69. In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region of SEQ ID NO:1: 5-20 and 62-77; 50-64; 37-53 and 59-72; 59-77; or 3-17 and 62-69.

[0203] In a further embodiment, the epitope comprises amino acid residues 3, 5, 9, 10, 12, 16, 17, 62, 64, 68, and 69 of SEQ ID NO:1, or suitably consists of amino acid residues 3, 5, 9, 10, 12, 16, 17, 62, 64, 68, and 69 of SEQ ID NO:1. In a further embodiment, the epitope comprises amino acid residues 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO:1, or suitably consists of amino acid residues 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO:1. In yet another embodiment, the epitope comprises, or suitably consists of, amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77 of SEQ ID NO:1. In a further embodiment, the epitope comprises, or suitably consists of, amino acid residues 50, 53, 59, 62, and 64 of SEQ ID NO:1. In a further embodiment, the epitope comprises, or suitably consists of, amino acid residues 59, 60, 68, and 72 of SEQ ID NO:1.

[0204] In one embodiment, the epitope comprises one or more amino acid residues within amino acid regions 5-20 and / or 62-77 of SEQ ID NO:1. In another embodiment, the epitope consists of one or more amino acid residues within amino acid regions 5-20 and 62-77 of SEQ ID NO:1. In yet another alternative embodiment, the epitope comprises one or more amino acid residues within amino acid regions 5-20 or 62-77 of SEQ ID NO:1. Antibodies or fragments thereof having such epitopes may have some or all of the sequence 1245_P01_E07, or such antibodies or fragments thereof may be derived from 1245_P01_E07. For example, antibodies or fragments thereof having one or more CDR sequences of 1245_P01_E07 or one or both of the VH and VL sequences of 1245_P01_E07 may bind to such epitopes.

[0205] In one embodiment, the epitope comprises one or more amino acid residues within amino acid region 50-64 of SEQ ID NO:1. In a further embodiment, the epitope consists of one or more amino acid residues within amino acid region 50-64 of SEQ ID NO:1. Antibodies or fragments thereof having such epitopes may have some or all of the sequence 1252_P01_C08, or such antibodies or fragments thereof may be derived from 1252_P01_C08. For example, antibodies or fragments thereof having one or more CDR sequences of 1252_P01_C08 or one or both of the VH and VL sequences of 1252_P01_C08 may bind to such epitopes.

[0206] In one embodiment, the epitope comprises one or more amino acid residues within amino acid regions 37-53 and / or 59-77 of SEQ ID NO:1. In another embodiment, the epitope consists of one or more amino acid residues within amino acid regions 37-53 and 59-77 of SEQ ID NO:1. In yet another alternative embodiment, the epitope comprises one or more amino acid residues within amino acid regions 37-53 or 59-77 of SEQ ID NO:1. Antibodies or fragments thereof having such epitopes may have some or all of the sequence 1245_P02_G04, or such antibodies or fragments thereof may be derived from 1245_P02_G04. For example, antibodies or fragments thereof having one or more CDR sequences of 1245_P02_G04 or one or both of the VH and VL sequences of 1245_P02_G04 may bind to such epitopes.

[0207] In one embodiment, the epitope comprises one or more amino acid residues within amino acid region 59-72 of SEQ ID NO:1. In a further embodiment, the epitope consists of one or more amino acid residues within amino acid region 59-72 of SEQ ID NO:1. Antibodies or fragments thereof having such epitopes may have some or all of the sequence 1251_P02_C05, or such antibodies or fragments thereof may be derived from 1251_P02_C05. For example, antibodies or fragments thereof having one or more CDR sequences of 1251_P02_C05 or one or both of the VH and VL sequences of 1251_P02_C05 may bind to such epitopes.

[0208] In one embodiment, the epitope does not contain amino acid residues in amino acid regions 11-21 of SEQ ID NO:1. In one embodiment, the epitope does not contain amino acid residues in amino acid regions 21-28 of SEQ ID NO:1. In one embodiment, the epitope does not contain amino acid residues in amino acid regions 59 and 60 of SEQ ID NO:1. In one embodiment, the epitope does not contain amino acid residues in amino acid regions 67-82 of SEQ ID NO:1.

[0209] In one implementation, the epitope is different from the epitope bound by commercially available anti-Vδ1 antibodies such as TS-1 or TS8.2. As described in WO2017197347, when the δ1 chain includes the Vδ1J1 and Vδ1J2 sequences but not the Vδ1J3 chain, the binding of TS-1 and TS8.2 to soluble TCRs is detected, indicating that the binding of TS-1 and TS8.2 involves key residues within the delta J1 and delta J2 regions.

[0210] The term "within" as used in this article includes the endpoints of the defined range. For example, "within amino acid region 5-20" means all amino acid residues from residue 5 to residue 20, and includes residues 5 and 20.

[0211] Various techniques are known in the art for determining which epitope is bound by an antibody. Exemplary techniques include, for example, conventional cross-blocking assays, alanine scanning mutation analysis, peptide blotting, peptide cleavage analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and antigen chemical modification can also be employed. Another method that can be used to identify amino acids within a peptide that interact with the antibody is hydrogen / deuterium exchange (as described in Example 9) detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterating a protein of interest, followed by binding an antibody to the deuterated protein. Next, the protein / antibody complex is transferred to water, where exchangeable protons within the amino acids protected by the antibody complex undergo deuterium-hydrogen reverse exchange at a slower rate than exchangeable protons within amino acids not belonging to the interface portion. Therefore, the amino acids forming the protein / antibody interface portion retain deuterium and thus exhibit a relatively higher quality compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal the deuterated residues corresponding to the specific amino acids interacting with the antibody.

[0212] antibody sequence

[0213] The isolated anti-Vδ1 antibody or its fragments can be described by referring to its CDR sequence.

[0214] In one embodiment, the anti-Vδ1 antibody or a fragment thereof comprises one or more of the following:

[0215] CDR3, which contains a sequence having at least 80% sequence identity with any one of SEQ ID NO: 2-25;

[0216] CDR2, which comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 26-37 and sequences A1-A12; and / or

[0217] CDR1 contains a sequence that has at least 80% sequence identity with any of SEQ ID NO: 38-61.

[0218] In one embodiment, the isolated anti-Vδ1 antibody or fragment thereof comprises CDR3, said CDR3 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 2-25. In one embodiment, the antibody or fragment thereof comprises CDR2, said CDR2 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 26-37 and sequences A1-A12 (Table 2). In one embodiment, the antibody or fragment thereof comprises CDR1, said CDR1 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 38-61.

[0219] In one embodiment, the antibody or fragment thereof comprises CDR3, said CDR3 comprising a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 2-25. In one embodiment, the antibody or fragment thereof comprises CDR2, said CDR2 comprising a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 26-37 and sequences: A1-A12 (Table 2). In one embodiment, the antibody or fragment thereof comprises CDR1, said CDR1 comprising a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 38-61.

[0220] In one embodiment, the antibody or fragment thereof comprises CDR3, said CDR3 consisting of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 2-25. In one embodiment, the antibody or fragment thereof comprises CDR2, said CDR2 consisting of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 26-37 and sequences: A1-A12 (Table 2). In one embodiment, the antibody or fragment thereof comprises CDR1, said CDR1 consisting of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 38-61.

[0221] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3 sequence sharing at least 80% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises a CDR3 sequence sharing at least 80% sequence identity with any of SEQ ID NO: 14-25. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3 consisting of a sequence sharing at least 80% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises a CDR3 consisting of a sequence sharing at least 80% sequence identity with any of SEQ ID NO: 14-25.

[0222] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3 consisting of a sequence having at least 90% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises a CDR3 consisting of a sequence having at least 90% sequence identity with any of SEQ ID NO: 14-25. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3 consisting of a sequence having at least 90% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises a CDR3 consisting of a sequence having at least 90% sequence identity with any of SEQ ID NO: 14-25.

[0223] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3 consisting of a sequence having at least 95% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises a CDR3 consisting of a sequence having at least 95% sequence identity with any of SEQ ID NO: 14-25. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3 consisting of a sequence having at least 95% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises a CDR3 consisting of a sequence having at least 95% sequence identity with any of SEQ ID NO: 14-25.

[0224] In one embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region comprises CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 14-25. In another embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region comprises CDR3 consisting of a sequence having at least 80% sequence identity with any of SEQ ID NO: 2-13, and the VL region comprises CDR3 consisting of a sequence having at least 80% sequence identity with any of SEQ ID NO: 14-25.

[0225] In one embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with SEQ ID NO: 2-7, particularly 2-6, such as any one of 2, 3, or 4, and the VL region comprising CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with SEQ ID NO: 14-19, particularly 14-18, such as any one of 14, 15, or 16. In another embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising CDR3, the CDR3 consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 2-7, particularly 2-6, such as any one of 2, 3, or 4, and the VL region comprising CDR3, the CDR3 consisting of a sequence having at least 80% sequence identity with SEQ ID NO: 14-19, particularly 14-18, such as any one of 14, 15, or 16.

[0226] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, the VH region comprising a CDR3, the CDR3 comprising a sequence having at least 90% sequence identity with SEQ ID NO: 2-7, particularly 2-6, such as any one of 2, 3, or 4, and the VL region comprising a CDR3, the CDR3 comprising a sequence having at least 90% sequence identity with SEQ ID NO: 14-19, particularly 14-18, such as any one of 14, 15, or 16. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, the VH region comprising a CDR3, the CDR3 consisting of a sequence having at least 90% sequence identity with SEQ ID NO: 2-7, particularly 2-6, such as any one of 2, 3, or 4, and the VL region comprising a CDR3, the CDR3 consisting of a sequence having at least 90% sequence identity with SEQ ID NO: 14-19, particularly 14-18, such as any one of 14, 15, or 16.

[0227] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, the VH region comprising a CDR3, the CDR3 comprising a sequence having at least 95% sequence identity with SEQ ID NO: 2-7, particularly 2-6, such as any one of 2, 3, or 4, and the VL region comprising a CDR3, the CDR3 comprising a sequence having at least 95% sequence identity with SEQ ID NO: 14-19, particularly 14-18, such as any one of 14, 15, or 16. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, the VH region comprising a CDR3, the CDR3 consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 2-7, particularly 2-6, such as any one of 2, 3, or 4, and the VL region comprising a CDR3, the CDR3 consisting of a sequence having at least 95% sequence identity with SEQ ID NO: 14-19, particularly 14-18, such as any one of 14, 15, or 16.

[0228] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, the VH region comprising CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 8-13, particularly 8, 9, 10, or 11, and the VL region comprising CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 20-25, particularly 20, 21, 22, or 23. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, the VH region comprising CDR3, the CDR3 consisting of a sequence having at least 80% sequence identity with any of SEQ ID NO: 8-13, particularly 8, 9, 10, or 11, and the VL region comprising CDR3, the CDR3 consisting of a sequence having at least 80% sequence identity with any of SEQ ID NO: 20-25, particularly 20, 21, 22, or 23.

[0229] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3, the CDR3 comprising a sequence having at least 90% sequence identity with SEQ ID NO: 8-13, particularly any one of 8, 9, 10, or 11, and the VL region comprises a CDR3, the CDR3 comprising a sequence having at least 90% sequence identity with SEQ ID NO: 20-25, particularly any one of 20, 21, 22, or 23. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3, the CDR3 consisting of a sequence having at least 90% sequence identity with SEQ ID NO: 8-13, particularly any one of 8, 9, 10, or 11, and the VL region comprises a CDR3, the CDR3 consisting of a sequence having at least 90% sequence identity with SEQ ID NO: 20-25, particularly any one of 20, 21, 22, or 23.

[0230] In one embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3, the CDR3 comprising a sequence having at least 95% sequence identity with any of SEQ ID NOs: 8-13, particularly 8, 9, 10, or 11, and the VL region comprises a CDR3, the CDR3 comprising a sequence having at least 95% sequence identity with any of SEQ ID NOs: 20-25, particularly 20, 21, 22, or 23. In another embodiment, the antibody or a fragment thereof comprises a VH region and / or a VL region, wherein the VH region comprises a CDR3, the CDR3 consisting of a sequence having at least 95% sequence identity with any of SEQ ID NOs: 8-13, particularly 8, 9, 10, or 11, and the VL region comprises a CDR3, the CDR3 consisting of a sequence having at least 95% sequence identity with any of SEQ ID NOs: 20-25, particularly 20, 21, 22, or 23.

[0231] The use of "at least 80%" or "80% or higher" in this document is to be understood as including all values ​​equal to or greater than 80%, such as 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity. In one embodiment, the antibody or a fragment thereof contains at least 85%, such as at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, sequence identity with a particular sequence.

[0232] As an alternative to percentage sequence identity, embodiments may also be defined by one or more amino acid changes, such as one or more additions, substitutions, and / or deletions. In one embodiment, the sequence may contain up to five amino acid changes, such as up to three amino acid changes, particularly up to two amino acid changes. In a further embodiment, the sequence may contain up to five amino acid substitutions, such as up to three amino acid substitutions, particularly up to one or two amino acid substitutions. For example, the CDR3 of an antibody or a fragment thereof comprises or is more suitably composed of a sequence having no more than two, more suitably no more than one, substitution compared to any of SEQ IDNO:2-25.

[0233] Suitablely, any residue of CDR1, CDR2, or CDR3 that differs from its corresponding residue in SEQ ID NO:2-61 and sequences A1-A12 is a conserved substitution relative to its corresponding residue. For example, any residue of CDR3 that differs from its corresponding residue in SEQ ID NO:2-25 is a conserved substitution relative to its corresponding residue.

[0234] In one implementation, the antibody or a fragment thereof comprises:

[0235] (i) VH region, which contains CDR3, said CDR3 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 2-13;

[0236] (ii) VH region, which contains CDR2, said CDR2 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 26-37;

[0237] (iii) VH region, which contains CDR1, said CDR1 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 38-49;

[0238] (iv) VL region, which contains CDR3, said CDR3 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 14-25;

[0239] (v) A VL region containing a CDR2, wherein the CDR2 contains a sequence having at least 80% sequence identity with any one of sequences A1-A12; and / or

[0240] (vi) VL region, which contains CDR1, said CDR1 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 50-61.

[0241] In one embodiment, the antibody or a fragment thereof comprises a heavy chain having:

[0242] (i) VH region, which contains CDR3, said CDR3 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 2-13;

[0243] (ii) A VH region comprising CDR2, wherein CDR2 comprises a sequence having at least 80% sequence identity with any of SEQ ID NO: 26-37; and

[0244] (iii) VH region, which contains CDR1, said CDR1 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 38-49.

[0245] In one embodiment, the antibody or a fragment thereof comprises a light chain having:

[0246] (i) VL region, which contains CDR3, said CDR3 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 14-25;

[0247] (ii) A VL region containing a CDR2, wherein the CDR2 contains a sequence having at least 80% sequence identity with any of sequences A1-A12; and

[0248] (iii) VL region, which contains CDR1, said CDR1 containing a sequence having at least 80% sequence identity with any of SEQ ID NO: 50-61.

[0249] In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VH region, said VH region comprising CDR3, said CDR3 comprising a sequence having at least 80% sequence identity with SEQ ID NO: 2, 3, 4, 5 or 6, such as 2, 3, 4 or 5, particularly any one of 2, 3 or 4. In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VH region, said VH region comprising CDR2, said CDR2 comprising a sequence having at least 80% sequence identity with SEQ ID NO: 26, 27, 28, 29 or 30, such as 26, 27, 28 or 29, particularly any one of 26, 27 or 28. In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VH region, the VH region comprising a CDR1, the CDR1 comprising a sequence having at least 80% sequence identity with SEQ ID NO: 38, 39, 40, 41 or 42, such as 38, 39, 40 or 41, particularly any one of 38, 39 or 40.

[0250] In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VH region, the VH region comprising CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 8, 9, 10, or 11. In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VH region, the VH region comprising CDR2, the CDR2 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 32, 33, 34, or 35. In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VH region, the VH region comprising CDR1, the CDR1 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 44, 45, 46, or 47.

[0251] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:2, CDR2 containing the sequence of SEQ ID NO:26, and CDR1 containing the sequence of SEQ ID NO:38. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:2, CDR2 consists of the sequence of SEQ ID NO:26, and CDR1 consists of the sequence of SEQ ID NO:38.

[0252] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:3, CDR2 containing the sequence of SEQ ID NO:27, and CDR1 containing the sequence of SEQ ID NO:39. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:3, CDR2 consists of the sequence of SEQ ID NO:27, and CDR1 consists of the sequence of SEQ ID NO:39.

[0253] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:4, CDR2 containing the sequence of SEQ ID NO:28, and CDR1 containing the sequence of SEQ ID NO:40. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:4, CDR2 consists of the sequence of SEQ ID NO:28, and CDR1 consists of the sequence of SEQ ID NO:40.

[0254] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:5, CDR2 containing the sequence of SEQ ID NO:29, and CDR1 containing the sequence of SEQ ID NO:41. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:5, CDR2 consists of the sequence of SEQ ID NO:29, and CDR1 consists of the sequence of SEQ ID NO:41.

[0255] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:6, CDR2 containing the sequence of SEQ ID NO:30, and CDR1 containing the sequence of SEQ ID NO:42. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:6, CDR2 consists of the sequence of SEQ ID NO:30, and CDR1 consists of the sequence of SEQ ID NO:42.

[0256] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:8, CDR2 containing the sequence of SEQ ID NO:32, and CDR1 containing the sequence of SEQ ID NO:44. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:8, CDR2 consists of the sequence of SEQ ID NO:32, and CDR1 consists of the sequence of SEQ ID NO:44.

[0257] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:9, CDR2 containing the sequence of SEQ ID NO:33, and CDR1 containing the sequence of SEQ ID NO:45. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:9, CDR2 consists of the sequence of SEQ ID NO:33, and CDR1 consists of the sequence of SEQ ID NO:45.

[0258] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:10, CDR2 containing the sequence of SEQ ID NO:34, and CDR1 containing the sequence of SEQ ID NO:46. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:10, CDR2 consists of the sequence of SEQ ID NO:34, and CDR1 consists of the sequence of SEQ ID NO:46.

[0259] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:11, CDR2 containing the sequence of SEQ ID NO:35, and CDR1 containing the sequence of SEQ ID NO:47. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:11, CDR2 consists of the sequence of SEQ ID NO:35, and CDR1 consists of the sequence of SEQ ID NO:47.

[0260] In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VL region, the VL region comprising CDR3, the CDR3 comprising a sequence having at least 80% sequence identity with SEQ ID NO:14-25, such as SEQ ID NO:14, 15, 16, 17 or 18, such as 14, 15, 16 or 17, particularly any one of 14, 15 or 16. In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VL region, the VL region comprising CDR2, the CDR2 comprising a sequence having at least 80% sequence identity with sequences A1-A12 (Table 2), such as sequences A1, A2, A3, A4 or A5, such as A1, A2, A3 or A4, particularly any one of A1, A2 or A3. In one embodiment, the antibody or a fragment thereof comprises (or consists of) a VL region, the VL region comprising a CDR1, the CDR1 comprising a sequence having at least 80% sequence identity with SEQ ID NO:50-61, such as SEQ ID NO:50, 51, 52, 53 or 54, such as 50, 51, 52 or 53, particularly any one of 50, 51 or 52.

[0261] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:14, CDR2 containing the sequence of sequence A1, and CDR1 containing the sequence of SEQ ID NO:50. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:14, CDR2 consists of the sequence of sequence A1, and CDR1 consists of the sequence of SEQ ID NO:50.

[0262] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:15, CDR2 containing the sequence of sequence A2, and CDR1 containing the sequence of SEQ ID NO:51. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:15, CDR2 consists of the sequence of sequence A2, and CDR1 consists of the sequence of SEQ ID NO:51.

[0263] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:16, CDR2 containing the sequence of sequence A3, and CDR1 containing the sequence of SEQ ID NO:52. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:16, CDR2 consists of the sequence of sequence A3, and CDR1 consists of the sequence of SEQ ID NO:52.

[0264] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:17, CDR2 containing the sequence of sequence A4, and CDR1 containing the sequence of SEQ ID NO:53. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:17, CDR2 consists of the sequence of sequence A4, and CDR1 consists of the sequence of SEQ ID NO:53.

[0265] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:18, CDR2 containing the sequence of sequence A5, and CDR1 containing the sequence of SEQ ID NO:54. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:18, CDR2 consists of the sequence of sequence A5, and CDR1 consists of the sequence of SEQ ID NO:54.

[0266] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:20, CDR2 containing the sequence of sequence A7, and CDR1 containing the sequence of SEQ ID NO:56. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:20, CDR2 consists of the sequence of sequence A7, and CDR1 consists of the sequence of SEQ ID NO:56.

[0267] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:21, CDR2 containing the sequence of sequence A8, and CDR1 containing the sequence of SEQ ID NO:57. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:21, CDR2 consists of the sequence of sequence A8, and CDR1 consists of the sequence of SEQ ID NO:57.

[0268] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:22, CDR2 containing the sequence of sequence A9, and CDR1 containing the sequence of SEQ ID NO:58. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:22, CDR2 consists of the sequence of sequence A9, and CDR1 consists of the sequence of SEQ ID NO:58.

[0269] In one embodiment, the VL region includes CDR3 containing the sequence of SEQ ID NO:23, CDR2 containing the sequence of sequence A10, and CDR1 containing the sequence of SEQ ID NO:59. In one embodiment, CDR3 consists of the sequence of SEQ ID NO:23, CDR2 consists of the sequence of sequence A10, and CDR1 consists of the sequence of SEQ ID NO:59.

[0270] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:2, CDR2 containing the sequence of SEQ ID NO:26, and CDR1 containing the sequence of SEQ ID NO:38, and the VL region includes CDR3 containing the sequence of SEQ ID NO:14, CDR2 containing the sequence of sequence A1, and CDR1 containing the sequence of SEQ ID NO:50. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:2, HCDR2 is composed of the sequence of SEQ ID NO:26, HCDR1 is composed of the sequence of SEQ ID NO:38, LCDR3 is composed of the sequence of SEQ ID NO:14, LCDR2 is composed of the sequence of sequence A1, and LCDR1 is composed of the sequence of SEQ ID NO:50.

[0271] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:3, CDR2 containing the sequence of SEQ ID NO:27, and CDR1 containing the sequence of SEQ ID NO:39, and the VL region includes CDR3 containing the sequence of SEQ ID NO:15, CDR2 containing the sequence of sequence A2, and CDR1 containing the sequence of SEQ ID NO:51. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:3, HCDR2 is composed of the sequence of SEQ ID NO:27, HCDR1 is composed of the sequence of SEQ ID NO:39, LCDR3 is composed of the sequence of SEQ ID NO:15, LCDR2 is composed of the sequence of sequence A2, and LCDR1 is composed of the sequence of SEQ ID NO:51.

[0272] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:4, CDR2 containing the sequence of SEQ ID NO:28, and CDR1 containing the sequence of SEQ ID NO:40, and the VL region includes CDR3 containing the sequence of SEQ ID NO:16, CDR2 containing the sequence of sequence A3, and CDR1 containing the sequence of SEQ ID NO:52. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:4, HCDR2 is composed of the sequence of SEQ ID NO:28, HCDR1 is composed of the sequence of SEQ ID NO:40, LCDR3 is composed of the sequence of SEQ ID NO:16, LCDR2 is composed of the sequence of sequence A3, and LCDR1 is composed of the sequence of SEQ ID NO:52.

[0273] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:5, CDR2 containing the sequence of SEQ ID NO:29, and CDR1 containing the sequence of SEQ ID NO:41, and the VL region includes CDR3 containing the sequence of SEQ ID NO:17, CDR2 containing the sequence of sequence A4, and CDR1 containing the sequence of SEQ ID NO:53. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:5, HCDR2 is composed of the sequence of SEQ ID NO:29, HCDR1 is composed of the sequence of SEQ ID NO:41, LCDR3 is composed of the sequence of SEQ ID NO:17, LCDR2 is composed of the sequence of sequence A4, and LCDR1 is composed of the sequence of SEQ ID NO:53.

[0274] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:6, CDR2 containing the sequence of SEQ ID NO:30, and CDR1 containing the sequence of SEQ ID NO:42, and the VL region includes CDR3 containing the sequence of SEQ ID NO:18, CDR2 containing the sequence of sequence A5, and CDR1 containing the sequence of SEQ ID NO:54. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:6, HCDR2 is composed of the sequence of SEQ ID NO:30, HCDR1 is composed of the sequence of SEQ ID NO:42, LCDR3 is composed of the sequence of SEQ ID NO:18, LCDR2 is composed of the sequence of sequence A5, and LCDR1 is composed of the sequence of SEQ ID NO:54.

[0275] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:7, CDR2 containing the sequence of SEQ ID NO:31, and CDR1 containing the sequence of SEQ ID NO:43, and the VL region includes CDR3 containing the sequence of SEQ ID NO:19, CDR2 containing the sequence of sequence A6, and CDR1 containing the sequence of SEQ ID NO:55. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:7, HCDR2 is composed of the sequence of SEQ ID NO:31, HCDR1 is composed of the sequence of SEQ ID NO:43, LCDR3 is composed of the sequence of SEQ ID NO:19, LCDR2 is composed of the sequence of sequence A6, and LCDR1 is composed of the sequence of SEQ ID NO:55.

[0276] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:8, CDR2 containing the sequence of SEQ ID NO:32, and CDR1 containing the sequence of SEQ ID NO:44, and the VL region includes CDR3 containing the sequence of SEQ ID NO:20, CDR2 containing the sequence of sequence A7, and CDR1 containing the sequence of SEQ ID NO:56. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:8, HCDR2 is composed of the sequence of SEQ ID NO:32, HCDR1 is composed of the sequence of SEQ ID NO:44, LCDR3 is composed of the sequence of SEQ ID NO:20, LCDR2 is composed of the sequence of sequence A7, and LCDR1 is composed of the sequence of SEQ ID NO:56.

[0277] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:9, CDR2 containing the sequence of SEQ ID NO:33, and CDR1 containing the sequence of SEQ ID NO:45, and the VL region includes CDR3 containing the sequence of SEQ ID NO:21, CDR2 containing the sequence of sequence A8, and CDR1 containing the sequence of SEQ ID NO:57. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:9, HCDR2 is composed of the sequence of SEQ ID NO:33, HCDR1 is composed of the sequence of SEQ ID NO:45, LCDR3 is composed of the sequence of SEQ ID NO:21, LCDR2 is composed of the sequence of sequence A8, and LCDR1 is composed of the sequence of SEQ ID NO:57.

[0278] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:10, CDR2 containing the sequence of SEQ ID NO:34, and CDR1 containing the sequence of SEQ ID NO:46, and the VL region includes CDR3 containing the sequence of SEQ ID NO:22, CDR2 containing the sequence of sequence A9, and CDR1 containing the sequence of SEQ ID NO:58. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:10, HCDR2 is composed of the sequence of SEQ ID NO:34, HCDR1 is composed of the sequence of SEQ ID NO:46, LCDR3 is composed of the sequence of SEQ ID NO:22, LCDR2 is composed of the sequence of sequence A9, and LCDR1 is composed of the sequence of SEQ ID NO:58.

[0279] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:11, CDR2 containing the sequence of SEQ ID NO:35, and CDR1 containing the sequence of SEQ ID NO:47, and the VL region includes CDR3 containing the sequence of SEQ ID NO:23, CDR2 containing the sequence of sequence A10, and CDR1 containing the sequence of SEQ ID NO:59. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:11, HCDR2 is composed of the sequence of SEQ ID NO:35, HCDR1 is composed of the sequence of SEQ ID NO:47, LCDR3 is composed of the sequence of SEQ ID NO:23, LCDR2 is composed of the sequence of sequence A10, and LCDR1 is composed of the sequence of SEQ ID NO:59.

[0280] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:12, CDR2 containing the sequence of SEQ ID NO:36, and CDR1 containing the sequence of SEQ ID NO:48, and the VL region includes CDR3 containing the sequence of SEQ ID NO:24, CDR2 containing the sequence of sequence A11, and CDR1 containing the sequence of SEQ ID NO:60. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:12, HCDR2 is composed of the sequence of SEQ ID NO:36, HCDR1 is composed of the sequence of SEQ ID NO:48, LCDR3 is composed of the sequence of SEQ ID NO:24, LCDR2 is composed of the sequence of sequence A11, and LCDR1 is composed of the sequence of SEQ ID NO:60.

[0281] In one embodiment, the VH region includes CDR3 containing the sequence of SEQ ID NO:13, CDR2 containing the sequence of SEQ ID NO:37, and CDR1 containing the sequence of SEQ ID NO:49, and the VL region includes CDR3 containing the sequence of SEQ ID NO:25, CDR2 containing the sequence of sequence A12, and CDR1 containing the sequence of SEQ ID NO:61. In one embodiment, HCDR3 is composed of the sequence of SEQ ID NO:13, HCDR2 is composed of the sequence of SEQ ID NO:37, HCDR1 is composed of the sequence of SEQ ID NO:49, LCDR3 is composed of the sequence of SEQ ID NO:25, LCDR2 is composed of the sequence of sequence A12, and LCDR1 is composed of the sequence of SEQ ID NO:61.

[0282] In one embodiment, the antibody or fragment thereof comprises one or more CDR sequences as described in Table 2. In a further embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1252_P01_C08 as described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P01_E07 as described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P02_G04 as described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P02_B07 as described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1251_P02_C05 as described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1139_P01_E04 as described in Table 2. In one alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P02_F07 as described in Table 2. In one alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P01_G06 as described in Table 2. In one alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P01_G09 as described in Table 2. In one alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1138_P01_B09 as described in Table 2. In one alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1251_P02_G10 as described in Table 2.

[0283] Suitably, each of the VH and VL regions described above comprises four frame regions (FR1-FR4). In one embodiment, the antibody or fragment thereof comprises frame regions (e.g., FR1, FR2, FR3, and / or FR4) containing a sequence having at least 80% sequence identity with a frame region of any of SEQ ID NO: 62-85. In one embodiment, the antibody or fragment thereof comprises frame regions (e.g., FR1, FR2, FR3, and / or FR4) containing a sequence having at least 90%, such as at least 95%, 97%, or 99%, sequence identity with a frame region of any of SEQ ID NO: 62-85. In one embodiment, the antibody or fragment thereof comprises frame regions (e.g., FR1, FR2, FR3, and / or FR4) containing a sequence of any of SEQ ID NO: 62-85. In one embodiment, the antibody or fragment thereof comprises frame regions (e.g., FR1, FR2, FR3, and / or FR4) consisting of a sequence of any of SEQ ID NO: 62-85.

[0284] The antibodies described herein can be defined by their complete light chain and / or heavy chain variable sequences. In one embodiment, the antibody or a fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62-85. In another embodiment, the antibody or a fragment thereof consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62-85.

[0285] In one embodiment, the antibody or a fragment thereof comprises a VH region, said VH region comprising an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62-73. In another embodiment, the antibody or a fragment thereof comprises a VH region consisting of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62-73. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62, 63, 64, 65, or 66, such as 62, 63, 64, or 65, particularly any of 62, 63, or 64. In a further embodiment, the VH region consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62, 63, 64, 65, or 66, such as 62, 63, 64, or 65, particularly any of 62, 63, or 64. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 68, 69, 70, 71, 72, or 73, for example, any one of 68, 69, 70, or 71. In a further embodiment, the VH region consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 68, 69, 70, 71, 72, or 73, for example, any one of 68, 69, 70, or 71.

[0286] In one embodiment, the antibody or a fragment thereof comprises a VL region having at least 80% sequence identity with any of SEQ ID NO: 74-85. In another embodiment, the antibody or a fragment thereof comprises a VL region consisting of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 74-85. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 74, 75, 76, 77 or 78, such as 74, 75, 76 or 77, particularly any of 74, 75 or 76. In a further embodiment, the VL region consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 74, 75, 76, 77 or 78, such as 74, 75, 76 or 77, particularly any of 74, 75 or 76. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 80, 81, 82, 83, 84, or 85, such as any one of 80, 81, 82, or 83. In a further embodiment, the VL region consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 80, 81, 82, 83, 84, or 85, such as any one of 80, 81, 82, or 83.

[0287] In a further embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62-73, and the VL region comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 74-85. In a further embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 62-73, and the VL region consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 74-85.

[0288] In one embodiment, the antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 63 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07). In an alternative embodiment, the antibody or fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region containing the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09).

[0289] In one embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 63 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07). In one alternative embodiment, the antibody or a fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06). In another alternative embodiment, the antibody or a fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09).

[0290] In one embodiment, the antibody or fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 75 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 74 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 81 (1245_P02_F07). In an alternative embodiment, the antibody or fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or a fragment thereof comprises a VL region containing the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0291] In one embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 75 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 74 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 81 (1245_P02_F07). In one alternative embodiment, the antibody or a fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In another alternative embodiment, the antibody or a fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0292] In one embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 63 (1252_P01_C08), and the VL region comprising the amino acid sequence of SEQ ID NO: 75 (1252_P01_C08). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07), and the VL region comprising the amino acid sequence of SEQ ID NO: 74 (1245_P01_E07). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04), and the VL region comprising the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In one alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04), and the VL region comprising the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In one alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07), and the VL region comprising the amino acid sequence of SEQ ID NO: 81 (1245_P02_F07). In one alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06), and the VL region comprising the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region comprising the amino acid sequence of SEQ ID NO: 71 (1245_P01_G06) and the VL region comprising the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0293] In one embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 63 (1252_P01_C08) and the VL region consists of the amino acid sequence of SEQ ID NO: 75 (1252_P01_C08). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07) and the VL region consists of the amino acid sequence of SEQ ID NO: 74 (1245_P01_E07). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04) and the VL region consists of the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In one alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04) and the VL region consists of the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In another alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07) and the VL region consists of the amino acid sequence of SEQ ID NO: 81 (1245_P02_F07). In yet another alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, wherein the VH region consists of the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06) and the VL region consists of the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or a fragment thereof comprises a VH region and a VL region, the VH region consisting of the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09) and the VL region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0294] For fragments containing both VH and VL regions, these regions can be covalently linked (e.g., via disulfide bonds or linkers) or non-covalently linked. The antibody fragments described herein may contain scFv, i.e., fragments containing VH and VL regions linked by a linker. In one embodiment, the VH and VL regions are linked via (e.g., a synthetically produced) peptide linker. The peptide linker may contain (Gly4Ser). n A linker, where n = 1 to 8, for example, 2, 3, 4, 5, or 7. The peptide linker may contain [(Gly4Ser]]. n(Gly3AlaSer) m ] p A connector, wherein n = 1 to 8, for example, 2, 3, 4, 5, or 7; m = 1 to 8, for example, 0, 1, 2, or 3; and p = 1 to 8, for example, 1, 2, or 3. In a further embodiment, the connector comprises SEQ ID NO: 98. In a further embodiment, the connector is composed of SEQ ID NO: 98.

[0295] In one embodiment, the antibody or fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 86-97. In a further embodiment, the antibody or fragment thereof comprises an amino acid sequence of any of SEQ ID NO: 86-97. In yet another embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 87 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 86 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 88 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 92 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 93 (1245_P02_F07). In one alternative embodiment, the antibody or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In another alternative embodiment, the antibody or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0296] In one embodiment, the antibody or fragment thereof consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 86-97. In a further embodiment, the antibody or fragment thereof consists of an amino acid sequence of any of SEQ ID NO: 86-97. In yet another embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 87 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 86 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 88 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 92 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 93 (1245_P02_F07). In one alternative embodiment, the antibody or a fragment thereof consists of the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In another alternative embodiment, the antibody or a fragment thereof consists of the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0297] Those skilled in the art will understand that scFv constructs including N-terminal and C-terminal modifications can be designed and prepared to aid translation, purification, and detection. For example, at the N-terminus of the scFv sequence, amino acid residues of methionine and / or alanine may be additionally included before the canonical VH sequence (e.g., the initiating QVQ or EVQ). At the C-terminus (i.e., the C-terminus ending with the canonical VL domain sequence as defined by IMGT), sequences such as (i) partial sequences of constant domains and / or (ii) additional synthetic sequences including tags, such as His-tag and Flag-tag, may be additionally included to aid purification and detection. In one embodiment, SEQ ID NO: 124 is added to the C-terminus of any of SEQ ID NO: 86, 88-90, 92-97. In one embodiment, SEQ ID NO: 125 is added to the C-terminus of any of SEQ ID NO: 86, 88-90, 92-97. In one embodiment, SEQ ID NO: 126 is added to the C-terminus of any of SEQ ID NO: 87 or 91. In one embodiment, SEQ ID NO: 127 is added to the C-terminus of either SEQ ID NO: 87 or 91. It is well known that the N- or C-terminal sequence of the scFv is optional and can be removed, modified, or substituted if alternative scFv design, translation, purification, or detection strategies are employed.

[0298] As described herein, the antibody can be in any format. In a preferred embodiment, the antibody is in IgG1 format. Therefore, in one embodiment, the antibody or a fragment thereof comprises an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 111-122. In a further embodiment, the antibody or a fragment thereof comprises the amino acid sequence of any of SEQ ID NO: 111-122. In yet another embodiment, the antibody or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 111-116, such as SEQ ID NO: 111-113 and 116. In yet another embodiment, the antibody or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 117-122, such as SEQ ID NO: 117-120. In yet another embodiment, the antibody or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 111, 112, 116-120, such as SEQ ID NO: 111, 112, or 116, or SEQ ID NO: 117-120.

[0299] In one embodiment, the antibody or fragment thereof consists of an amino acid sequence having at least 80% sequence identity with any of SEQ ID NO: 111-122. In a further embodiment, the antibody or fragment thereof consists of an amino acid sequence of any of SEQ ID NO: 111-122. In yet another embodiment, the antibody or fragment thereof consists of an amino acid sequence of SEQ ID NO: 111-116, such as SEQ ID NO: 111-113 and 116. In yet another embodiment, the antibody or fragment thereof consists of an amino acid sequence of SEQ ID NO: 117-122, such as SEQ ID NO: 117-120. In yet another embodiment, the antibody or fragment thereof consists of an amino acid sequence of SEQ ID NO: 111, 112, 116-120, such as SEQ ID NO: 111, 112, or 116, or SEQ ID NO: 117-120.

[0300] In one embodiment, the antibody binds to or competes with an antibody or fragment thereof as defined herein for the same or substantially the same epitope. Whether an antibody binds to or competes with a reference anti-Vδ1 antibody for the same epitope can be readily determined using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-Vδ1 antibody, the reference antibody is bound to the Vδ1 protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the Vδ1 chain is evaluated. If the test antibody binds to Vδ1 after saturation binding with the reference anti-Vδ1 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-Vδ1 antibody. On the other hand, if the test antibody does not bind to the Vδ1 chain after saturation binding with the reference anti-Vδ1 antibody, the test antibody may bind to the same epitope as the one bound by the reference anti-Vδ1 antibody.

[0301] This invention also includes anti-Vδ1 antibodies that compete with antibodies or fragments thereof as defined herein, or antibodies having the CDR sequence of any of the exemplary antibodies described herein, for binding to Vδ1. For example, competitive assays can be performed using antibodies to determine which proteins, antibodies, and other antagonists compete with the antibody for binding to the Vδ1 chain and / or share epitopes. These assays are well known to those skilled in the art; they assess competition between antagonists or ligands for a limited number of binding sites on a protein (e.g., Vδ1). The antibody (or fragment thereof) is fixed or insoluble before or after the competition, and the sample bound to the Vδ1 chain is separated from the unbound sample, for example, by decantation (where the antibody is insoluble beforehand) or centrifugation (where the antibody precipitates after the competitive reaction). Furthermore, competitive binding can be determined by whether the function is altered by the binding or non-binding of the antibody to the protein, for example, by whether the antibody molecule inhibits or enhances, for example, the activity of a labeled enzyme. As known in the art and described herein, ELISA and other functional assays can be used.

[0302] If each of two antibodies competitively inhibits (blocks) the binding of the other to a target antigen, then the two antibodies bind to the same or overlapping epitopes. That is, as measured in a competitive binding assay, an excess of 1, 5, 10, 20, or 100 times that of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%. Alternatively, if substantially all amino acid mutations in the target antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope.

[0303] Further routine experiments (e.g., peptide mutation and binding assays) can then be performed to confirm whether the observed loss of binding to the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether the observed loss of binding is due to steric hindrance (or other phenomena). Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0304] In some embodiments, antibodies or fragments thereof are made to contain modified effector functions by altering the sugar linked to Asn 297 (Kabat numbering scheme). In a further modification, Asn 297 is not fucosylated or exhibits reduced fucosylation (i.e., defucosylated antibodies or non-fucosylated antibodies). Fucosylation involves adding fucose to the molecule, for example, linking fucose to N-glycans, O-glycans, and glycolipids. Thus, in defucosylated antibodies, fucose is not linked to the carbohydrate chain in the constant region. Antibodies can be modified to prevent or inhibit fucosylation of the antibody. Typically, glycosylation modification involves expressing the antibody or fragment thereof in host cells with alternating glycosylation processing capabilities through targeted engineering or by targeted or incidental host or clonal selection. These and other effector modifications are discussed further in recent reviews, such as Xinhua Wang et al. (2018) Protein & Cell 9:63-73 and Pereira et al. (2018) mAbs 10(5):693-711, which are incorporated herein by reference.

[0305] Antibody sequence modification

[0306] Antibodies and their fragments can be modified using known methods. Those skilled in the art can readily incorporate sequence modifications into the antibody molecules described herein. The following examples are non-limiting.

[0307] During antibody discovery and sequence recovery from phage libraries, the desired antibody variable domains can be reformatted into full-length IgG via subcloning. To accelerate this process, restriction endonucleases are typically used to transfer the variable domains. These unique restriction sites can introduce additional / alternative amino acids and deviate from the canonical sequence (e.g., such canonical sequences can be found in the international ImMunoGeneTics [IMGT] information system, see http: / / www.imgt.org). These can be introduced as kappa or lambda light chain sequence modifications.

[0308] Kappa light chain embellishment

[0309] During the reformulation into full-length IgG, a variable kappa light chain sequence can be cloned using restriction sites (e.g., Nhe1-Not1). More specifically, an additional Ala-Ser sequence is introduced at the N-terminus of the kappa light chain to support cloning. Preferably, this additional Ala-Ser sequence is then removed during further development to produce a canonical N-terminal sequence. Thus, in one embodiment, the antibodies containing the kappa light chain described herein do not contain the Ala-Ser sequence at their N-terminus; that is, SEQ ID NO: 74, 76-78, and 80-85 do not include the initial Ala-Ser sequence. In a further embodiment, SEQ ID NO: 74 and 76-78 do not include the initial Ala-Ser sequence. It should be understood that this embodiment also applies to other sequences included herein that contain this sequence (e.g., SEQ ID NO: 86, 88-90, and 92-97).

[0310] Further amino acid alterations can be made to support cloning. For example, for the antibody described herein, a valine-to-alanine change was introduced at the kappa light chain variable domain / constant domain boundary to support cloning. This resulted in modification of the kappa constant domain. Specifically, this led to the modification of the constant domain-initiated RT. A AAPS (from the NotI restriction site). Preferably, this sequence can be modified during further development to produce RT. V The canonical kappa light chain constant region begins with AAPS. Therefore, in one embodiment, the antibody containing the kappa light chain described herein contains a constant structural domain beginning with the sequence RTV. Thus, in one embodiment, the sequence RTV of SEQ ID NO: 111-114 and 117-122... A AAPS was sequence RT V AAPS replacement.

[0311] Lambda light chain embellishment

[0312] Similar to the kappa example above, the lambda light chain variable domain can also be cloned by introducing a restriction site (e.g., Nhe1-Not1) during reformatting to full-length IgG. More specifically, an additional Ala-Ser sequence can be introduced at the N-terminus of the lambda light chain to support cloning. Preferably, this additional AS sequence is then removed during further development to produce a canonical N-terminal sequence. Thus, in one embodiment, the antibodies containing the lambda light chain described herein do not contain an AS sequence at their N-terminus; that is, SEQ ID NO:75 and 79 do not include the initial AS sequence. It should be understood that this embodiment also applies to other sequences included herein that contain this sequence (e.g., SEQ ID NO:87, 91, 115, and 116). In one embodiment, SEQ ID NO:75 does not contain the initial six residues; that is, the ASSYEL sequence is removed.

[0313] As another example, for the antibody described in this paper, a lysine-to-alanine change was introduced at the lambda light chain variable / constant domain boundary to support cloning. This resulted in modification of the lambda constant domain. Specifically, this led to a GQP at the constant domain initiation. A AAPS (from the NotI restriction site). Preferably, this sequence can be modified during further development to produce GQP. K The canonical lambda light chain constant region begins with AAPS. Therefore, in one embodiment, the antibody containing the lambda light chain described herein contains a constant structural domain beginning with the sequence GQPK. Thus, in one embodiment, the sequence GQPK of SEQ ID NO: 115 or 116... A AAPS was sequenced by GQP K AAPS replacement.

[0314] Heavy chain modification

[0315] Typically, human variable heavy chain sequences begin with basic glutamine (Q) or acidic glutamate (E). However, both sequences are known to convert to acidic amino acid residues, pyroglutamic acid (pE). The conversion from Q to pE results in a change in the antibody's charge, while the conversion from E to pE does not change the antibody's charge. Therefore, to avoid charge changes over time, one option is to first modify the starting heavy chain sequence from Q to E. Thus, in one embodiment, the antibody heavy chain described herein contains a Q-to-E modification at its N-terminus. Specifically, the initial residues of SEQ ID NO:62, 64, and / or 67-71 can be modified from Q to E. It should be understood that this embodiment is also applicable to other sequences including this sequence (e.g., SEQ ID NO:86, 88, 91-97 and 111, 112, 115, 117-120).

[0316] Furthermore, the C-terminus of the constant domain of IgG1 is terminated with PGK. However, the terminal basic lysine (K) is typically cleaved during expression (e.g., in CHO cells). This, in turn, leads to alterations in the antibody's charge through varying losses of C-terminal lysine residues. Therefore, one option is to remove the lysine first, resulting in a uniform and consistent C-terminal sequence of the heavy chain terminated with PG. Thus, in one embodiment, the heavy chain of the antibody described herein has a terminal K removed from its C-terminus. In particular, the antibodies of the present invention may comprise any of SEQ ID NO: 111-122, wherein the terminal lysine residue has been removed.

[0317] Optional homomorphic modifications

[0318] During antibody discovery, specific human allotypes can be used. Optionally, antibodies can be converted to different human allotypes during development. As a non-limiting example, for the kappa chain, there are three human allotypes, referred to as Km1, Km1,2, and Km3, which define three Km alleles (using allotype numbering): Km1 is associated with valine 153 (IMGTV45.1) and leucine 191 (IMGT L101); Km1,2 is associated with alanine 153 (IMGT A45.1) and leucine 191 (IMGTL101); and Km3 is associated with alanine 153 (IMGT A45.1) and valine 191 (IMGT V101). Optionally, the sequence can therefore be modified from one allotype to another using standard cloning methods. For example, L191V (IMGTL101V) changes the Km1,2 allotype to the Km3 allotype. For further reference on such allotypes, see Jefferis and Lefranc (2009) MAbs 1(4):332-8, which is incorporated herein by reference.

[0319] Therefore, in one embodiment, the antibody described herein contains an amino acid substitution derived from another human allotype of the same gene. In a further embodiment, the antibody contains an L191V (IMGT L101V) substitution of the kappa chain to convert the c-domain from the km1,2 to the km3 allotype.

[0320] antibody binding

[0321] As measured by surface plasmon resonance, the antibody or its fragments can be smaller than 1.5 x 10⁻⁶. -7 The binding affinity (KD) of M (i.e., 150 nM) binds to the Vδ1 chain of γδTCR. In a preferred embodiment, the KD is less than 1.5 x 10⁻⁶. -7 M (i.e., 150 nM). In another embodiment, KD is 1.3 x 10⁻⁶. -7 M (i.e., 130nM) or smaller, such as 1.0x10 -7 M (i.e., 100nM) or smaller. In yet another embodiment, KD is less than 5.0 x 10⁻⁶. -8 M (i.e., 50nM), for example, less than 4.0 x 10 -8 M (i.e., 40nM) is less than 3.0 x 10 -8 M (i.e., 30nM) or less than 2.0x10 -8 M (i.e., 20 nM). For example, according to one aspect, a human anti-Vδ1 antibody is provided, as measured by surface plasmon resonance, which has a molecular weight of less than 1.5 x 10⁻⁶. -7 The binding affinity (KD) of M (i.e., 150 nM) binds to the Vδ1 chain of γδTCR.

[0322] In one implementation, as measured by surface plasmon resonance, the antibody or a fragment thereof is less than 4.0 x 10⁻⁶. -8 M (i.e., 40nM) is less than 3.0 x 10 -8 M (i.e., 30nM) or less than 2.0x10 -8 The binding affinity (KD) of M (i.e., 20nM) binds to the Vδ1 chain of γδTCR.

[0323] In one embodiment, the binding affinity of the antibody or a fragment thereof is established by coating the antibody or a fragment thereof onto a sensor surface (e.g., an amine high-capacity chip or equivalent) directly or indirectly (e.g., by capture with anti-human IgG Fc), wherein the target bound by the antibody or fragment thereof (i.e., the Vδ1 chain of γδTCR) flows through the chip to detect binding. Suitablely, the MASS-2 instrument (also known as the Sierra SPR-32) is used at 25°C in PBS + 0.02% Tween 20 running buffer at a rate of 30 μl / min.

[0324] This article describes other assays that can be used to define antibody function. For example, the antibodies or fragments thereof described herein can be evaluated by γδTCR binding, for example, by measuring the downregulation of γδTCR after antibody binding. For example, the surface expression of γδTCR after application of the antibody or fragment thereof (optionally present on the cell surface) can be measured by flow cytometry. The antibodies or fragments thereof described herein can also be evaluated by measuring γδT cell degranulation. For example, the expression of the cell degranulation marker CD107a after application of the antibody or fragment thereof (optionally present on the cell surface) to γδT cells can be measured by flow cytometry. The antibodies or fragments thereof described herein can also be evaluated by measuring γδT cell killing activity (to test whether the antibody has an effect on the killing activity of γδT cells). For example, target cells and γδT cells can be incubated in the presence of the antibody or fragment thereof (optionally present on the cell surface). After incubation, the culture can be stained with a cell viability dye to distinguish between live and dead target cells. Then, for example, the proportion of dead cells can be measured by flow cytometry.

[0325] As described herein, antibodies or fragments thereof used in the assay can be present on surfaces, such as cell surfaces, including the surface of cells containing Fc receptors. For example, antibodies or fragments thereof can be present on THP-1 cells, such as TIB-202. TM The surface of cells (available from the American Type Culture Collection (ATCC)). Alternatively, antibodies or fragments thereof can be used directly in the assay.

[0326] In this type of functional assay, the output, also known as "EC50" or "50% effective concentration," can be measured by calculating the half-maximal concentration. The term "IC50" refers to the inhibitory concentration. Both EC50 and IC50 can be measured using methods known in the art, such as flow cytometry. For the avoidance of doubt, the EC50 values ​​in this application are provided using antibodies in IgG1 format. These values ​​can be readily converted to equivalent values ​​based on the molecular weight of the antibody form, as follows:

[0327] (μg / ml) / (MW in kDa) = μM

[0328] Upon antibody (or fragment) binding, the EC50 of γδTCR downregulation can be less than 0.50 μg / ml, for example, less than 0.40 μg / ml, 0.30 μg / ml, 0.20 μg / ml, 0.15 μg / ml, 0.10 μg / ml, or 0.05 μg / ml. In a preferred embodiment, the EC50 of γδTCR downregulation upon antibody (or fragment) binding is less than 0.10 μg / ml. Specifically, the EC50 of γδTCR downregulation upon antibody (or fragment) binding can be less than 0.06 μg / ml, for example, less than 0.05 μg / ml, 0.04 μg / ml, or 0.03 μg / ml. Specifically, the EC50 value is measured when the antibody is in IgG1 format. For example, the EC50 γδTCR downregulation value can be measured using flow cytometry (e.g., as described in the assay of Example 6).

[0329] After antibody (or fragment) binding, the EC50 of γδT cell degranulation can be less than 0.050 μg / ml, for example, less than 0.040 μg / ml, 0.030 μg / ml, 0.020 μg / ml, 0.015 μg / ml, 0.010 μg / ml, or 0.008 μg / ml. Specifically, after antibody (or fragment) binding, the EC50 of γδT cell degranulation can be less than 0.005 μg / ml, for example, less than 0.002 μg / ml. In a preferred embodiment, after antibody (or fragment) binding, the EC50 of γδT cell degranulation is less than 0.007 μg / ml. Specifically, the EC50 value is measured when the antibody is in IgG1 format. For example, the EC50 value of γδT cell degranulation can be measured by detecting CD107a expression (i.e., a marker of cell degranulation) using flow cytometry (e.g., as described in the assay of Example 7). In one implementation, CD107a expression is measured using an anti-CD107a antibody, such as anti-human CD107a BV421 (clone H4A3) (BD Biosciences).

[0330] Upon antibody (or fragment) binding, the EC50 for γδT cell killing can be less than 0.50 μg / ml, for example, less than 0.40 μg / ml, 0.30 μg / ml, 0.20 μg / ml, 0.15 μg / ml, 0.10 μg / ml, or 0.07 μg / ml. In a preferred embodiment, upon antibody (or fragment) binding, the EC50 for γδT cell killing is less than 0.10 μg / ml. Specifically, upon antibody (or fragment) binding, the EC50 for γδT cell killing can be less than 0.060 μg / ml, for example, less than 0.055 μg / ml, particularly less than 0.020 μg / ml or 0.010 μg / ml. Specifically, the EC50 value is measured when the antibody is in IgG1 form. For example, the EC50 γδT cell killing value can be measured by detecting the proportion of dead cells using flow cytometry (i.e., using a cell viability dye) after incubation of the antibody, γδT cells, and target cells (e.g., as described in the assay in Example 8). In one embodiment, the cell viability dye used to measure target cell death is Viability Dye eFluor. TM 520 (ThermoFisher).

[0331] In the assays described in these aspects, the antibody or a fragment thereof may be present on the surface of cells, for example, in THP-1 cells, such as TIB-202. TM The surface of (ATCC). THP-1 cells are optionally labeled with dyes, such as CellTracker. TM Orange CMTMR (ThermoFisher) tag.

[0332] Antibodies (or fragments) can be obtained and manipulated using techniques disclosed, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012), 4th edition, Cold Spring Harbour Laboratory Press.

[0333] Monoclonal antibodies can be produced using hybridoma technology, which involves fusing B cells that produce specific antibodies with myeloma (B-cell carcinoma) cells that are capable of growing in tissue culture and do not have antibody chain synthesis.

[0334] For example, monoclonal antibodies against a specific antigen can be obtained in the following ways:

[0335] a) Using immortalized cells, preferably myeloma cells, to immortalize lymphocytes obtained from the peripheral blood of animals previously immunized with a defined antigen, in order to form a hybridoma.

[0336] b) Immortalized cells (hybridomas) formed through culture are recovered to produce antibodies with the desired specificity.

[0337] Alternatively, hybridoma cells are not required. Antibodies capable of binding to the target antigens described herein can be isolated from a suitable antibody library using routine practices, such as phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Therefore, monoclonal antibodies can be obtained, for example, by a method comprising the following steps:

[0338] a) Cloning DNA or cDNA sequences obtained from lymphocytes, especially peripheral blood lymphocytes, of animals (appropriately previously immunized with a defined antigen) into vectors, especially bacteriophages, and even more so filamentous bacteriophages.

[0339] b) Transform prokaryotic cells using the above-mentioned vector, under conditions that allow antibody production.

[0340] c) Select antibodies by antigen affinity selection.

[0341] d) Recover antibodies with the required specificity.

[0342] Pharmaceutical Composition

[0343] According to another aspect of the invention, a composition comprising a population of Vδ1T cells obtained by the methods defined herein is provided. In one embodiment, the Vδ1T cell population is an expanded population of Vδ1T cells. In such embodiments, the composition may comprise cells, optionally in combination with other excipients. Also included is a composition comprising one or more additional active agents (e.g., active agents suitable for treating the diseases mentioned herein).

[0344] Pharmaceutical compositions may comprise Vδ1T cells as described herein, particularly expanded Vδ1T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions that can be used for the pharmaceutical compositions of the present invention include, for example, DMSO. For example, the compositions may be formulated for intravenous administration.

[0345] In one embodiment, the pharmaceutical composition is substantially free of, for example, contaminants such as endotoxins or mycoplasma, at detectable levels.

[0346] The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal). In one preferred embodiment, the composition is administered by intravenous infusion or injection. In another preferred embodiment, the composition is administered by intramuscular or subcutaneous injection.

[0347] The pharmaceutical compositions of the present invention may be used as adjuncts to or in combination with other established therapies commonly used to treat such diseases, or as part of a treatment method for the diseases described herein, within the scope of the present invention.

[0348] In another aspect of the invention, cell populations, compositions, or pharmaceutical compositions are administered sequentially, simultaneously, or separately with at least one active agent.

[0349] Therapies using cell populations

[0350] According to another aspect of the invention, cell populations obtained by the methods defined herein are provided for use as pharmaceuticals. According to another aspect of the invention, expanded cell populations as defined herein are provided for use as pharmaceuticals. References herein to cell populations “used” as pharmaceuticals or for treatment are limited to administering the cell populations to a subject. Such use does not include the direct administration of antibodies or fragments thereof to a patient, i.e., where said antibodies are used as therapeutic agents.

[0351] In one embodiment, the cell population is used to treat cancer, infectious diseases, or inflammatory diseases. In a further embodiment, the cell population is used to treat cancer.

[0352] In one embodiment, the cell population used as the drug comprises more than 50% Vδ1T cells, such as more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% Vδ1T cells. In another embodiment, the cell population used as the drug consists of Vδ1T cells.

[0353] In one embodiment, the cell population used as the drug comprises less than 10% αβT cells, for example, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, or less than 3% αβT cells. In one embodiment, the cell population used as the drug comprises less than 10% Vδ2T cells, for example, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, or less than 3% Vδ2T cells. In one embodiment, the cell population used as the drug comprises less than 50% NK cells, for example, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% NK cells. In one embodiment, less than 50% of the cells present in the cell population used as the drug express CD56, for example, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the cells express CD56.

[0354] According to another aspect of the invention, a pharmaceutical composition is provided for use as a medicine, said pharmaceutical composition comprising a cell population as defined herein. In one embodiment, the pharmaceutical composition comprising a cell population is used to treat cancer, an infectious disease, or an inflammatory disease. In a further embodiment, the pharmaceutical composition comprising a cell population is used to treat cancer.

[0355] According to another aspect of the invention, a method for modulating an immune response in a subject in need is provided, the method comprising administering a therapeutically effective amount of a cell population as defined herein.

[0356] According to another aspect of the invention, a method is provided for treating cancer, infectious diseases, or inflammatory diseases in a subject with such need, the method comprising administering a therapeutically effective amount of a cell population as defined herein. Alternatively, administering a therapeutically effective amount of a pharmaceutical composition comprising a cell population.

[0357] According to other aspects of the invention, the use of cell populations as defined herein in the preparation of medicaments, such as medicaments for treating cancer, infectious diseases, or inflammatory diseases, is provided.

[0358] Adoptive T-cell therapy

[0359] γδT cells obtained by the expansion method of the present invention can be used as drugs, for example, as drugs for adoptive T-cell therapy. This involves transferring γδT cells into a patient. The treatment can be autologous, i.e., γδT cells can be transferred back to the same patient from which they were obtained, or the treatment can be allogeneic, i.e., γδT cells from one person can be transferred into a different patient. In cases involving allogeneic transfer, the γδT cells may be substantially devoid of αβT cells. For example, after expansion, αβT cells can be depleted from the γδT cell population using any suitable method known in the art (e.g., by negative selection, e.g., using magnetic beads). One treatment method may include: providing a sample obtained from a donor individual (e.g., a non-hematopoietic tissue sample); culturing the γδT cells obtained from the sample as described herein, e.g., to produce an expanded population; and administering the γδT cell population to a recipient individual.

[0360] Patients or subjects to be treated are preferably human cancer patients (e.g., human cancer patients undergoing treatment for solid tumors) or patients with viral infections (e.g., patients with CMV or HIV infection). In some cases, patients have been and / or are currently receiving treatment for solid tumors. Because Vδ1T typically resides in non-hematopoietic tissues, tissue-resident Vδ1T is more likely to home to and remain within the tumor mass compared to its systemic blood-based counterpart, and adoptive transfer of these cells may more effectively target solid tumors and potentially other non-hematopoietic tissue-related immunopathologies.

[0361] Because γδT cells are non-MHC-restricted, they cannot be recognized as foreign by the host to which they are transferred, meaning they are unlikely to cause graft-versus-host disease. This means they can be used "off the job" and transferred to any recipient, for example, for allogeneic adoptive T-cell therapy.

[0362] γδT cells obtained by the methods described herein can express NKG2D and respond to NKG2D ligands (e.g., MICA), which are closely associated with malignant tumors. They may also express a cytotoxic profile without any activation, and therefore may be effective at killing tumor cells. For example, γδT cells obtained as described herein may express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granzyme A and B, and perforin in the absence of any activation. IL-17A may not be expressed.

[0363] In some embodiments, a method of treating an individual with a tumor may include: providing a sample of the tumor obtained from a donor individual, culturing γδT cells obtained from the sample as described above, and administering a population of γδT cells to the individual with the tumor. In a further embodiment, a method of treating an individual with a tumor in non-hematopoietic tissue may include: providing a sample of the non-hematopoietic tissue obtained from a donor individual, culturing γδT cells obtained from the sample as described above, and administering a population of γδT cells to the individual with the tumor.

[0364] In some cases, a therapeutically effective amount of γδT cells obtained by any of the methods described above can be administered to a subject in a therapeutically effective amount (e.g., for the treatment of cancer, such as solid tumors). In some cases, a therapeutically effective amount of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) is less than 10 x 10^6 cells. 12 Cells per dose (e.g., less than 9 x 10) 12 Cells per dose, less than 8 x 10 12 Cells per dose, less than 7 x 10 12 Cells per dose, less than 6 x 10 12 Cells per dose, less than 5 x 10 12 Cells per dose, less than 4 x 10 12 Cells per dose, less than 3 x 10 12 Cells per dose, less than 2 x 10 12 Cells per dose, less than 1x10 12 Cells per dose, less than 9 x 10 11 Cells per dose, less than 8 x 10 11 Cells per dose, less than 7 x 10 11Cells per dose, less than 6 x 10 11 Cells per dose, less than 5 x 10 11 Cells per dose, less than 4 x 10 11 Cells per dose, less than 3 x 10 11 Cells per dose, less than 2 x 10 11 Cells per dose, less than 1x10 11 Cells per dose, less than 9 x 10 10 Cells per dose, less than 7.5 x 10-1 10 Cells per dose, less than 5 x 10 10 Cells per dose, less than 2.5 x 10-1 10 Cells per dose, less than 1x10 10 Cells per dose, less than 7.5 x 10-1 9 Cells per dose, less than 5 x 10 9 Cells per dose, less than 2.5 x 10-1 9 Cells per dose, less than 1x10 9 Cells per dose, less than 7.5 x 10-1 8 Cells per dose, less than 5 x 10 8 Less than 2.5 x 10 cells per dose. 8 Cells per dose, less than 1x10 8 Cells per dose, less than 7.5 x 10-1 7 Cells per dose, less than 5 x 10 7 Less than 2.5 x 10 cells per dose. 7 Cells per dose, less than 1x10 7 Cells per dose, less than 7.5 x 10-1 6 Cells per dose, less than 5 x 10 6 Less than 2.5 x 10 cells per dose. 6 Cells per dose, less than 1x10 6 Cells per dose, less than 7.5 x 10-1 5 Cells per dose, less than 5 x 10 5 Less than 2.5 x 10 cells per dose. 5 Cells per dose, or less than 1x10 5 (cells per dose).

[0365] In some implementations, the therapeutically effective amount of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) is less than 10 x 10^6 cells during the treatment course. 12 Cells (e.g., less than 9x10 during the treatment course) 12 1 cell, less than 8 x 10 12 1 cell, less than 7 x 1012 1 cell, less than 6 x 10 12 1 cell, less than 5 x 10 12 1 cell, less than 4 x 10 12 1 cell, less than 3 x 10 12 1 cell, less than 2 x 10 12 1 cell, less than 1x10 12 1 cell, less than 9x10 11 1 cell, less than 8 x 10 11 1 cell, less than 7 x 10 11 1 cell, less than 6 x 10 11 1 cell, less than 5 x 10 11 1 cell, less than 4 x 10 11 1 cell, less than 3 x 10 11 1 cell, less than 2 x 10 11 1 cell, less than 1x10 11 1 cell, less than 9x10 10 1 cell, less than 7.5 x 10 10 1 cell, less than 5 x 10 10 1 cell, less than 2.5 x 10 10 1 cell, less than 1x10 10 1 cell, less than 7.5 x 10 9 1 cell, less than 5 x 10 9 1 cell, less than 2.5 x 10 9 1 cell, less than 1x10 9 1 cell, less than 7.5 x 10 8 1 cell, less than 5 x 10 8 1 cell, less than 2.5 x 10 8 1 cell, less than 1x10 8 1 cell, less than 7.5 x 10 7 1 cell, less than 5 x 10 7 1 cell, less than 2.5 x 10 7 1 cell, less than 1x10 7 1 cell, less than 7.5 x 10 6 1 cell, less than 5 x 10 6 1 cell, less than 2.5 x 10 6 1 cell, less than 1x10 6 1 cell, less than 7.5 x 10 5 1 cell, less than 5 x 10 5 1 cell, less than 2.5 x 10 5 1 cell or less than 1x10 5 (cells).

[0366] In some embodiments, a dose of γδT cells as described herein (e.g., skin-derived γδT cells and / or Vδ1T cells) contains approximately 1 x 102 6 1.1x10 6 2x10 6 3.6x10 6 5x10 6 1x10 7 1.8x10 7 2x10 7 5x10 7 1x10 8 2x10 8 or 5x10 8 Cells / kg. In some embodiments, one dose of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) contains up to about 1 x 10⁻⁶ cells / kg. 6 1.1x10 6 2x10 6 3.6x10 6 5x10 6 1x10 7 1.8x10 7 2x10 7 5x10 7 1x10 8 2x10 8 or 5x10 8 Cells / kg. In some embodiments, one dose of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) contains approximately 1.1 x 10⁻⁶ cells / kg. 6 -1.8x10 7 Cells / kg. In some embodiments, one dose of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) contains approximately 1 x 10⁻⁶ cells / kg. 7 2x10 7 5x10 7 1x10 8 2x10 8 5x10 8 1x10 9 2x10 9 or 5x10 9 1 x 103 cells. In some embodiments, one dose of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) contains at least about 1 x 103 cells. 7 2x10 7 5x10 7 1x10 8 2x10 85x10 8 1x10 9 2x10 9 or 5x10 9 1 x 103 cells. In some embodiments, one dose of γδT cells (e.g., skin-derived γδT cells and / or Vδ1T cells) contains up to about 1 x 103 cells. 7 2x10 7 5x10 7 1x10 8 2x10 8 5x10 8 1x10 9 2x10 9 or 5x10 9 Each cell.

[0367] In one implementation scheme, the subject was administered 10 4 Up to 10 6 γδT cells (e.g., 10 per kilogram of subject body weight) 4 Up to 10 6 (10 skin-derived γδT cells and / or Vδ1T cells). In one implementation, the subject receives an initial dose of γδT cell population (e.g., 10 cells per kilogram of body weight initially administered to the subject). 4 Up to 10 6 γδT cells, for example, 10 per kilogram of body weight initially administered to the subject. 4 Up to 10 5 One or more γδT cells), and one or more subsequent administrations of γδT cells (e.g., 2, 3, 4 or 5 times per kilogram of body weight per subject). 4 Up to 10 6 γδT cells, for example, 10 per kilogram of body weight in the subject. 4 Up to 10 5 (Number of γδT cells). In one embodiment, one or more subsequent administrations are administered less than 15 days after a previous administration, for example, less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after a previous administration, for example, less than 4, 3, or 2 days after a previous administration. In one embodiment, the subject receives approximately 10 γδT cell populations per kilogram of subject body weight in total during at least three administrations. 6 One γδT cell, for example, the subject receives an initial dose of 1x10 5 γδT cells, 3x10 for the second administration 5 10 γδT cells, and the third administration of 6x10 5 1,000 γδT cells, and, for example, each application is given less than 4, 3 or 2 days after the previous application.

[0368] In some implementations, one or more additional therapeutic agents may be administered to the subject. These additional therapeutic agents may be selected from immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, anti-angiogenic agents, or combinations of two or more thereof. The additional therapeutic agent may be administered concurrently with, before, or after γδT cells. The additional therapeutic agent may be an immunotherapeutic agent that acts on a target (e.g., the subject's own immune system) and / or metastatic γδT cells within the subject.

[0369] The composition can be administered in any convenient manner. The composition described herein can be administered to the patient via artery, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous, or intraperitoneal injection, for example, via intradermal or subcutaneous injection. Components of γδT cells can be injected directly into tumors, lymph nodes, or sites of infection.

[0370] Genetic engineering

[0371] The γδT cells obtained by the method of this invention can also be genetically engineered to enhance therapeutic properties, such as for chimeric antigen receptor T-cell (CAR-T) therapy. This involves generating engineered T-cell receptors (TCRs) to reprogram T cells with novel specificities, such as the specificity of monoclonal antibodies. Engineered TCRs can make T cells specific for malignant cells and thus usable for cancer immunotherapy. For example, T cells can recognize cancer cells expressing tumor antigens, such as tumor-associated antigens not expressed by normal somatic cells from a subject's tissue. Therefore, CAR-modified T cells can be used, for example, in adoptive T-cell therapy for cancer patients.

[0372] Other uses of antibodies or fragments thereof

[0373] According to another aspect of the invention, the use of the anti-Vδ1 antibody or fragment thereof described herein in studying the antigen recognition, activation, signal transduction, or function of γδT cells (particularly Vδ1T cells) is provided. As described herein, the antibody has been shown to be active in assays that can be used to study γδT cell function. Such antibodies can also be used to induce the proliferation of γδT cells, and therefore can be used in methods for expanding γδT cells (e.g., Vδ1T cells).

[0374] Antibodies that bind to the Vδ1 chain can be used to detect γδT cells (i.e., as markers). Preferably, the antibody used as a marker does not stimulate cell proliferation, so that the target Vδ1T cells are unaffected after antibody binding. For example, the antibody can be a detectable marker or reporter molecule marker, or used as a capture ligand to selectively detect and / or isolate Vδ1T cells in a sample. The labeled antibody can be used in many methods known in the art, such as immunohistochemistry and ELISA.

[0375] The detectable marker or reporter molecule can be a radioactive isotope, for example 3 H, 14 C 32 P, 35 S, or 125 l; a fluorescent or chemiluminescent component, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. The fluorescent labeling applied to the antibodies of this invention can then be used in the fluorescent activated cell sorting (FACS) method.

[0376] Polynucleotides and expression vectors

[0377] Also provided are polynucleotides encoding the anti-Vδ1 antibody or fragment of the present invention. In one embodiment, the anti-Vδ1 antibody or fragment is encoded by a polynucleotide comprising, or consisting of, a sequence having at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99% sequence identity with SEQ ID NO: 99-110. In one embodiment, the anti-Vδ1 antibody or fragment is encoded by an expression vector comprising the VH region of SEQ ID NO: 99-110. In another embodiment, the anti-Vδ1 antibody or fragment is encoded by an expression vector comprising the VL region of SEQ ID NO: 99-110. In a further embodiment, the polynucleotide comprises, or consists of, SEQ ID NO: 99-110. In another aspect, cDNA comprising said polynucleotide is provided.

[0378] In one embodiment, the polynucleotide comprises, or is composed of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO: 99-110. In one embodiment, the expression vector comprises the VH region of SEQ ID NO: 99-110. In another embodiment, the expression vector comprises the VL region of SEQ ID NO: 99-110. In a further embodiment, the polynucleotide comprises, or is composed of, SEQ ID NO: 99-110. In another aspect, cDNA comprising said polynucleotide is provided.

[0379] In one embodiment, the polynucleotide comprises, or is composed of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity with SEQ ID NO: 99-101 or 105-108. In one embodiment, the expression vector comprises the VH region of SEQ ID NO: 99-101 or 105-108. In another embodiment, the expression vector comprises the VL region of SEQ ID NO: 99-101 or 105-108. In a further embodiment, the polynucleotide comprises, or is composed of, SEQ ID NO: 99-101 or 105-108. In another aspect, cDNA comprising said polynucleotide is provided.

[0380] In one embodiment, the polynucleotide comprises, or is composed of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, sequence identity with SEQ ID NO: 99-101. In one embodiment, the expression vector comprises the VH region of SEQ ID NO: 99-101. In another embodiment, the expression vector comprises the VL region of SEQ ID NO: 99-101. In a further embodiment, the polynucleotide comprises, or is composed of, SEQ ID NO: 99-101. In another aspect, cDNA comprising said polynucleotide is provided.

[0381] In one embodiment, the polynucleotide comprises, or is composed of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, sequence identity with any portion of SEQ ID NO: 99-110, said portion encoding CDR1, CDR2, and / or CDR3 of the encoded immunoglobulin chain variable domain. In one embodiment, the polynucleotide comprises, or is composed of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, sequence identity with any portion of SEQ ID NO: 99-101 or 105-108, said portion encoding CDR1, CDR2, and / or CDR3 of the encoded immunoglobulin chain variable domain. In one embodiment, the polynucleotide comprises, or consists of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with any portion of SEQ ID NO: 99-101, said any portion encoding CDR1, CDR2 and / or CDR3 of the encoded immunoglobulin chain variable domain.

[0382] In one embodiment, the polynucleotide comprises, or consists of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, sequence identity with any portion of SEQ ID NO: 99-110, said portion encoding FR1, FR2, FR3, and / or FR4 of the encoded immunoglobulin chain variable domain. In one embodiment, the polynucleotide comprises, or consists of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, sequence identity with any portion of SEQ ID NO: 99-101 or 105-108, said portion encoding FR1, FR2, FR3, and / or FR4 of the encoded immunoglobulin chain variable domain. In one embodiment, the polynucleotide comprises, or consists of, a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with any portion of SEQ ID NO: 99-101, said any portion encoding FR1, FR2, FR3 and / or FR4 of the encoded immunoglobulin chain variable domain.

[0383] The polynucleotides and expression vectors of the present invention can also be described with reference to the encoded amino acid sequences. Thus, in one embodiment, the polynucleotide comprises, or is composed of, a sequence encoding an amino acid sequence of any one of SEQ ID NO: 62 to 85. In one embodiment, the expression vector comprises a sequence encoding an amino acid sequence of any one of SEQ ID NO: 62 to 73. In another embodiment, the expression vector comprises a sequence encoding an amino acid sequence of any one of SEQ ID NO: 74 to 85.

[0384] To express an antibody or a fragment thereof, a polynucleotide encoding the portion or full-length light and heavy chains as described herein is inserted into an expression vector, such that the gene is operatively linked to transcriptional and translational control sequences. Therefore, in one aspect of the invention, an expression vector comprising a polynucleotide sequence as defined herein is provided. In one embodiment, the expression vector comprises a VH region of SEQ ID NO: 99-110, such as SEQ ID NO: 99, 100, 101, 105, 106, 107, or 108. In another embodiment, the expression vector comprises a VL region of SEQ ID NO: 99-110, such as SEQ ID NO: 99, 100, 101, 105, 106, 107, or 108.

[0385] It should be understood that the nucleotide sequences described herein include additional sequences encoding amino acid residues to aid in translation, purification, and detection; however, depending on the expression system used, alternative sequences may be used. For example, the initial (5'-terminal) nine nucleotides of SEQ ID NO: 99-110 and the final (3'-terminal) 36 nucleotides of SEQ ID NO: 99-100, 102-103, 105-110, or the final (3'-terminal) 39 nucleotides of SEQ ID NO: 101 and 104 are optional sequences. These optional sequences may be deleted, modified, or substituted if an alternative design, translation, purification, or detection strategy is employed.

[0386] Mutations can be made in the DNA or cDNA encoding polypeptides. These mutations silence the polypeptide's amino acid sequence but provide preferred codons for translation in a particular host. Preferred codons for nucleic acid translation in, for example, *E. coli* and *Saccharomyces cerevisiae*, as well as in mammals, particularly humans, are known.

[0387] Mutations in polypeptides can be achieved, for example, by substituting, adding, or deleting nucleic acids encoding the polypeptide. Substitution, addition, or deletion of nucleic acids encoding the polypeptide can be introduced through numerous methods, including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive integration mutagenesis, exponential integration mutagenesis, site-specific mutagenesis, gene recombination, artificial gene synthesis, site-saturated mutagenesis (GSSM), synthetic ligation recombination (SLR), or combinations thereof. Modification, addition, or deletion of nucleic acids can also be introduced through: recombination, recursive sequence recombination, phosphate-thioester modified DNA mutagenesis, uracil-containing template mutagenesis, nick duplex mutagenesis, site-mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiation-induced mutagenesis, deletion mutagenesis, restriction selection mutagenesis, restriction purification mutagenesis, integration mutagenesis, chimeric nucleic acid multimer production, or combinations thereof.

[0388] Specifically, artificial gene synthesis can be used. The gene encoding the polypeptide of the present invention can be synthesized, for example, through solid-phase DNA synthesis. The entire gene can be synthesized de novo without precursor template DNA. To obtain the desired oligonucleotide, building units are sequentially coupled to the growing oligonucleotide chain according to the required product sequence. After chain assembly, the product is released from the solid phase into solution, deprotected, and collected. The product can be separated by high-performance liquid chromatography (HPLC) to obtain the desired high-purity oligonucleotide.

[0389] Expression vectors include, for example, plasmids, retroviruses, kinases, yeast artificial chromosomes (YAC), and Epstein-Barr virus (EBV)-derived episomes. A polynucleotide is ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating polynucleotide transcription and translation. Expression and / or control sequences may include promoters, enhancers, transcription terminators, a start codon (i.e., ATG) at the 5' end of the coding sequence, intron splicing signals, and stop codons. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. SEQ ID NO:99-110 contains nucleotide sequences encoding single-stranded variable fragments of the present invention, comprising VH and VL regions linked by a synthetic adapter (e.g., encoding SEQ ID NO:98). It should be understood that the polynucleotides or expression vectors of the present invention may contain VH regions, VL regions, or both (optionally including adapters). Therefore, polynucleotides encoding VH and VL regions can be inserted into separate vectors, or sequences encoding both regions can be inserted into the same expression vector. The polynucleotide is inserted into the expression vector using standard methods (e.g., ligating complementary restriction sites on the polynucleotide and the vector, or ligating blunt ends if no restriction sites are present).

[0390] A convenient vector is one that encodes a fully functional human CH or CL immunoglobulin sequence with suitable engineered restriction sites, allowing for the easy insertion and expression of any VH or VL sequence, as described herein. Expression vectors can also encode signal peptides that promote the secretion of antibodies (or fragments thereof) from host cells. Polynucleotides can be cloned into the vector to link the signal peptide within the N-terminal frame of the antibody. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0391] The host cell may contain a first vector encoding a light chain of an antibody or a fragment thereof, and a second vector encoding a heavy chain of an antibody or a fragment thereof. Alternatively, the heavy and light chains encoded on the same expression vector may be introduced into the host cell. In one embodiment, the polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to an antibody or a fragment thereof, wherein the antibody or a fragment thereof is present on the extracellular surface of the host cell.

[0392] Transformation can be performed using any known method to introduce polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well-known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, gene gun injection, and direct microinjection of DNA into the cell nucleus. Furthermore, nucleic acid molecules can be introduced into mammalian cells via viral vectors.

[0393] Mammalian cell lines suitable for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, 3T3 cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Antigen-binding fragments of antibodies, such as scFv and Fv fragments, can be isolated and expressed in *E. coli* using methods known in the art.

[0394] Antibodies are produced by culturing host cells for a period of time sufficient to allow antibody expression in the host cells, or more preferably, sufficient to allow antibody secretion into the culture medium in which the host cells grow. The antibodies can be recovered from the culture medium using standard protein purification methods.

[0395] The antibodies (or fragments) of the present invention can be obtained and manipulated using techniques disclosed, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012), 4th edition, Cold Spring Harbour Laboratory Press.

[0396] Monoclonal antibodies can be produced using hybridoma technology, which involves fusing B cells that produce specific antibodies with myeloma (B-cell carcinoma) cells that are capable of growing in tissue culture and do not have antibody chain synthesis.

[0397] For example, monoclonal antibodies against a specific antigen can be obtained in the following ways:

[0398] a) Using immortalized cells, preferably myeloma cells, to immortalize lymphocytes obtained from the peripheral blood of animals previously immunized with a defined antigen, in order to form a hybridoma.

[0399] b) Immortalized cells (hybridomas) formed through culture are recovered to produce antibodies with the desired specificity.

[0400] Alternatively, hybridoma cells are not required. Antibodies capable of binding to the target antigens described herein can be isolated from a suitable antibody library using routine practices, such as phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Therefore, monoclonal antibodies can be obtained, for example, by a method comprising the following steps:

[0401] a) Cloning DNA or cDNA sequences obtained from lymphocytes, especially peripheral blood lymphocytes, of animals (appropriately previously immunized with a defined antigen) into vectors, especially bacteriophages, and even more so filamentous bacteriophages.

[0402] b) Transform prokaryotic cells using the above-mentioned vector, under conditions that allow antibody production.

[0403] c) Select antibodies by antigen affinity selection.

[0404] d) Recover antibodies with the required specificity.

[0405] It should be understood that all embodiments described herein can be applied to all aspects of the present invention.

[0406] Other features and advantages of the invention will be apparent from the description provided herein. However, it should be understood that while the description and specific examples indicate preferred embodiments of the invention, they are given by way of illustration only, as various changes and modifications will be apparent to those skilled in the art. The invention will now be described using the following non-limiting embodiments:

[0407] Example

[0408] Example 1: Materials and Methods

[0409] Human antibody discovery

[0410] Human phage display was used to generate human anti-human variable Vδ1+ domain antibodies as described herein. The library was constructed according to the description in Schofield et al. (Genome biology 2007, 8(11):R254) and contained a library of approximately 40 billion human clones displaying single-chain variable fragments (scFv). The library was screened using antigen, methods, selection, deselection, screening, and characterization strategies as described herein.

[0411] Antigen preparation

[0412] The design of the soluble γδTCR heterodimer containing TCRα and TCRβ constant regions used in the following examples is based on that produced by Xu et al. (2011) PNAS 108:2414-2419. The Vγ or Vδ domain is fused within the TCRα or TCRβ constant region frame lacking a transmembrane domain, followed by a leucine zipper sequence or Fc sequence and a histidine tag / connector.

[0413] The expression construct was transiently transfected into mammalian EXPI HEK293 suspension cells (as a heterodimer, either as a single transfection or a co-transfection). The secreted recombinant protein was recovered and purified from the culture supernatant by affinity chromatography. To ensure good recovery of the monomeric antigen, the sample was further purified using preparative size exclusion chromatography (SEC). The purity of the purified antigen was analyzed by SDS-PAGE, and the aggregation state was analyzed by analytical SEC.

[0414] Antigen function verification

[0415] The specificity of antigens containing the delta variable 1 (Vδ1) chain was confirmed in the DELFIA immunoassay (Perkin Elmer) and in a flow cytometry-based assay competing with γδT cells using the REA173-Miltenyi Biotec anti-Vδ1 antibody.

[0416] Dissociation-enhanced fluorescence immunoassay for lanthanides (DELFIA)

[0417] To confirm the specificity of the antigen, the DELFIA immunoassay was performed using the antigen directly coated on the plate. 3 μg / mL antigen was incubated overnight at 4°C in 50 μL PBS (Nunc #437111), and the primary antibody was serially diluted starting at 300 nM. DELFIA Eu-N1 anti-human IgG (Perkin Elmer #1244-330) was used as the secondary antibody, diluted 1 / 500 in 50 μL 3% MPBS (PBS + 3% (w / v) skim milk powder) for detection. Colorimetric development was performed using 50 μL of DELFIA enhancement solution (Perkin Elmer #4001-0010).

[0418] Affinity ranking of the target antibodies was performed using the DELFIA immunoassay, in which the antibodies were captured by protein G coated on the plate, and soluble biotinylated L1 (DV1-GV4) antigen was added at 5 nM to 50 μL (3M PBS). For detection, 50 μL of streptavidin-Eu (1:500 in assay buffer, Perkin Elmer) was used, and a signal was generated using DELFIA enhancement solution. D1.3 hIgG1 (described in England et al. (1999) J. Immunol. 162:2129-2136) was used as a negative control.

[0419] Phage display-selected subclones were fed into the scFv expression vector pSANG10 (Martin et al. (2006) BMC Biotechnol. 6:46). Soluble scFv was expressed and screened for binding to directly immobilized targets in DELFIA. A hit was defined as a DELFIA signal exceeding 3000 fluorescent units.

[0420] Antibody preparation

[0421] A selected scFv subclone was inserted into the IgG1 framework using a commercially available plasmid. The plasmid was then used to transfect expi293F suspension cells for antibody expression. For convenience, unless otherwise stated, the antibodies characterized in these examples refer to antibodies selected from phage-displayed IgG1 format as scFv. However, the antibodies of the present invention can be any antibody format as described above.

[0422] Antibody purification

[0423] IgG antibodies were purified in batches from the supernatant using protein A chromatography. The concentrated protein A eluent was then purified using size exclusion chromatography (SEC). The quality of the purified IgG was analyzed using ELISA, SDS-PAGE, and SEC-HPLC.

[0424] Preparation of γδT cells

[0425] Enriched γδT cell populations were prepared according to the methods described in WO2016 / 198480 (i.e., blood-derived γδT cells) or WO2020 / 095059 (i.e., skin-derived γδT cells). Briefly, for blood-derived γδT cells, PBMCs were obtained from blood and subjected to magnetic depletion of αβT cells. The αβ-depleted PBMCs were then cultured for 7 days in CTS OpTmiser medium (ThermoFisher) in the presence of OKT-3 (or the corresponding anti-Vδ1 antibody), IL-4, IFN-γ, IL-21, and IL-1β. On day 7 of culture, the medium was supplemented with OKT-3 (or the corresponding anti-Vδ1 antibody), IL-21, and IL-15, and maintained for another 4 days. On day 11 of culture, the medium was supplemented with OKT-3 (or the corresponding anti-Vδ1 antibody) and IL-15, and maintained for another 3 days. On day 14 of culture, half of the culture medium was replaced with fresh, complete OpTmiser and supplemented with OKT-3 (or the corresponding anti-Vδ1 antibody), IL-15, and IFN-γ. Starting from day 17 of culture, OKT-3 (or the corresponding anti-Vδ1 antibody) and IL-15 were supplemented into the culture every 3 to 4 days; every 7 days, half of the culture medium was replaced with fresh culture medium.

[0426] For skin-derived γδT cells, skin samples were prepared by removing subcutaneous fat and then punched multiple times using a 3mm biopsy punch. The punch was placed on a carbon matrix grid and inserted into the wells of a G-REX6 (Wilson Wolf) plate. Each well was filled with complete separation medium containing AIM-V medium (Gibco, Life Technologies), CTS immune serum substitute (Life Technologies), IL-2, and IL-15. For the first 7 days of culture, complete separation medium containing amphotericin B (Life Technologies) (“+AMP”) was used. Every 7 days, the medium was changed by gently aspirating the top medium and replacing it with 2X complete separation medium (without AMP), taking care not to disturb the cells at the bottom of the plate or bioreactor. After more than 3 weeks of culture, the resulting egressed cells were passaged into fresh tissue culture containers and fresh medium (e.g., AIM-V medium or TexMAX medium (Miltenyi)) with recombinant IL-2, IL-4, IL-15, and IL-21, and then harvested. Optionally, αβT cells also present in the culture may be removed with the aid of an αβT cell depletion kit and related protocols (e.g., those provided by Miltenyi). Further reference is available in WO2020 / 095059.

[0427] γδT cell binding assay

[0428] The binding of the antibody to γδT cells was tested by incubating 250,000 γδT cells with a fixed concentration of purified antibody. This incubation was performed under closed conditions to prevent nonspecific binding of the antibody via the Fc receptor. Detection was performed by adding a secondary, fluorescently conjugated anti-human IgG1 antibody. For negative controls, cells were prepared using a) only isotype antibody (recombinant human IgG), b) only fluorescently conjugated anti-human IgG antibody, and c) a combination of a) and b). Control wells with completely unstained cells were also prepared and analyzed. As positive controls, purified mouse monoclonal IgG2 anti-human CD3 antibody and purified mouse monoclonal IgG1 anti-human TCR Vδ1 antibody were used at two different concentrations and stained with a fluorescently conjugated goat anti-mouse secondary antibody. The assay was accepted if the mean fluorescence intensity of the lower concentration of the positive control in the FITC channel was at least 10 times that of the highest negative control.

[0429] SPR measurement method

[0430] SPR assays were performed using a MASS-2 instrument (Sierra Sensors, Germany) equipped with an amine high-capacity chip. 15 nM IgG was captured into the amine high-capacity chip via Protein G (100 nM for TS8.2). L1 (DV1-GV4) antigen, serially diluted 1:2 from 2000 nM to 15.625 nM, was flowed through cells with the following parameters: 180 sec binding, 600 sec dissociation, flow rate 30 μL / min, and run buffer PBS + 0.02% Tween 20. All experiments were performed at room temperature on the MASS-2 instrument. Steady-state fit was determined using Sierra Analyzer 3.2 software based on Langmuir 1:1 binding.

[0431] Comparator antibodies

[0432] As described, the antibody is compared with a commercially available antibody in the assay.

[0433]

[0434] γδTCR downregulation and threshing assay

[0435] Using CellTracker TM Orange CMTMR (ThermoFisher, C2927) labeled with THP-1 (TIB-202) loaded or unloaded with the test antibody TMγδT cells were incubated with ATCC target cells at a 2:1 ratio in the presence of CD107a antibody (anti-human CD107a BV421 (clone H4A3) BDBiosciences 562623). After 2 hours of incubation, the surface expression of γδTCR on γδT cells (to measure TCR downregulation) and the expression of CD107a (to measure degranulation) were assessed by flow cytometry.

[0436] Lethality test

[0437] Gamma delta T cell cytotoxic activity was assessed by flow cytometry, and the effect of antibodies on γδ T cell cytotoxic activity was tested. γδ T cells were co-cultured in vitro at a 20:1 ratio with CellTracker. TM Orange CMTMR (ThermoFisher, C2927) labeled THP-1 cells (with or without antibody) were used 4 hours later with Viability DyeeFluor TM 520 (ThermoFisher, 520 65-0867-14) staining was used to distinguish between live and dead target THP-1 cells. During sample collection, target cells were stained with CellTracker... TM Gating was performed on Orange CMTMR-positive cells, and cell death was examined based on Viability Dye uptake. CMTMR and eFluor TM 520 double-positive cells were identified as target cells for death. The cytotoxic activity of γδT cells is expressed as the percentage of target cells that died.

[0438] Epitope plotting

[0439] The protein integrity and aggregation levels of all protein samples used for epitope mapping (antigen L1 (DV1-GV4) and antibodies 1245_P01_E07, 1245_P02_G04, 1252_P01_C08, 1251_P02_C05 and 1141_P01_E01) were analyzed using high-quality MALDI.

[0440] To determine the epitopes of the L1(DV1-GV4) / 1245_P01_E07, L1(DV1-GV4) / 1245_P02_G04, L1(DV1-GV4) / 1252_P01_C08, L1(DV1-GV4) / 1251_P02_C05, and L1(DV1-GV4) / 1141_P01_E01 complexes at high resolution, the protein complexes were incubated with a deuterated cross-linking agent and subjected to multienzymatic proteolysis using trypsin, chymotrypsin, Asp-N, elastase, and thermophilic protease. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-LTQ-Orbitrap MS), and the generated data were analyzed using XQuest and Stavrox software.

[0441] SYTOX-Flow Cytometry Kill Assay

[0442] The SYTOX assay allows for the quantification of T cell-mediated target cell lysis using flow cytometry. This is achieved through the use of dead cell staining agents (…). AADvanced TM Life Technologies (S10274) detects dead / dying cells. This staining agent only penetrates into cells with damaged plasma membranes but cannot cross the intact cell membranes of healthy cells. CTV dye (Cell Trace Violet) is used to detect these cells. TM NALM-6 is labeled with Life Technologies (C34557) to target cells, thus distinguishing them from unlabeled effector T cells. Dead / dying target cells are identified by dual staining with dead cell dye and cell-tracking dye.

[0443] Effector cells and CTV-labeled target cells were co-cultured in vitro for 16 hours at a specified effector-to-target ratio (E:T, 1:1, or 10:1), and then... AADvanced TM Staining cells and in FACSLyric TM Data was collected on (BD). Kill results are expressed as a % reduction in target cells, calculated considering the number of viable target cells in the test sample (sample count) relative to the number of viable target cells in the control wells without effector cells (maximum count):

[0444]

[0445] Example 2 Antigen Design

[0446] Gamma delta (γδ) T cells are polyclonal cells with CDR3 polyclonality. To avoid the selection of antibodies against the CDR3 sequence (since the CDR3 sequence differs between TCR clones), antigen design involves maintaining CDR3 consistency across different formats. This design aims to generate antibodies that recognize sequences within variable domains, which are germline-encoded and therefore identical across all clones, thus providing antibodies that recognize a broader subset of γδ T cells.

[0447] Another important aspect of antigen preparation is designing antigens suitable for protein expression. γδTCR is a complex protein consisting of heterodimers with inter- and intra-chain disulfide bonds. Soluble TCR antigens were generated using leucine zipper (LZ) and Fc formats for selection of phage display. Both LZ and Fc formats expressed TCRs well and successfully displayed TCRs (especially heterodimer TCRs, e.g., Vδ1Vγ4).

[0448] The CDR3 sequence of γδTCR from a public database entry was found to be well expressed as a protein (RCSB ProteinData Bank entries: 3OMZ). Therefore, it was selected for antigen preparation.

[0449] Antigens containing a delta variable 1 chain are expressed in LZ format as heterodimers (i.e., combined with different gamma variable chains—“L1”, “L2”, “L3”) and in Fc format as heterodimers (“F1”, “F2”, “F3”) or homodimers (i.e., combined with another delta variable 1 chain—“Fc1 / 1”). All antigens contain a 3OMZ CDR3 in their delta variable 1 chains. Another series of γδTCR antigens using a similar format are designed to contain different delta variable chains (e.g., delta variable 2 and delta variable 3) and are used to deselect antibodies with nonspecific or off-target binding (“L4”, “F9”, “Fc4 / 4”, “Fc8 / 8”). These antigens are also designed to include a 3OMZ CDR3 to ensure that antibodies binding in the CDR3 region are also deselected.

[0450] Antigen function validation was performed to confirm that the designed antigen would be suitable for generating anti-TRDV1 (TCR delta variable 1) antibodies. Detection was only performed on antigens containing the δ1 domain. Figure 1 ).

[0451] Example 3: Phage Display

[0452] In rounds 1 and 2, phage display selection was performed on the human scFv library using the heterodimeric LZ TCR format, with heterodimeric LZ TCR deselected in both rounds. Alternatively, round 1 was performed using the homodimeric Fc fusion TCR, with human IgG1 Fc deselected, followed by round 2 using the heterodimeric LZ TCR, with heterodimeric LZ TCR deselected (see Table 1).

[0453] Table 1 summarizes the phage display selection.

[0454] target Round 1 selection Cancel selection in round 1 Round 2 selection Cancel selection in round 2 DV1 bt-L1(DV1-GV4) L4(DV2-GV4) bt-L3(DV1-GV8) L4(DV2-GV4) DV1 bt-Fc1 / 1(DV1-DV1) Fc bt-L1(DV1-GV4) L4(DV2-GV4)

[0455] bt = Biotin.

[0456] Selection was performed using 100 nM biotinylated protein in the solution phase. Deselection was performed using 1 μM non-biotinylated protein.

[0457] Successful phage display selection was analyzed using a polyclonal phage ELISA (DELFIA). All DV1 selection outputs showed the expected binding to the targets Fc 1 / 1, L1, L2, L3, F1, and F3. Binding to varying degrees to non-targets L4, F9, Fc 4 / 4, Fc 8 / 8, and Fc was detected (see [link to DELFIA]). Figure 2 (A and B).

[0458] Example 4 Antibody Selection

[0459] The hits obtained in Example 3 were sequenced (using standard methods known in the art). 130 unique clones were identified, exhibiting unique combinations of VH and VL CDR3. Of these 130 unique clones, 125 exhibited unique VH CDR3 and 109 exhibited unique VL CDR3.

[0460] The unique clones were rearranged and their specificity was analyzed by ELISA (DELFIA). A group of 94 unique human scFv conjugates were identified from the selection that bind to TRDV1 (L1, L2, L3, F1, F2, F3) but not to TRDV2 (L4).

[0461] The affinity ranking of the selected conjugates was included to aid in the selection of subsequent clones. A large number of conjugates showed affinity in the nanomolar range, reacting with 25 to 100 nM biotinylated antigens. A few conjugates showed strong reactions with 5 nM antigens, indicating the possibility of single-digit nanomolar affinities. Some conjugates did not react with 100 nM antigens, indicating affinity in the micromolar range.

[0462] To select clones for IgG conversion, the aim is to include as many pedigrees and as many different CDR3s as possible. Furthermore, sequence biases such as glycosylation, integrin binding sites, CD11c / CD18 binding sites, and unpaired cysteine ​​residues are avoided. In addition, multiple affinities are also considered.

[0463] Clones selected were screened using skin-derived γδT cells obtained from different donors and bound to γδTCRs expressed on the surface of the cells. Clones selected to convert to IgG are shown in Table 2.

[0464]

[0465] Example 5: Antibody SPR Analysis

[0466] The prepared IgG antibodies were analyzed using a γδ cell binding assay, and five optimal binding compounds were selected for further functional and biophysical characterization. SPR analysis was performed to determine the equilibrium dissociation constant (K0). D The sensor plot showing the interaction between the antibody and the analyte, and the steady-state fit (if any) displayed. Figure 3 No binding of TS8.2 to 80RU IgG captured on the chip was detected. The results are summarized in Table 3.

[0467] Table 3 Results of IgG capture

[0468] Analytes Cloned ID <![CDATA[K D (nM)]]> <![CDATA[K D (M)]]> L1(DV1-GV4) 1245_P01_E07 12.4 1.24e-08 L1(DV1-GV4) 1252_P01_C08 100 1.00e-07 L1(DV1-GV4) 1245_P02_G04 126 1.26e-07 L1(DV1-GV4) 1245_P01_B07 341 3.41e-07 L1(DV1-GV4) 1251_P02_C05 1967* 1.97e-06 L1(DV1-GV4) 1139_P01_E04 251 2.51e-07 L1(DV1-GV4) 1245_P02_F07 193 1.93e-07 L1(DV1-GV4) 1245_P01_G06 264 2.64e-07 L1(DV1-GV4) 1245_P01_G09 208 2.08e-07 L1(DV1-GV4) 1138_P01_B09 290 2.90e-07 L1(DV1-GV4) 1251_P02_G10 829 8.29e-07 L1(DV1-GV4) TS8.2 (commercial anti-Vδ1 antibody) 44 4.40e-08

[0469] The binding of *1252_P02_C05 has not reached saturation, therefore the extrapolated data...

[0470] Example 6: TCR binding assay

[0471] The inventors designed several assays for the functional characterization of selected antibodies. The first assay assesses γδTCR binding by measuring the downregulation of γδTCRs after antibody binding. The selected antibody is tested against commercially available anti-CD3 and anti-Vδ1 antibodies used as positive controls, or against 1252_P01_C08 (for 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) used as a positive control. A commercially available anti-panγδ antibody is used as a negative control because it is a panγδ antibody that recognizes all γδT cells, regardless of the variable chain, and therefore may have a different mode of action.

[0472] The assay was performed using skin-derived γδT cells obtained from three different donor samples (sample purities of 94%, 80%, and 57%). Results showed... Figure 4 The EC50 values ​​are summarized in Table 4 below.

[0473] Example 7: T cell degranulation assay

[0474] The second assay assessed the degranulation of γδT cells. γδT cells are thought to potentially mediate target cell killing through perforin-granzyme-mediated apoptotic activation. Lysogenic granules within the cytoplasm of γδT cells can be released to target cells upon T cell activation. Therefore, labeling target cells with CD107a antibody and measuring its expression by flow cytometry can be used to identify degranulated γδT cells.

[0475] For Example 6, selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies used as positive controls, or against 1252_P01_C08 (for 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) used as positive controls. IgG2a, IgG1, and D1.3 antibodies were used as negative controls. Assays were performed using skin-derived γδT cells obtained from three different donor samples (sample purities of 94%, 80%, and 57%). Results were shown... Figure 5 The EC50 values ​​are summarized in Table 4 below.

[0476] Example 8: Lethality Test

[0477] The third assay assessed the ability of γδT cells activated with the selected antibody to kill target cells.

[0478] For Example 6, selected antibodies were tested against commercial anti-CD3 and anti-Vδ1 antibodies used as positive controls, or against 1252_P01_C08 (for 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) used as positive controls, and against anti-panγδ used as a negative control. Antibodies with IgG2a, IgG1, and D1.3 were also used as isotype controls. Assays were performed using skin-derived γδ T cells (94% and 80% purity) obtained from two donors, and results showed... Figure 6 middle.

[0479] The results of the three functional assays tested in Examples 6-8 are summarized in Table 4.

[0480] Table 4 Summary of results obtained from the functional testing method

[0481]

[0482]

[0483] N / D: Undeterminable; N / D*: Undeterminable, titration curve did not reach plateau; N / D**: Killing curve decreased, EC50 not established.

[0484] Example 9: Epitope Mapping

[0485] To identify epitopes of the antigen / antibody complex at high resolution, the protein complex was incubated with a deuterated cross-linking agent and subjected to multi-enzyme cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-LTQ-Orbitrap MS), and the generated data were analyzed using XQuest (version 2.0) and Stavrox (version 3.6) software.

[0486] After proteolytic hydrolysis of the protein complex L1(DV1-GV4) / 1245_P01_E07 with deuterated d0d12 by trypsin, chymotrypsin, Asp-N, elastase, and thermophilic protease, nLC-orbitrap MS / MS analysis detected 13 cross-linked peptides between L1(DV1-GV4) and antibody 1245_P01_E07. The results showed Figure 7 middle.

[0487] After proteolytic hydrolysis of the protein complex L1(DV1-GV4) / 1252_P01_C08 containing deuterated d0d12 by trypsin, chymotrypsin, Asp-N, elastase, and thermophilic protease, nLC-orbitrap MS / MS analysis detected five cross-linked peptides between L1(DV1-GV4) and antibody 1252_P01_C08. The results showed Figure 8 middle.

[0488] After proteolytic hydrolysis of the protein complex L1(DV1-GV4) / 1245_P02_G04 with deuterated d0d12 by trypsin, chymotrypsin, Asp-N, elastase, and thermophilic protease, nLC-orbitrap MS / MS analysis detected 20 cross-linked peptides between L1(DV1-GV4) and antibody 1245_P02_G04. The results showed Figure 9 middle.

[0489] After proteolytic hydrolysis of the protein complex L1(DV1-GV4) / 1251_P02_C05 containing deuterated d0d12 by trypsin, chymotrypsin, Asp-N, elastase, and thermophilic protease, nLC-orbitrap MS / MS analysis detected five cross-linked peptides between L1(DV1-GV4) and antibody 1252_P01_C05. The results showed Figure 10 middle.

[0490] Epitope binding with another antibody, clone ID 1141_P01_E01, was also tested. Following proteolysis of the protein complex L1(DV1-GV4) / 1141_P01_E01 with deuterated d0d12 by trypsin, chymotrypsin, Asp-N, elastase, and thermophilic protease, nLC-orbitrap MS / MS analysis detected 20 cross-linked peptides between L1(DV1-GV4) and antibody 1141_P01_E01. Results showed... Figure 11 middle.

[0491] The results of the epitope plotting are summarized in Table 5.

[0492] Table 5. Epitope mapping results of antigen / antibody complexes

[0493]

[0494]

[0495] Example 10: Expansion of Vδ1T cells

[0496] The expansion of isolated γδT cells was studied in the presence of selected antibodies and comparator antibodies. The comparator antibodies were selected from: OKT3 anti-CD3 antibody as a positive control, no antibody as a negative control, or IgG1 antibody as an isotype control. Commercially available anti-Vδ1 antibody, TS-1, and TS8.2 were also tested for comparison.

[0497] Experiment 1:

[0498] As described in "Preparation of γδT Cells" for blood-derived γδT cells in Example 1, preliminary studies were conducted by seeding 70,000 cells / well and using a complete optimizer and cytokines. Selected antibodies and comparator antibodies were tested at various concentrations ranging from 4.2 ng / ml to 420 ng / ml. This experiment was performed using tissue culture plates, which allow antibodies to be bound / immobilized on plastic.

[0499] Cells were harvested on days 7, 14, and 18, and the total cell count was determined using a cell counter (NC250, ChemoMetec). Results are as follows: Figure 12 As shown. Cell viability of Vδ1T cells was also measured at each harvest, and all antibodies indicated that cell viability was maintained throughout the experiment (data not shown). On day 18, the percentage, cell count, and fold change of Vδ1T cells were also analyzed. Results are as follows... Figure 13 As shown.

[0500] exist Figure 12As can be seen, the total number of cells produced in the antibody-containing culture steadily increased throughout the culture process, and was comparable to or better than that of commercial anti-Vδ1 antibodies. On day 18, the proportion of Vδ1 positive cells was greater in the presence of antibodies at most of the tested concentrations of 1245_P02_G04 (“G04”), 1245_P01_E07 (“E07”), 1245_P01_B07 (“B07”), and 1252_P01_C08 (“C08”) than in cultures containing OKT3, TS-1, or TS8.2 control antibodies (see [link to relevant documentation]). Figure 13 A).

[0501] Experiment 2:

[0502] As described in “Preparation of γδT Cells” in Example 1, the isolated cells were subjected to subsequent experiments in a culture vessel containing cytokines. A different culture vessel was used compared to Experiment 1, with a surface unfavorable for antibody binding / fixation. Selected antibodies and comparator antibodies were tested at various concentrations from 42 pg / ml to 42 ng / ml. In Experiment 2, results were obtained from triplicate experiments.

[0503] Cells were harvested on days 7, 14, and 17, and the total cell count was determined using a cell counter as previously described. Results are as follows: Figure 14 As shown. On day 17, the percentage, cell count, and fold change of Vδ1T cells were also analyzed. The results are as follows. Figure 15 As shown.

[0504] Cell composition, including non-Vδ1 cells, was also measured in Experiment 2. Cells were harvested on day 17 and the surface expression of Vδ1, Vδ2, and αβTCR was analyzed by flow cytometry. The proportion of each cell type in each culture is shown graphically. Figure 16 The percentage values ​​are provided in Table 6.

[0505] Table 6 Cell composition on day 17 - Percentage of viable cells in each subgroup

[0506] αβ-γδ- Vδ1 Vδ2 Non-Vδ1 / Vδ2 αβ No AB 63.00 18.17 0.86 7.10 0.37 OKT-3 25.63 50.43 0.25 20.13 1.13 IgG1 65.77 15.59 1.11 6.91 0.42 TS8.2 42ng / ml 30.60 53.57 3.59 7.46 0.14 TS-1 42ng / ml 18.77 65.90 0.91 9.51 0.12 C08 42ng / ml 0.79 96.43 0.08 2.51 0.05 C08 4.2ng / ml 1.91 94.67 0.18 2.63 0.05 C08 420pg / ml 8.47 80.57 0.28 8.42 0.04 C08 42pg / ml 35.97 25.93 3.04 19.50 0.31 B07 42ng / ml 0.94 95.57 0.46 2.73 0.05 B07 4.2ng / ml 1.79 94.10 0.40 3.28 0.01 B07 420pg / ml 3.08 91.80 0.29 3.94 0.02 B07 42pg / ml 17.93 62.90 0.85 9.16 0.07 E07 42ng / ml 2.29 85.13 0.19 11.65 0.04 E07 4.2ng / ml 2.15 91.23 0.13 5.77 0.04 E07 420pg / ml 9.25 73.90 0.42 13.05 0.02 E07 42pg / ml 49.23 18.67 2.17 7.70 0.43 G04 42ng / ml 1.90 88.53 0.47 8.09 0.05 G04 4.2ng / ml 4.25 89.67 0.93 3.98 0.02 G04 420pg / ml 25.97 50.60 1.45 12.72 0.11 G04 42pg / ml 44.00 13.77 2.33 26.30 0.32 C05 42ng / ml 25.00 42.03 3.75 13.67 1.32 C05 4.2ng / ml 46.87 22.03 2.58 16.46 0.38 C05 420pg / ml 33.53 44.60 2.23 11.13 0.22 C05 42pg / ml 36.83 25.23 6.16 18.00 0.30

[0507] These results show that cultures containing B07, C08, E07, and G04 had a higher proportion of Vδ1-positive cells compared to the OKT3, TS-1, or TS8.2 controls. Therefore, the tested antibodies were more effective than commercially available antibodies in generating and amplifying Vδ1-positive cells, even when present at low concentrations in the cultures.

[0508] Other cellular markers from cells on day 17 of Experiment 2 were also analyzed, including CD3-CD56+, to identify natural killer (NK) cells and the presence of Vδ1T cells expressing CD27 (i.e., CD27+). The results are summarized in Table 7.

[0509] Table 7. Cell composition on day 17 - percentage of NK and CD27+ cells.

[0510]

[0511]

[0512] SEM: Standard error of the mean

[0513] Example 11: Functional properties of Vδ1T cells

[0514] Vδ1T cells expanded in the presence of selective antibodies retained a polyclonal library of the CDR3 region, and their functionality was also tested using a SYTOX flow cytometry killing assay. Results of cells obtained in Experiment 1 on day 14 using an effector-target (E:T) ratio of 10:1 are shown. Figure 17 A), and the results of cells obtained in Experiment 2, on day 17 (after freeze-thaw), using E:T ratios of 1:1 and 10:1. Figure 17 B).

[0515] exist Figure 17 The results show that Vδ1-positive cells amplified in the presence of all antibodies effectively lyse target cells, indicating that they remain functional even after freezing and thawing.

[0516] Example 12: Functional properties of stored cells

[0517] The functionality of cells after a freeze-thaw storage procedure was also investigated. A subset of cells was removed from the culture on day 17 of Experiment 2 and frozen. The cells were then thawed and further expanded in culture with IL-15. Figure 18 This shows the total cell count of cultures that were contacted with antibodies B07, C08, E07, G04, or OKT-3 before freezing, 7 days after freeze-thaw. All cultures showed proliferative capacity after storage. Culture continued until day 42, during which time the total cell count was monitored (results are shown in [image missing]). Figure 19 In cultures previously exposed to the selected antibody, the total cell number remains or increases.

[0518] Example 13: Anti-Vδ1 antibody confers regulation and proliferation of immune cells in TIL

[0519] Studies were conducted to explore the regulation and proliferation of human tumor-infiltrating lymphocytes (TILs) embodied by anti-Vδ1 antibodies. For these studies, human renal cell carcinoma (RCC) tumor biopsy samples were shipped fresh and processed upon receipt. Specifically, tissue was cut into pieces approximately 2 mm in size. 2Add up to 1g of tissue along with 4.7mL of RPMI and enzymes from the Miltenyi Tumor Dissociation Kit at the manufacturer's recommended concentration to each Miltenyi C tube, except for Enzyme R, which is used at a 0.2x concentration to prevent cleavage of relevant cell surface molecules. Place the C tubes in a gentleMACS heater. TM On Octo Dissociator. The program 37C_h_TDK_1 for isolating soft tumors was selected. The digest was then filtered through a 70 mM filter to produce a single-cell suspension. RPMI containing 10% FBS was added to the digest to quench enzyme activity. Cells were washed twice with RPMI / 10% FBS and resuspended for counting. Derived cells were then seeded at 2.5 x 10e6 cells per well in TC wells (24-well G-REX, WilsonWolf). Cells were then incubated for 18 days with or without cytokines and with or without antibodies. Antibodies included in the study are outlined in […]. Figure 20 These antibodies included OKT3 (up to 50 ng / ml) and 1252_P01_C08, also referred to herein as "C08" (up to 500 ng / ml). When included, these antibodies were added in single-bolus additions on days 0, 7, 11, and 14. During the incubation period, the medium was replaced with fresh medium on days 11 and 14. Flow cytometry analysis was performed on days 0 and 18 to determine lymphocyte phenotype and fold changes in cell number. Cells were first gated on live CD45+ cells, then as shown. In the group including recombinant cytokines, the following were added: Day 0: IL-4, IFN-γ, IL-21, IL-1β. Additional IL-15 was included on days 7, 11, and 14. Additional IL-21 and IFN-γ were included on days 7 and 14, respectively. Figure 20 (A) shows the fold increase in TIL Vδ1+ cells after 18 days of culture in the presence of CO8 or OKT3, with and without cytokine support (CK). These results demonstrate that the use of CO8 or the comparator OKT3 antibody significantly increases the fold increase of TIL Vδ1+ cells in the presence of cytokines, compared to antibodies or cytokines alone. Figure 20 (B) shows an increase in the total number of Vδ1 cells after harvest. These results demonstrate that, in the presence of cytokines, culture with CO8 or comparator OKT3 antibodies significantly increases the number of TIL Vδ1+ cells compared to antibodies or cytokines alone. Figure 20(C) illustrates an example gating strategy used for flow cytometry analysis. Lymphocyte gating was applied to cells from a live CD45+ cell population based on their forward and side-scattering characteristics (not shown), and γδT cells were then separated from αβT cells by staining for T cell receptors. Finally, the proportion of Vδ1 cells in the total γδT cell population was determined. Example data from day 18 showed the following under the two conditions (+ / -1252_P01_C08): 64.3% of the cells were CD45+, of which 53.1% were γδ+, and among the γδ cells, 89.7% were Vδ1+. Figure 20 (D) shows the cell surface phenotypic profile of TIL Vδ1+ cells at harvest. Higher levels of CD69 were observed after culturing with the C08 antibody. Figure 20 (E) shows the fractionation of live CD45-positive TILs at harvest, including γδ-negative and CD8-positive lymphocytes. In summary, the combined results highlight the regulatory role of the anti-Vδ1 antibody of the present invention in conferring TIL populations.

Claims

1. A method for expanding Vδ1 T cells in vitro, the method comprising administering an anti-Vδ1 antibody or a fragment thereof to a cell population containing Vδ1 T cells, said anti-Vδ1 antibody or fragment thereof comprising: The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 38, CDR2 with the sequence shown in SEQ ID NO: 26, and CDR3 with the sequence shown in SEQ ID NO: 2; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 50, CDR2 with the sequence shown in VAS, and CDR3 with the sequence shown in SEQ ID NO: 14; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 39, CDR2 with the sequence shown in SEQ ID NO: 27, and CDR3 with the sequence shown in SEQ ID NO: 3; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 51, CDR2 with the sequence YDS, and CDR3 with the sequence shown in SEQ ID NO: 15; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 40, CDR2 with the sequence shown in SEQ ID NO: 28, and CDR3 with the sequence shown in SEQ ID NO: 4; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 52, CDR2 with the sequence of DAS, and CDR3 with the sequence shown in SEQ ID NO: 16; The VH region contains CDR1 with the sequence shown in SEQ ID NO: 41, CDR2 with the sequence shown in SEQ ID NO: 29, and CDR3 with the sequence shown in SEQ ID NO: 5; and the VL region contains CDR1 with the sequence shown in SEQ ID NO: 53, CDR2 with the sequence AAS, and CDR3 with the sequence shown in SEQ ID NO: 17; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 42, CDR2 with the sequence shown in SEQ ID NO: 30, and CDR3 with the sequence shown in SEQ ID NO: 6; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 54, CDR2 with the sequence of DAS, and CDR3 with the sequence shown in SEQ ID NO: 18; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 43, CDR2 with the sequence shown in SEQ ID NO: 31, and CDR3 with the sequence shown in SEQ ID NO: 7; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 55, CDR2 with the sequence shown in EVS, and CDR3 with the sequence shown in SEQ ID NO: 19; The VH region contains CDR1 with the sequence shown in SEQ ID NO: 44, CDR2 with the sequence shown in SEQ ID NO: 32, and CDR3 with the sequence shown in SEQ ID NO: 8; and the VL region contains CDR1 with the sequence shown in SEQ ID NO: 56, CDR2 with the sequence of DAS, and CDR3 with the sequence shown in SEQ ID NO: 20; The VH region contains CDR1 with the sequence shown in SEQ ID NO: 45, CDR2 with the sequence shown in SEQ ID NO: 33, and CDR3 with the sequence shown in SEQ ID NO: 9; and the VL region contains CDR1 with the sequence shown in SEQ ID NO: 57, CDR2 with the sequence of DAS, and CDR3 with the sequence shown in SEQ ID NO: 21; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 46, CDR2 with the sequence shown in SEQ ID NO: 34, and CDR3 with the sequence shown in SEQ ID NO: 10; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 58, CDR2 with the sequence AAS, and CDR3 with the sequence shown in SEQ ID NO: 22; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 47, CDR2 with the sequence shown in SEQ ID NO: 35, and CDR3 with the sequence shown in SEQ ID NO: 11; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 59, CDR2 with the sequence of DAS, and CDR3 with the sequence shown in SEQ ID NO: 23; The VH region comprises CDR1 with the sequence shown in SEQ ID NO: 48, CDR2 with the sequence shown in SEQ ID NO: 36, and CDR3 with the sequence shown in SEQ ID NO: 12; and the VL region comprises CDR1 with the sequence shown in SEQ ID NO: 60, CDR2 with the sequence of DAS, and CDR3 with the sequence shown in SEQ ID NO: 24; or The VH region contains CDR1 with the sequence shown in SEQ ID NO: 49, CDR2 with the sequence shown in SEQ ID NO: 37, and CDR3 with the sequence shown in SEQ ID NO: 13; and the VL region contains CDR1 with the sequence shown in SEQ ID NO: 61, CDR2 with the sequence AAS, and CDR3 with the sequence shown in SEQ ID NO:

25.

2. The method of claim 1, wherein the antibody or a fragment thereof comprises: The VH region, whose amino acid sequence is shown in SEQ ID NO: 62, and the VL region, whose amino acid sequence is shown in SEQ ID NO: 74; The VH region, whose amino acid sequence is shown in SEQ ID NO: 63, and the VL region, whose amino acid sequence is shown in SEQ ID NO: 75; The VH region, whose amino acid sequence is shown in SEQ ID NO: 64, and the VL region, whose amino acid sequence is shown in SEQ ID NO: 76; The VH region, whose amino acid sequence is shown in SEQ ID NO: 68, and the VL region, whose amino acid sequence is shown in SEQ ID NO: 80; The VH region, whose amino acid sequence is shown in SEQ ID NO: 69, and the VL region, whose amino acid sequence is shown in SEQ ID NO: 81; The VH region, whose amino acid sequence is shown in SEQ ID NO: 70, and the VL region, whose amino acid sequence is shown in SEQ ID NO: 82; or The VH region has an amino acid sequence as shown in SEQ ID NO: 71, and the VL region has an amino acid sequence as shown in SEQ ID NO:

83.

3. The method according to claim 1 or 2, wherein, as measured by surface plasmon resonance, the antibody or fragment thereof has a density of less than 1.5 x 10⁻⁶. -7 The binding affinity of M (KD) binds to the variable delta 1 (Vδ1) chain of the γδT cell receptor (TCR).

4. The method according to claim 1 or 2, wherein the antibody or fragment thereof is scFv, Fab, Fab', F(ab')2, Fv, biantibody, microantibody, or full-length antibody.

5. The method according to claim 1 or 2, wherein the method provides an expanded population of Vδ1 T cells comprising more than 85% Vδ1 T cells.

6. The method of claim 5, wherein the method provides an expanded population of Vδ1 T cells comprising more than 90% Vδ1 T cells.

7. The method according to claim 1 or 2, wherein the method comprises culturing the cell population for at least 5 days.

8. The method according to claim 1 or 2, wherein the method comprises culturing the cell population in the presence of at least one cytokine.

9. The method according to claim 8, wherein the cytokine is selected from: interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 21 (IL-21), or mixtures thereof.

10. The method according to claim 1 or 2, wherein the method comprises culturing the cell population in the presence of IL-2, IL-9 and / or IL-15.

11. The method of claim 1 or 2, wherein the method comprises culturing the cell population in the presence of IL-21.

12. The method of claim 1 or 2, wherein the method comprises culturing the cell population in the presence of IL-4.

13. The method of claim 1 or 2, wherein the method comprises culturing a cell population in a first culture medium containing IL-4 and then culturing the cell population in a second culture medium containing IL-15.

14. The method of claim 13, wherein the first culture medium is free of IL-15, IL-2 and / or IL-7.

15. The method of claim 13, wherein the second culture medium is free of IL-4.

16. The method of claim 13, wherein the first or second culture medium or both culture media contain one or more additional cytokines.

17. The method of claim 16, wherein the additional cytokine is selected from IL-21, IFN-γ, and IL-1β.

18. The method of claim 7, wherein the cell population does not come into direct contact with stromal cells and / or epithelial cells during culture.

19. The method of claim 18, wherein the cell population does not come into direct contact with fibroblasts during culture.

20. The method of claim 7, wherein the cell population does not come into direct contact with tumor cells and / or feeder cells during culture.

21. The method of claim 1 or 2, wherein the method comprises culturing the cell population in a serum-free culture medium.

22. The method of claim 1 or 2, wherein the T cells of the cell population are enriched prior to administration of the antibody or a fragment thereof.

23. The method of claim 1 or 2, wherein the γδT cells of the cell population are enriched prior to administration of the antibody or a fragment thereof.

24. The method according to claim 1 or 2, wherein the αβ T cells or NK cells of the cell population are depleted before the antibody or a fragment thereof is administered.

25. The method of claim 1 or 2, wherein the cell population is obtained from a hematopoietic sample or a fraction thereof.

26. The method according to claim 25, wherein the hematopoietic sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, lymph node tissue or fractions thereof.

27. The method of claim 25, wherein the hematopoietic sample comprises low-density mononuclear cells (LDMCs) or peripheral blood mononuclear cells (PBMCs).

28. The method according to claim 1 or 2, wherein the cell population is obtained from a non-hematopoietic tissue sample.

29. The method of claim 28, wherein the non-hematopoietic tissue is skin, colon, intestine, breast, lung, prostate, liver, pancreas, uterus, vagina or other skin, mucous membrane or serous membrane sample.

30. The method of claim 28, wherein the cell population is obtained from a non-hematopoietic tissue sample by culturing the sample on a synthetic scaffold, the synthetic scaffold being configured to facilitate cell expulsion from the non-hematopoietic tissue sample.

31. The method according to claim 1 or 2, wherein the cell population is obtained from a cancer tissue sample.

32. The method according to claim 1 or 2, wherein the cell population is obtained from human or non-human animal tissue.

33. The method according to claim 1 or 2, wherein the cell population is isolated from the sample prior to the administration of the anti-Vδ1 antibody or a fragment thereof.

Images