Ex vivo γδ T cell population

Abstract

The present invention relates to an ex vivo method for modulating V51 T cells using an anti-V51 antibody or a fragment thereof.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a population of gamma delta T cells contacted with an anti-TCR delta variable 1 (anti-Vδ1) antibody. [Background technology]

[0002] BACKGROUND OF THE INVENTION Growing interest in T cell immunotherapy of cancer has focused on the apparent ability of subsets of CD8+ and CD4+ alphabeta (αβ) T cells to recognize cancer cells and mediate host-protective functional potential, particularly when disinhibited by clinically mediated antagonism of inhibitory pathways mediated by PD-1, CTLA-4, and other receptors. However, αβ T cells are MHC-restricted, which can lead to graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) represent a subset of T cells that express a distinct, definitive γδ T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain, each of which undergoes chain rearrangement but has a limited number of V genes compared to αβ T cells. The main TRG V gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV8, TRGV9, and TRGV11, as well as the nonfunctional TRGV10, TRGV11, TRGVA, and TRGVB. The most common TRG V gene segments encode Vδ1, Vδ2, and Vδ3, as well as several V segments with both Vδ and Vα designations (Adams et al., Cell Immunol. 296:30-40 (2015)). Human γδ T cells can be broadly classified based on their TCR chain, as particular γ and δ types are more commonly, if not exclusively, found in cells in one or more tissue types. For example, most blood-resident γδ T cells express a Vδ2 TCR, commonly Vγ9Vδ2, whereas this is less common among tissue-resident γδ T cells, e.g., in the skin, which in the gut more frequently use a Vδ1 TCR paired with a gamma chain, e.g., often paired with Vγ4.

[0004] To utilize γδ T cells for immunotherapy, either in situ expansion of the cells or ex vivo expansion before harvesting and reinjecting them is required. The latter approach has previously been described using the addition of exogenous cytokines. See, for example, WO2017 / 072367 and WO2018 / 212808. Methods for expanding a patient's own γδ T cells have been described using pharmacologically modified forms of hydroxymethylbut-2-enyl pyrophosphate (HMBPP) or clinically approved aminobisphosphonates. These approaches have apparently safely treated over 250 cancer patients, with rare occurrences of complete remission. However, there remains a need for activating agents with a proven ability to expand large numbers of γδ T cells. Summary of the Invention

[0005] (Summary of the Invention) According to a first aspect of the present invention, there is provided an ex vivo method for modulating V51 T cells, comprising administering to said patient a gene encoding a V51 T cell comprising the amino acid region: (i) SEQ ID NO: 1, 3 to 20; and / or (ii) 37 to 77 of SEQ ID NO: 1 The present invention provides an ex vivo method comprising administering to a population of cells comprising V51 T cells a human anti-TCR delta variable 1 (anti-V51) antibody or fragment thereof that binds to an epitope of the variable delta 1 (V51) chain of the gamma delta T cell receptor (TCR) comprising one or more amino acid residues within

[0006] According to a further aspect of the invention there is provided an ex vivo method for modulating V51 T cells comprising the steps of: a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 25; CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26 to 37 and sequences A1 to A12 (in Table 2); and / or CDR1 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61 an anti-V51 antibody or fragment thereof comprising one or more of:

[0007] According to a further aspect of the present invention there is provided a population of V51 T cells obtained by the ex vivo method defined herein.

[0008] According to a further aspect of the invention there is provided a composition comprising a population of V51 T cells as defined herein.

[0009] According to a further aspect of the invention there is provided a pharmaceutical composition comprising a V51 T cell population as defined herein.

[0010] According to a further aspect of the invention there is provided a method of treating cancer, an infectious disease or an inflammatory disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a V51 T cell population or pharmaceutical composition as defined herein. [Brief explanation of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: ELISA detection of directly coated antigens with anti-Vδ1 Ab (REA173, Miltenyi Biotec). Detection was only seen with antigens containing the Vδ1 domain. The leucine zipper (LZ) format appears to be more potent than the Fc format, which is consistent with cell-based flow competition assays (data not shown). [Figure 2]Figure 2: Polyclonal phage DELFIA data for DV1 selection. A) Heterodimer selection: heterodimeric LZ TCR format in rounds 1 and 2, deselected with heterodimeric LZ TCR in both rounds. B) Homodimer selection: Round 1 is performed with homodimeric Fc fusion TCR and deselected with human IgG1 Fc, followed by round 2 with heterodimeric LZ TCR and deselected with heterodimeric LZ TCR. To represent selection from different libraries, each graph contains two bars for each target. [Figure 3] Figure 3: IgG capture: Left) Sensorgram of the interaction of anti-L1 IgG with L1, Right) Steady-state fit (where possible). All experiments were performed at room temperature on a MASS-2 instrument. Steady-state fitting with Langmuir 1:1 binding. [Figure 4] Figure 4: Results of TCR down-modulation assay for 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). [Figure 5] Figure 5: Results of T cell degranulation assay for 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). [Figure 6]Figure 6: Results of killing assay (THP-1 flow-based assay) for 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). [Figure 7] Figure 7: Epitope mapping data for 1245_P01_E07. Graphical representation of the epitope binding site of 1245_P01_E07 on SEQ ID NO: 1. [Figure 8] Figure 8: Epitope mapping data for 1252_P01_C08. Graphical representation of the epitope binding site of 1252_P01_C08 on SEQ ID NO: 1. [Figure 9] Figure 9: Epitope mapping data for 1245_P02_G04. Graphical representation of the epitope binding site of 1245_P02_G04 on SEQ ID NO: 1. [Figure 10] Figure 10: Epitope mapping data for 1251_P02_C05. Graphical representation of the epitope binding site of 1251_P02_C05 on SEQ ID NO: 1. [Figure 11] Figure 11: Epitope mapping data for 1141_P01_E01. Graphical representation of the epitope binding site of 1141_P01_E01 on SEQ ID NO: 1. [Figure 12] Figure 12: Total cell counts for experiment 1 of Example 10. Samples were cultured with various concentrations of anti-V51 antibodies described herein and compared to samples cultured with a comparator antibody or control. Graphs show total cell counts at (A) day 7, (B) day 14, and (C) day 18. [Figure 13] Figure 13: Analysis of V51 T cells in experiment 1 of example 10. Graphs show (A) percentage of V51 T cells, (B) V51 T cell count, and (C) V51 fold change in day 18 samples. [Figure 14]Figure 14: Total cell counts for experiment 2 of Example 10. Samples were cultured with various concentrations of anti-V51 antibodies described herein and compared to samples cultured with a comparator antibody or control. Graphs show total cell counts at (A) day 7, (B) day 11, (C) day 14, and (D) day 17. [Figure 15] Figure 15: Analysis of V51 T cells in experiment 2 of Example 10. Graphs show (A) percentage of V51 T cells, (B) V51 T cell count, and (C) V51 fold change in day 17 samples. [Figure 16] Figure 16: Cell composition analysis. The cell types present in the samples (including non-V51 cells) were determined on day 17 of experiment 2. Cells were harvested and analyzed by flow cytometry for surface expression of V51, V52, and αβ TCR. Percentage values ​​are also provided in Table 6. [Figure 17] Figure 17: SYTOX-flow killing assay results. Cell functionality was tested using the SYTOX-flow killing assay. Results are presented for (A) experiment 1 on day 14 using cells at an effector-to-target (E:T) ratio of 10:1, and (B) experiment 2 on day 17 (post-freeze-thaw) using cells at E:T ratios of 1:1 and 10:1. [Figure 18] Figure 18: Total cell counts after freeze-thaw. The graph shows total cell counts 7 days after freeze-thawing for cultures that were contacted with B07, C08, E07, G04, or OKT-3 antibodies before freezing. [Figure 19] Figure 19: Monitoring cell expansion. Total cell counts were monitored for cells cultured after freeze-thawing up to 42 days. [Figure 20] Figure 20: Anti-V51 antibodies conferred modulation and proliferation of tumor infiltrating lymphocytes (TIL) in human tumors. Study on renal cell carcinoma (RCC) + / - antibodies. A) Fold increase in TIL V51+ cells. B) Total number of TIL V51+ cells. C) Example of gating strategy. D) Comparative cell surface phenotypic profile of TIL V51+ cells. E) Analysis of TIL V51-negative gated fraction. DETAILED DESCRIPTION OF THE INVENTION

[0012] (Detailed Description of the Invention) (definition) Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0013] Gamma delta (γδ) T cells represent a small subset of T cells that express a distinctive, definitive T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain. Each chain contains a variable (V) region, a constant (C) region, a transmembrane region, and a cytoplasmic tail. The V region contains the antigen-binding site. There are two main subtypes of human γδ T cells: those that predominate in peripheral blood and those that predominate in non-hematopoietic tissues. The two subtypes can be defined by the type of δ and / or γ present on the cell. For example, γδ T cells that predominate in peripheral blood express predominantly the delta variable 2 chain (Vδ2). γδ T cells that predominate in non-hematopoietic tissues (i.e., are tissue-resident) express predominantly the delta variable 1 (Vδ1) chain. Reference to "Vδ1 T cells" refers to γδ T cells that have a Vδ1 chain, i.e., Vδ1. + Refers to T cells.

[0014] Reference to "delta variable 1" may be referred to as V51 or Vd1, while the nucleotides encoding the TCR chain containing this region may be referred to as "TRDV1". Any antibody or fragment thereof that interacts with the V51 chain of a γδ TCR is effectively an antibody or fragment thereof that binds to V51 and may be referred to as an "anti-TCR delta variable 1 antibody or fragment thereof" or an "anti-V51 antibody or fragment thereof".

[0015] Further reference may be made herein to other delta chains, such as the "delta variable 2" chain. These may be referred to in a similar manner. For example, the delta variable 2 chain may be referred to as V52, while the nucleotides encoding the TCR chain containing this region may be referred to as "TRDV2." In preferred embodiments, antibodies or fragments thereof that interact with the V51 chain of a γδ TCR do not interact with other delta chains, such as V52.

[0016] Reference may also be made herein to a "gamma variable chain." These may be referred to as γ-chains or Vγ, while the nucleotides encoding the TCR chain containing this region may be referred to as TRGV. For example, TRGV4 refers to the Vγ4 chain. In preferred embodiments, antibodies or fragments thereof that interact with the Vδ1 chain of a γδ TCR do not interact with a gamma chain such as Vγ4.

[0017] The term "antibody" includes any antibody protein construct comprising at least one antibody variable domain containing at least one antigen-binding site (ABS). Antibodies include, but are not limited to, immunoglobulins of the types IgA, IgG, IgE, IgD, and IgM (and their subtypes). The overall structure of immunoglobulin G (IgG) antibodies, assembled from two identical heavy (H) chain polypeptides and two identical light (L) chain polypeptides, is well established and highly conserved among mammals (Padlan (1994) Mol. Immunol. 31:169-217).

[0018] A conventional antibody or immunoglobulin (Ig) is a protein comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy (H) chain variable domain is abbreviated herein as VH, and the light (L) chain variable domain is abbreviated herein as VL. These domains, domains related to them, and domains derived from them may be referred to herein as immunoglobulin chain variable domains. The VH and VL domains (also referred to as VH and VL regions) can be further divided into regions called "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The framework and complementarity-determining regions have been precisely defined (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services (1991) NIH Publication Number 91-3242). Other numbering conventions for CDR sequences exist, such as those presented in Chothia et al. (1989) Nature 342: 877-883. In conventional antibodies, each VH and VL consists of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed, for example, using heavy and light immunoglobulin chains interconnected by disulfide bonds, with the heavy chains similarly connected. The heavy chain constant region contains three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The heavy chain variable domain and the light chain variable domain are binding domains that interact with antigens. The constant regions of the antibodies typically mediate 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.

[0019] As used herein, a fragment of an antibody (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 a target, the delta variable 1 (V51) chain of the γδ T-cell receptor (e.g., a molecule in which one or more immunoglobulin chains are not full length, but which specifically binds to a target). Examples of binding fragments encompassed by the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain); (ii) F(ab')2 fragment (a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragment (consisting of a VH domain and a CH1 domain); (iv) Fv fragment (consisting of the VL and VH domains of a single arm of an antibody); (v) single-chain variable fragments, scFv (consisting of VL and VH domains connected by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH domains pair to form a monovalent molecule); (vi) VH (immunoglobulin chain variable domain consisting of a VH domain); (vii) VL (immunoglobulin chain variable domain consisting of a VL domain); (viii) domain antibodies (dAbs consisting of either a VH domain or a VL domain); (ix) a minibody (consisting of a pair of scFv fragments linked via a CH3 domain); and (x) diabodies (consisting of noncovalent dimers of scFv fragments consisting of a VH domain from one antibody connected by a small peptide linker to a VL domain from another antibody); Examples include:

[0020] A "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human subject administered such a human antibody does not develop a cross-species antibody response (e.g., a HAMA response—called human anti-mouse antibodies) against primary amino acids contained within the antibody. Such a human antibody may contain amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis or by somatic mutation) not encoded by human germline immunoglobulin sequences, for example, in the CDRs, particularly CDR3. However, this term is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences. Human antibodies that are prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector introduced into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences, can also be referred to as "recombinant human antibodies."

[0021] The replacement of at least one amino acid residue in a framework region of a non-human immunoglobulin variable domain with the corresponding residue from a human variable domain is called "humanization." Humanization of variable domains can reduce immunogenicity in humans.

[0022] "Specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or fragment thereof can bind. Antibody specificity is the antibody's ability to recognize a particular antigen as a unique molecular entity and distinguish it from others. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts with a particular target antigen or epitope with greater frequency, rapidity, duration, and / or greater affinity than it reacts with other targets. An antibody "specifically binds" to a target antigen or epitope if it binds with greater affinity, avidity, rapidity, and / or duration than it binds to other substances.

[0023] "Affinity," expressed by the equilibrium constant (KD) for dissociation of an antigen with an antigen-binding polypeptide, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antibody (or fragment thereof): the smaller the KD value, the greater the binding strength between an antigenic determinant and an antigen-binding polypeptide. Alternatively, affinity can be expressed as an affinity constant (KA), which is 1 / KD. Affinity can be determined by known methods depending on the specific antigen of interest.

[0024] 10 -6 Any KD value less than 0.05 is considered to indicate binding. Specific binding of an antibody or fragment thereof to an antigen or antigenic determinant can be determined by any suitable known method, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays), as well as various modifications thereof known in the art.

[0025] "Avidity" is a measure of the strength of binding between an antibody or fragment thereof and the relevant antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antibody and the number of relevant binding sites on the antibody.

[0026] "Human tissue V51+ cells," "hematopoietic and blood V51+ cells," and "tumor-infiltrating lymphocyte (TIL) V51+ cells" are defined as V51+ cells contained in or derived from human tissues or the hematopoietic system or human tumors, respectively. All such cell types can be identified by their (i) location or where they originate from, and (ii) their expression of the V51+ TCR.

[0027] A "modulatory antibody" is an antibody that confers a measurable change, including but not limited to, a measurable change in cell cycle and / or cell number, and / or cell viability, and / or one or more cell surface markers, and / or secretion of one or more secreted molecules (e.g., cytokines, chemokines, leukotrienes, etc.), and / or function (e.g., cytotoxicity against target cells or diseased cells), upon contact with or binding to cells expressing the target to which the antibody binds.

[0028] A method of "modulating" a cell or population thereof refers to a method of inducing at least one measurable change in the cell or cells, secretions therefrom, to produce one or more "modulated cells."

[0029] An "immune response" is a measurable change in at least one cell, or one cell type, or one endocrine pathway, or one exocrine pathway of the immune system upon addition of a regulatory antibody (including, but not limited to, a cell-mediated response, a humoral response, a cytokine response, or a chemokine response).

[0030] "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 lymphoid cells, natural killer (NK) cells, and gamma delta T cells. Immune cells are typically classified using combinatorial cell surface molecular analysis (e.g., by flow cytometry) to identify, group, or cluster immune cells, differentiating them into subpopulations. These can then be further subdivided by further analysis. For example, CD45+ lymphocytes can be further subdivided into vδ-positive and vδ-negative populations.

[0031] A "model system" is a biological model or biological description designed to aid in the understanding of how a drug, such as an antibody or fragment thereof, may function as a pharmaceutical agent in ameliorating the signs or symptoms of a disease. Such models typically involve the use of in vitro, ex vivo, and in vivo diseased, non-diseased, healthy, effector cells, and tissues, etc., and in which the performance of the drug can be studied and compared.

[0032] A "disease cell" exhibits a phenotype associated with the progression of a disease, such as cancer, an infection, e.g., a viral infection, or an inflammatory disease or disorder. For example, a disease cell can be a tumor cell, an autoimmune tissue cell, or a virally infected cell. Thus, the disease cell can be defined as neoplastic, virally infected, or inflammatory.

[0033] "Healthy cells" refer to normal cells that are not diseased. They may also be referred to as "normal" or "non-disease" cells. Non-disease cells include non-cancerous or non-infected or non-inflammatory cells. Such cells are often utilized in conjunction with related disease cells to determine the disease cell specificity conferred by a drug and / or to better understand the therapeutic index of a drug.

[0034] "Disease cell specificity" is a measure of how effectively an effector cell or population thereof (e.g., a population of V51 cells) identifies and kills diseased cells, such as cancer cells, while sparing non-diseased or healthy cells. This potential can be measured in a model system and can involve comparing the tendency of an effector cell or population of effector cells to selectively kill or lyse diseased cells with the potential of the effector cells to kill or lyse non-diseased or healthy cells. The disease cell specificity can inform the potential therapeutic index of a drug.

[0035] "Enhanced disease cell specificity" describes a phenotype of an effector cell, e.g., a V51+ cell or population thereof, that has been modulated to further increase its ability to specifically kill disease cells. This enhancement can be measured in a variety of ways, including fold change or percentage increase in disease cell killing specificity or selectivity.

[0036] Preferably, the antibodies or fragments thereof (i.e., polypeptides) of the present invention are isolated. An "isolated" polypeptide is one that has been removed from its original environment. The term "isolated" can be used to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to Vδ1 or a fragment thereof is substantially free of antibodies that bind to antigens other than Vδ1). The term "isolated" can also refer to a preparation in which the isolated antibody is sufficiently pure to be administered therapeutically when formulated as the active ingredient of a pharmaceutical composition, or is 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.

[0037] Preferably, the polynucleotide used in the present invention is isolated. An "isolated" polynucleotide is a polynucleotide that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated, for example, if it is cloned into a vector that is not part of its natural environment or if it is contained in a cDNA.

[0038] An antibody or fragment thereof may be a "functionally active variant," including naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As known in the art, an allelic variant is an alternative form of a (poly)peptide characterized by one or more amino acid substitutions, deletions, or additions that do not essentially alter the biological function of the polypeptide. As a non-limiting example, such functionally active variants can still function when the framework containing the CDR is modified, when the CDR itself is modified, when the CDR is grafted onto another framework, or when an N- or C-terminal extension is incorporated. Furthermore, a CDR-containing binding domain can be paired with a different partner chain, e.g., a chain shared with another antibody. When shared with a so-called "common" light chain or a "common" heavy chain, the binding domain can still function. Furthermore, the binding domain can function when multimerized. Furthermore, "antibody or fragment thereof" can also include functional variants in which the VH or VL or constant domains have been altered away from or towards different canonical sequences (e.g., listed on IMGT.org) and still function.

[0039] For comparison of two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence can be calculated using NCBI BLAST v2.0 using standard settings for polypeptide sequences (BLASTP). For comparison of two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using NCBI BLAST v2.0 using standard settings for nucleotide sequences (BLASTN).

[0040] A polypeptide or polynucleotide sequence is said to be the same as or "identical" to another polypeptide or polynucleotide sequence if they share 100% sequence identity over their entire length. Residues in a sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus for polypeptides; from the 5' end to the 3' end for polynucleotides.

[0041] 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 otherwise identical to a first sequence (100% sequence identity) results in a decrease in percent sequence identity. For example, if an identical sequence is 9 amino acid residues long, a single substitution in the second sequence results in 88.9% sequence identity. If a first and second polypeptide sequence are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity).

[0042] Alternatively, for purposes 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 to generate the second sequence can be ascertained. An "addition" is the addition of one amino acid residue to the sequence of a first polypeptide (including additions at either end of the first polypeptide). A "substitution" is the replacement of one amino acid residue in the first polypeptide sequence with a different amino acid residue. The substitution may be conservative or non-conservative. A "deletion" is the removal of one amino acid residue from the sequence of a first polypeptide (including deletions at either end of the first polypeptide).

[0043] "Conservative" amino acid substitutions are those in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and are expected to have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions are preferably those in which one amino acid residue within the following group is replaced with another amino acid residue from the same group: [Table 1]

[0044] Preferably, the hydrophobic amino acid residue is a non-polar amino acid. More preferably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W, or C.

[0045] As used herein, the numbering of polypeptide sequences and the definitions of CDRs and FRs are as defined according to the Kabat system (Kabat et al., 1991, incorporated herein by reference in its entirety). "Corresponding" amino acid residues between a first polypeptide sequence and a second polypeptide sequence are amino acid residues in the first sequence that share the same position as the amino acid residues in the second sequence according to the Kabat system, while the amino acid residues in the second sequence may differ in content from the first. Preferably, corresponding residues will share the same number (and letter) if the framework and CDRs are the same length according to the Kabat definition. Alignment can be achieved manually or by using known computer algorithms for sequence alignment, for example, NCBI BLAST v2.0 (BLASTP or BLASTN), using standard settings.

[0046] References herein to an "epitope" refer to the portion of a target that is specifically bound by an antibody or fragment thereof. An epitope can also be referred to as an "antigenic determinant." An antibody binds to "essentially the same epitope" as another antibody if both recognize the same or sterically overlapping epitope. A commonly used method for determining whether two antibodies bind to the same or overlapping epitope is a competition assay, which uses either labeled antigen or labeled antibody and can be configured in several different formats (e.g., well plates using radioactive or enzyme labels, or flow cytometry on antigen-expressing cells).

[0047] Epitopes found on protein targets can be defined as "linear epitopes" or "conformational epitopes." Linear epitopes are formed by contiguous amino acid sequences in the protein antigen. Conformational epitopes are formed from amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0048] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain 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"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are most often used in the form of vectors, the terms "plasmid" and "vector" can be used interchangeably herein. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions, and also includes bacteriophage and phagemid systems. As used herein, the term "recombinant host cell" (or simply, "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such cells, where the progeny are used to generate cell lines or cell banks that are subsequently stored, provided, sold, transported, or used at will to produce the antibodies or fragments thereof described herein.

[0049] References to a "subject," "patient," or "individual" refer to a subject to be treated, particularly a mammalian subject. Mammalian subjects include humans, non-human primates, farm animals (e.g., cows), sport animals, or pets, 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.

[0050] The term "sufficient amount" means an amount sufficient to produce a desired effect. The term "therapeutically effective amount" is an amount that is effective in ameliorating the symptoms of a disease or disorder. Where prevention can be considered a form of therapy, a therapeutically effective amount can be a "prophylactically effective amount."

[0051] As used herein, the term "about" as used herein includes values ​​up to 10% (inclusive) greater than and less than the specified value, preferably up to 5% (inclusive) greater than and less than the specified value, particularly the specified value. The term "between" includes values ​​in the specified range.

[0052] A disease or disorder is "improved" if the severity of a sign or symptom of the disease or disorder, the frequency with which such sign or symptom is experienced by a subject, or both, are reduced.

[0053] As used herein, "treating a disease or disorder" means reducing the frequency and / or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0054] As used herein, "cancer" refers to the abnormal growth or division of cells. Typically, the growth and / or lifespan of cancer cells exceeds and is out of step with the growth and / or lifespan of surrounding normal cells and tissues. Cancers can be benign, pre-malignant, or malignant. Cancers arise in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, reproductive system (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0055] (Method for modulating gamma delta T cells) According to a first aspect of the present invention there is provided an ex vivo method of modulating delta variable 1 chain (V51) T cells, the ex vivo method comprising administering an anti-V51 antibody or fragment thereof as defined herein to a population of cells comprising V51 T cells. It will be understood that "administering" the antibody or fragment thereof includes "contacting" the V51 T cells.

[0056] Vδ1 T cells are regulated by: - expansion of Vδ1 T cells, for example by selectively increasing the number of Vδ1 T cells or promoting the survival of Vδ1 T cells; - for example, increased Vδ1 T cell strength, i.e., stimulation of Vδ1 T cells with increased target cell killing; -Prevention of Vδ1 T cell exhaustion, for example, by increasing Vδ1 T cell persistence; -Vδ1 T cell degranulation; - immunosuppression of V51 T cells, for example by downregulating V51 TCR cell surface expression, i.e. by causing a decrease in V51 TCR internalization or V51 TCR protein expression or by blocking V51 TCR binding; - a reduction in V51 T cell numbers, for example by inhibiting V51 T cell proliferation or by reducing V51 T cell death (i.e. killing of V51 T cells) may include:

[0057] Such modulation of V51 T cells can include, for example, V51 T cell activation or V51 T cell inhibition. In one embodiment, V51 T cells are activated by administering an anti-V51 antibody, or a fragment thereof, as defined herein. In an alternative embodiment, V51 T cells are inhibited by administering an anti-V51 antibody, or a fragment thereof, as defined herein. In an alternative embodiment, V51 T cells are not inhibited by administering an anti-V51 antibody, or a fragment thereof, as defined herein to the patient.

[0058] In one embodiment, modulating V51 T cells comprises administering an anti-TCR delta1 variable antibody or fragment thereof to V51 T cells in culture (i.e. in vitro or ex vivo). V51 T cells may be present in a mixed cell population, for example in a cell population comprising other lymphoid cell types (e.g. αβ T cells or NK cells).

[0059] In one embodiment, the cell population comprising V51 T cells is isolated (i.e., from a sample as described herein) prior to administration of the anti-V51 antibody or fragment thereof. In a further embodiment, the cell population is enriched for T cells prior to administration of the anti-V51 antibody or fragment thereof. In yet a further embodiment, the cell population is enriched for γδ T cells prior to administration of the anti-V51 antibody or fragment thereof.

[0060] The method can also be performed on a cell population comprising a purified fraction of γδ T cells. In such embodiments, the cell population is depleted of cell types other than γδ T cells present in the sample, such as αβ T cells and / or NK cells, before administration of an anti-Vδ1 antibody or a fragment thereof. Additionally or alternatively, the cell population can be enriched for cell types that may contain Vδ1, such as T cells and / or γδ cells, before administration of an anti-Vδ1 antibody or a fragment thereof. For example, before culturing the sample, the sample can be enriched for T cells, or γδ T cells, or the sample can be depleted of αβ T cells or non-γδ T cells. In one embodiment, the sample is first depleted of αβ T cells, and then enriched for CD3+ cells. Enrichment or depletion can be achieved using techniques known in the art, for example, using magnetic beads coated with antibodies that bind to molecules on the cell surface associated with the phenotype to be enriched / depleted.

[0061] The presence of cell types other than lymphocytes in cell culture can inhibit V51 cell expansion. Such cells, for example, stromal, epithelial, tumor, and / or feeder cells, can be removed before culturing. Thus, in one embodiment, the cell population is not in direct contact with stromal cells during culturing. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematopoietic tumor cells. Preferably, lymphocytes are not in direct contact with fibroblasts during culturing. In one embodiment, the cell population is not in direct contact with epithelial cells during culturing. In one embodiment, the cell population is not in direct contact with tumor cells and / or feeder cells during culturing.

[0062] In one embodiment, the method comprises culturing V51 T cells in the absence of substantial stromal cell contact. In a further embodiment, the method comprises culturing V51 T cells in the absence of substantial fibroblast cell contact.

[0063] In one embodiment, the method comprises culturing V51 T cells in a medium that is substantially free of serum (e.g., serum-free medium or medium containing serum replacement (SR)). Accordingly, in one embodiment, the method comprises culturing in serum-free medium. Such serum-free medium can also include serum replacement medium, in which the serum replacement is based on chemically defined components to avoid the use of human or animal-derived serum. In an alternative embodiment, the method comprises culturing in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the medium contains a serum replacement. In one embodiment, the medium does not contain animal-derived products.

[0064] It will be appreciated that samples cultured in serum-free medium have the advantage of avoiding issues related to serum filtration, precipitation, contamination, and supply. Furthermore, animal-derived products are not preferred for use in the clinical-grade manufacture of human therapeutics. The use of serum-free medium for cells, particularly V51 T cells, significantly increases the number of cells obtained from a sample compared to the use of medium containing AB serum.

[0065] In one embodiment, the anti-V51 antibody or fragment thereof is in soluble or immobilized form. For example, the antibody or fragment thereof can be administered to V51 T cells in soluble form. Alternatively, the antibody or fragment thereof can be administered to V51 T cells when the antibody or fragment thereof is bound or covalently bound to a surface such as a bead or plate (i.e., in immobilized form). In one embodiment, the antibody is immobilized on a surface such as an Fc-coated well. Alternatively, the antibody or fragment thereof is bound to the surface of a cell (e.g., immobilized on the surface of an antigen-presenting cell (APC)). In another embodiment, the antibody is not immobilized on a surface when the cell population is contacted with the antibody.

[0066] The cell population contacted by the anti-V51 antibody or fragment thereof can be obtained from various sample types (methods of isolation are further described below). In one embodiment, the sample is a non-hematopoietic tissue sample. Reference herein to "non-hematopoietic tissue" or "non-hematopoietic tissue sample" includes skin (e.g., human skin) and intestine (e.g., human intestine). Non-hematopoietic tissue is tissue other than blood, bone marrow, lymphatic 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., γδ T cells) is not obtained from a specific type of biological fluid sample, such as blood or synovial fluid. In some embodiments, the cell population (e.g., γδ T cells) is obtained from skin (e.g., human skin), which can be obtained by methods known in the art. For example, the cell population can be obtained from a non-hematopoietic tissue sample by culturing the non-hematopoietic tissue sample on a synthetic scaffold configured to promote cell migration from the non-hematopoietic tissue sample. Alternatively, the method may be used to treat tissue in the digestive tract (e.g., colon or intestine), mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina, and other skin tissues. skin, It can be applied to cell populations obtained from mucosa or serosal membranes (eg, γδ T cells).

[0067] In alternative embodiments, 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). References herein to a "hematopoietic sample" or "hematopoietic tissue sample" include blood (e.g., peripheral blood or umbilical cord blood), bone marrow, lymphoid tissue, lymph node tissue, thymus tissue, and fractions or enriched portions thereof. The sample is preferably peripheral blood or umbilical cord blood, or blood containing fractions thereof, including buffy coat cells, leukapheresis products, 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 a blood sample using techniques known in the art, such as density gradient centrifugation. For example, whole blood can be layered onto an equal volume of FICOLL-HYPAQUE, followed by centrifugation at 400 x g at room temperature for 15-30 minutes. The interface material contains low density mononuclear cells, which can be collected, washed in culture medium, and centrifuged at 200 xg for 10 minutes at room temperature.

[0068] The cell population can be obtained from a cancer tissue sample, e.g., a breast or prostate tumor (i.e., γδ T cells can also be present in cancer tissue samples). In some embodiments, the cell population can be obtained from a human cancer tissue sample (e.g., solid tumor tissue). In other embodiments, the cell population can be derived from a sample other than human cancer tissue (e.g., tissue that does not contain a significant number of tumor cells). For example, the cell population can be derived from an area of ​​skin (e.g., healthy skin) away from nearby or adjacent cancer tissue. Thus, in some embodiments, the cell population is not obtained from cancer tissue (e.g., human cancer tissue).

[0069] The cell population can be obtained from human or non-human animal tissue. Thus, the method can further comprise obtaining the cell population from human or non-human animal tissue. In one embodiment, the sample is obtained from a human. In an alternative embodiment, the sample is obtained from a non-human animal subject.

[0070] (expansion of gamma delta T cells) In one embodiment, the modulation comprises activation of V51 T cells, in particular expansion of V51 T cells. Thus, according to one aspect of the present invention, there is provided an ex vivo method for expanding V51 T cells, comprising administering an anti-V51 antibody or fragment thereof as defined herein to a cell population comprising V51 T cells. Such expansion of V51 T cells can be achieved by selectively increasing the number of V51 T cells and / or by promoting the survival of V51 T cells. In one embodiment, the expansion of V51 T cells comprises administering an anti-TCR delta1 variable antibody or fragment thereof to V51 T cells in culture (i.e., in vitro or ex vivo). V51 T cells may be present in a mixed cell population, for example in a cell population comprising other lymphoid cell types (e.g., αβ T cells or NK cells).

[0071] Thus, the present invention provides an ex vivo method for producing an enriched population of γδ T cells (e.g., V51 T cells). The enriched population can be produced from an isolated mixed cell population (e.g., obtained from a sample taken from a patient / donor) by a method comprising contacting the mixed cell population or a purified fraction thereof with an antibody or fragment thereof. The antibody (or fragment thereof) selectively expands V51 T cells by binding to an epitope specific for the V51 chain of the γδ TCR.

[0072] Also provided is an expanded V51 T cell population obtained according to the method defined herein. It will be appreciated that according to this aspect of the invention, such an expanded population of V51 T cells may be obtained and / or expanded in vitro or ex vivo. In one aspect, there is provided an expanded V51 population obtained according to the method defined herein, in which the V51 population is isolated and expanded in vitro or ex vivo.

[0073] The antibodies or fragments thereof described herein can be used in methods for expanding γδ T cells (e.g., Vδ1 T cells). These methods can be performed in vitro. When the expansion method is performed in vitro, the antibody (or fragment thereof) can be applied to isolated γδ T cells (e.g., Vδ1 T cells) obtained as described above. In some embodiments, γδ T cells are expanded from a cell population isolated from a non-hematopoietic tissue sample. In an alternative embodiment, γδ T cells are expanded from a cell population isolated from a hematopoietic tissue sample, e.g., a blood sample.

[0074] Expansion of γδ T cells (e.g., Vδ1 T cells) can include culturing the sample in the presence of an antibody or fragment thereof described herein and a cytokine. Cytokines can include interleukins, lymphokines, interferons, colony-stimulating factors, and chemokines. In one embodiment, the cytokine is selected from the group consisting of 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 will be understood that references to cytokines described herein can include any compound that has the same activity as the cytokine in terms of its ability to promote a similar physiological effect on V51 T cells in culture, and includes, but is not limited to, a mimetic, or any functional equivalent thereof.

[0075] In one embodiment, the cytokine is a common cytokine receptor gamma chain (γ c In a further embodiment, the γ c- the cytokines are selected from: IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-21, or a mixture thereof.

[0076] The cytokine (e.g., interleukin) used may be of human or animal origin, preferably of human origin. It may be a wild-type protein or any biologically active fragment or variant, i.e., capable of binding to its receptor. Such binding is capable of inducing activation of γδ T cells under the conditions of the method according to the invention. More preferably, the cytokine may be in soluble form, for example, fused or complexed with another molecule, such as a peptide, polypeptide, or biologically active protein. Preferably, human recombinant cytokines are used. More preferably, the interleukin concentration may vary in the range of 1 to 10,000 U / ml, even more preferably, 100 to 1,000 U / ml.

[0077] In a further embodiment, the cytokine is a chemokine. It will further be appreciated that the chemokine will vary and be selected depending on the sample used to obtain the γδ T cells.

[0078] In one embodiment, the method comprises culturing the cell population in the presence of IL-2, IL-9, and / or IL-15. In a further embodiment, the method comprises culturing the cell population in the presence of IL-2 and / or IL-15 (i.e., IL-2, IL-15, or a combination thereof). In an alternative embodiment, the method comprises culturing the cell population in the presence of IL-9 and / or IL-15 (i.e., IL-9, IL-15, or a combination thereof). In one embodiment, the method comprises culturing the cell population in the presence of IL-2, IL-9, and / or IL-15 and an additional growth factor (e.g., IL-21). In other embodiments, the method comprises culturing the cell population in medium lacking growth factors other than IL-2 and / or IL-15. In an alternative embodiment, the method comprises culturing the cell population in medium lacking growth factors other than IL-9 and / or IL-15. In a further embodiment, the method comprises culturing the cell population in a medium consisting of basal medium supplemented with IL-2, IL-9, and / or IL-15. In a further embodiment, the method comprises culturing the cell population in a medium consisting of basal medium supplemented with IL-2 and / or IL-15.

[0079] In one embodiment, the method comprises culturing the cell population in the presence of IL-21.

[0080] In one embodiment, the method comprises culturing the cell population in the presence of IL-4. The physiological effects of IL-4 on V51 T cells (described in WO2016 / 198480) include a decrease in NKG2D and NCR expression levels, inhibition of cytotoxic function, and improved selective survival. Furthermore, it has previously been shown that the absence of IL-4 during the latter stages of culture can alter the physiological properties of the cells to a phenotype more suitable for use as an antitumor or antiviral treatment. Therefore, in one embodiment, the expansion method further comprises culturing the sample in the absence of a growth factor with IL-4-like activity, e.g., IL-4. In one embodiment, the expansion method comprises culturing the sample in the absence of IL-4.

[0081] In one embodiment, the cytokine is a growth factor with interleukin-15-like activity, i.e., any compound with the same activity as IL-15 in terms of its ability to promote similar physiological effects on cultured V51 T cells, including, but not limited to, IL-15 and IL-15 mimetics, or any functional equivalent of IL-15, including IL-2 and IL-7. The physiological effects on cultured V51 T cells promoted by IL-15, IL-2, and IL-7 (described in WO 2016 / 198480) are essentially equivalent, i.e., the induction of cell differentiation toward a more cytotoxic phenotype. Furthermore, it has previously been shown that the absence of IL-2, IL-7, and IL-15 during the initial period of culture contributed to starvation and apoptosis of contaminating cells (including TCRαβ+ T and V52+ T cells) that are highly dependent on these cytokines for survival. Therefore, in one embodiment, the expansion method initially involves culturing the sample in the absence of a growth factor with IL-15-like activity.

[0082] Thus, in one embodiment, the method comprises culturing a cell population in a first culture medium comprising IL-4, and thereafter culturing the cell population in a second culture medium comprising IL-15.

[0083] In one embodiment, the first culture medium lacks IL-15, IL-2, and / or IL-7. In one embodiment, the second culture medium lacks IL-4.

[0084] Thus, in one embodiment, the present expansion method comprises: (1) culturing cells in the sample in a first culture medium containing an antibody or fragment thereof described herein and IL-4 in the absence of IL-15, IL-2, and IL-7; and (2) culturing the cells obtained in step (1) in a second culture medium containing an antibody or fragment thereof described herein and IL-15 in the absence of IL-4. Contains:

[0085] As described herein, the culture medium can also contain other growth factors, including cytokines, that can further enhance the expansion of V51 T cells. 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 can be added include costimulatory molecules, such as human anti-SLAM antibodies, any soluble ligand of CD27, or any soluble ligand of CD7. Any combination of these growth factors can be included in the medium.

[0086] In one embodiment, the first or second culture medium, or both culture media, comprise one or more additional cytokines. The first and / or second culture medium may comprise a second, third, and / or fourth cytokine. In a further embodiment, the additional cytokine is selected from IL-21, IFN-γ, and IL-1β.

[0087] In one embodiment, the method comprises culturing the cell population in the presence of IL-15 and a factor selected from the group consisting of 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).

[0088] Expansion of γδ T cells can include culturing the sample in the presence of at least one additional T cell mitogen. The term "T cell mitogen" (which can also be referred to as "γδ TCR agonist") refers to any agent capable of stimulating T cells through TCR signaling, including, but not limited to, plant lectins such as phytohemagglutinin (PHA) and concanavalin A (ConA), as well as 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-acetate (TPA) and related compounds such as mezerein, or bacterial compounds (e.g., staphylococcal enterotoxin A (SEA) and streptococcal protein A). The T cell mitogen can be soluble or immobilized, and multiple T cell mitogens can be used in the expansion method.

[0089] As used herein, reference to "expanded" or "expanded population of γδ T cells" includes a population of cells that is larger than a non-expanded population or that contains a greater number of cells than a non-expanded population. Such a population may be a large number, a small number, or a mixed population with expansion of a portion or specific cell type within the population. It will be understood that the term "expansion method" refers to a process that results in an expanded or expanded population. Thus, an expanded or expanded population may be a larger number or contain more cells compared to a population without an expansion step or prior to any expansion step. It will be further understood that any number provided herein to indicate expansion (e.g., fold increase or fold expansion) is indicative of an increase in the number or size of a population of cells or number of cells, and is indicative of the amount of expansion.

[0090] In one embodiment, the method comprises 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 comprises culturing the cell population for at least 7 days, e.g., at least 11 days or at least 14 days.

[0091] In further embodiments, the method comprises culturing the cell population for a sustained period (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., between 5 and 40 days, between 7 and 35 days, between 14 and 28 days, or about 21 days) in an amount effective to produce an expanded population of γδ T cells.

[0092] In one embodiment, the cell population is cultured for 5 to 60 days, e.g., at least 7 to 45 days, 7 to 21 days, or 7 to 18 days. Where the method includes an isolation culture period (e.g., 1 to 40 days, e.g., 14 to 21 days), the isolation and expansion steps can, in some embodiments, last 21 to 39 days.

[0093] The method may include periodic addition of an anti-V51 antibody or fragment thereof and / or a growth factor during the culture period. For example, the anti-V51 antibody or fragment thereof and / or growth factor can be added every 2 to 5 days, more preferably every 3 to 4 days. In one embodiment, the anti-V51 antibody or fragment thereof and / or growth factor is added after 7 days of culture and every 3 to 4 days thereafter.

[0094] The expansion method provides a population of expanded γδ T cells that is more numerous than a reference population. In some embodiments, the expanded population of γδ T cells (e.g., Vδ1 T cells) is more numerous than the population of isolated γδ T cells prior to the expansion step (e.g., at least 2-fold in number, at least 5-fold in number, at least 10-fold in number, at least 25-fold in number, at least 50-fold in number, at least 60-fold in number, at least 70-fold in number, at least 80-fold in number, at least 90-fold in number, at least 100-fold in number, at least 200-fold in number, at least 300-fold in number, at least 400-fold in number, at least 500-fold in number, 600-fold in number, at least 1,000-fold in number, or more, relative to the population of isolated γδ T cells prior to the expansion step). In one embodiment, the expanded population of γδ T cells (e.g., Vδ1 T cells) is more numerous than a population cultured for the same time in the absence of the antibody or fragment thereof. In one embodiment, the expanded population of γδ T cells (e.g., Vδ1 T cells) is more numerous than a population cultured for the same time in the presence of TS8.2 or TS-1.

[0095] The expansion methods provide a population of expanded V51 T cells having a higher percentage of V51 T cells than the reference population. In some embodiments, the expanded population of V51 T cells contains more than about 50% V51 T cells, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, or 95% V51 T cells. In further embodiments, the expanded population of V51 T cells contains more than about 85% V51 T cells, such as more than about 90% V51 T cells.

[0096] In some embodiments, the expanded population of γδ T cells (e.g., V51 T cells) contains less than about 10% αβ T cells, e.g., less than about 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% αβ T cells. In further embodiments, the expanded population of V51 T cells contains less than about 1% αβ T cells. T cells bearing αβ receptors are highly reactive, and therefore, cell populations suitable for administration to patients in the context of the present invention can only contain low levels of αβ T cells. The antibodies described herein can be used to selectively expand V51 T cell populations, thereby reducing the need for extensive purification methods after expansion to remove αβ T cells.

[0097] In some embodiments, the expanded population of γδ T cells (e.g., V51 T cells) contains less than about 10% V52 T cells, such as less than about 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% V52 T cells. In further embodiments, the expanded population of V51 T cells contains less than about 0.5% V52 T cells.

[0098] In some embodiments, the expanded population of γδ T cells (e.g., V51 T cells) contains less than about 10% natural killer (NK) cells (also referred to as CD56+CD3− cells), such as less than about 5%, 4%, 3%, 2.5%, 2%, 1.5%, or 1% NK cells. In further embodiments, the expanded population of V51 T cells contains less than about 2% NK cells.

[0099] Increased or decreased expression of cell surface markers, including CD27, CD69, TIGIT, PD-1, and TIM-3, can additionally or alternatively be used to characterize one or more expanded populations of V51 T cells. In some embodiments, the expanded population of V51 T cells expresses high levels of CD27 (CD27 high). For example, more than about 70%, e.g., more than about 80%, 85%, 90% of the expanded population of V51 T cells express CD27 (i.e., CD27+). In some embodiments, the expanded population of V51 T cells has a greater average expression of CD27, e.g., compared to the population of isolated V51 T cells prior to expansion. In some embodiments, the expanded population of V51 T cells expresses low levels of CD69, TIGIT, PD-1, and / or TIM-3. For example, less than about 40%, e.g., less than about 30%, of the expanded population of V51 T cells express CD69, TIGIT, PD-1, and / or TIM-3. In some embodiments, the expanded population of V51 T cells has a lower average expression of one or more markers selected from the group consisting of CD69, TIGIT, PD-1, and TIM-3, compared to the population of isolated V51 T cells.

[0100] Many basal culture media suitable for use in the expansion of γδ T cells are available, particularly media in the presence of serum or plasma, such as AIM-V, Iscoves' medium, and RPMI-1640 (Life Technologies), EXVIVO-10, EXVIVO-15, or EXVIVO-20 (Lonza). The medium may be supplemented with other media factors, such as serum, serum proteins, and selection agents, such as antibiotics, as defined herein. 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×MEM Non-Essential Amino Acids (Life Technologies)), and 10 μl / L β-mercaptoethanol. In alternative embodiments, the AIM-V medium may be supplemented with serum replacement and amphotericin B from CTS Immune. In certain embodiments, the medium may be further supplemented with IL-2, IL-4, IL-9, and / or IL-15 as described herein. Advantageously, during isolation and / or expansion, cells are cultured in a suitable culture medium at 37° C. in a humidified atmosphere containing 5% CO2.

[0101] The addition of other factors in the expansion culture of γδ T cells can also be used. In one embodiment, such factors are used in the expansion to selectively promote the expansion of γδ T cells. For example, the expansion can further include the addition of exogenous cytokines to the expansion culture, such as interleukins. Such expansion can include culturing γδ T cells in the presence of IL-2 and IL-15. Alternatively, the expansion can include culturing γδ T cells in the presence of IL-9 and IL-15. It will be understood that any expansion step is carried out for a period of time effective to produce an expanded population of γδ T cells.

[0102] Methods of expanding γδ T cells can include a population doubling time 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).

[0103] (Method for isolating γδ T cells) As described herein, the antibody (or fragment thereof) can be applied to γδ T cells in culture, i.e., γδ T cells obtained from a sample. In one embodiment, a population of cells is isolated from the sample before administering the anti-V51 antibody or fragment thereof. Thus, provided is a method of modulating (in particular expanding) V51 T cells, comprising administering an anti-V51 antibody or fragment thereof as defined herein to a population of γδ T cells isolated from a sample (e.g., a cell population comprising V51 T cells).

[0104] γδ T cells that are predominant in non-hematopoietic tissues (i.e., tissue-resident) contain primarily the delta variable 1 chain, and therefore the anti-Vδ1 antibodies described herein are of particular use in γδ T cells isolated from non-hematopoietic tissues. Thus, in one embodiment, the sample is a non-hematopoietic tissue sample, e.g., skin. Alternatively, the methods of the invention can be used to expand the population of Vδ1 T cells in a sample that does not primarily contain the Vδ1 chain, e.g., a blood sample. Thus, the methods can be used to increase the number of Vδ1 T cells in a sample.

[0105] References herein to "isolation" or "isolating" cells, particularly γδ T cells, refer to methods or processes by which cells are removed, separated, purified, enriched, or otherwise removed from a tissue or pool of cells. Such references will be understood to include the terms "isolated," "depleted," "purified," "enriched," and similar terms. Isolation of γδ T cells includes isolation or separation of cells from an intact non-hematopoietic tissue sample or from stromal cells of non-hematopoietic tissue (e.g., fibroblasts or epithelial cells). Such isolation may alternatively or additionally include isolation or separation of γδ T cells from other hematopoietic cells (e.g., αβ T cells or other lymphocytes). Isolation can be of a defined duration, e.g., beginning when the tissue explant or biopsy is placed in isolation culture and ending when the cells are recovered from the culture, e.g., by centrifugation or other means to transfer the isolated cell population to expansion culture or used for other purposes, or when the original tissue explant or biopsy is removed from the culture. The isolation process can be at least about 3 days to about 45 days. In one embodiment, the isolation process is at least about 10 days to at least 28 days. In a further embodiment, the isolation process is at least 14 days to at least 21 days. Therefore, the isolation process may 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, about 35, about 40, or about 45 days. During this isolation process, cell proliferation may be insignificant, but it is understood that this does not necessarily mean that cell proliferation is absent. In fact, those skilled in the art will recognize that isolated cells may begin to divide, generating multiple such cells within the isolation container containing the sample.

[0106] Accordingly, reference herein to "isolated γδ T cells", "isolated γδ T cell population", or "population of isolated γδ T cells" will be understood to refer to γδ cells that have been isolated, separated, depleted, purified or enriched from a sample, e.g., a non-hematopoietic tissue sample of origin, such that the cells are not in substantial contact with cells comprised in an intact (non-hematopoietic tissue) sample. Reference herein to "isolated V51 T cells", "isolated V51 T cell population", "population of isolated V51 T cells", "isolated V51 T cell", "isolated V51 T cell population", or "population of isolated V51 T cells" will be understood to refer to V51 T cells that have been isolated, separated, depleted, purified or enriched from a sample, e.g., a non-hematopoietic tissue sample of origin, such that the cells are not in substantial contact with cells comprised in the intact (non-hematopoietic tissue) sample.

[0107] The cell population can be obtained by any suitable method that allows the isolation of lymphocytes, particularly V51 T cells, from a human or non-human animal sample, such as a non-hematopoietic tissue sample. 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 explant can be attached to a synthetic scaffold to promote lymphocyte migration from the explant onto the scaffold. A synthetic scaffold refers to a non-natural three-dimensional structure suitable for supporting cell growth. Synthetic scaffolds can be constructed from materials such as polymers (e.g., natural or synthetic polymers, e.g., polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (tantalum, titanium, platinum, and metals in the same elemental group as platinum, niobium, hafnium, tungsten, and alloy combinations thereof). Biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, antibodies and fragments thereof, cytokines (e.g., IL-2 or IL-15, and combinations thereof) can be coated onto the scaffold surface or encapsulated within the scaffold material according to methods known in the art to enhance cell adhesion, migration, survival, or proliferation. This and other methods can be used to isolate cell populations from several other non-hematopoietic tissue types, e.g., intestine, prostate, and breast. Another example of a suitable isolation method utilizes a "crawling" method, which can include culturing a cell population and / or sample in the presence of cytokines and / or chemokines sufficient to induce the isolation or separation of γδ T cells, particularly Vδ1 T cells. Isolation of γδ T cells from a sample (e.g., a non-hematopoietic tissue sample) can include culturing the sample in the presence of IL-2 and IL-15.

[0108] Non-hematopoietic tissue-resident lymphocytes can be harvested and separated from stromal cells, e.g., skin fibroblasts, by, for example, vigorous pipetting. The lymphocyte harvest can be further washed through a 40 μm nylon mesh to retain fibroblast aggregates that may become disintegrated during the process. Lymphocytes can also be isolated using, for example, fluorescence- or magnetic-associated cell sorting using CD45 antibodies.

[0109] Alternatively, isolation of γδ T cells from a sample (e.g., a hematopoietic tissue sample) can be achieved by treating the sample with a T cell mitogen (e.g., a γδ TCR agonist) and cytokines (particularly common cytokine receptor gamma chain (γ c As another alternative, isolating γδ T cells from a sample (e.g., a hematopoietic tissue sample) may comprise culturing the sample in the presence of a T cell mitogen and a cytokine as described in WO2016 / 198480.

[0110] Isolating γδ T cells can include culturing the sample in the presence of at least one cytokine. For example, the method can include culturing the sample in the presence of at least an agent, such as a chemokine. It will be further understood that the chemokine will be selected depending on the γδ T cells to be isolated. Furthermore, the chemokine will vary and be selected depending on the sample used to isolate the γδ T cells.

[0111] Isolating γδ T cells may further comprise culturing the sample in the presence of at least one cytokine, which may be different from the cytokine used in the initial culture.

[0112] Isolation methods may include culturing the sample. References herein to "culturing" include the addition of a sample containing cells isolated, separated, removed, purified, or enriched from the sample to a medium containing growth factors and / or essential nutrients required and / or preferred by the cells and / or sample. It will be understood that such culture conditions may be adapted according to the cells or cell population to be isolated from the sample, or the cells or cell population to be isolated and expanded from the sample.

[0113] In certain embodiments, the sample is cultured for a period sufficient to isolate γδ T cells from the sample. In certain embodiments, the culture period is at least 14 days. In certain embodiments, the culture period is less than 45 days, e.g., less than 30 days, e.g., less than 25 days. In further embodiments, the culture period is 14 to 35 days, e.g., 14 to 21 days. In yet further embodiments, the culture period is about 21 days.

[0114] In certain embodiments, γδ T cells are recovered from the culture of the sample after culturing the sample. Recovery of γδ T cells may include physical recovery of γδ T cells from the culture, isolation of γδ T cells from other lymphocytes (e.g., αβ T cells and / or NK cells), or isolation and / or separation of γδ T cells from other cells present in the sample, e.g., stromal cells, e.g., fibroblasts. In one embodiment, γδ T cells are recovered by mechanical means (e.g., pipetting). In a further embodiment, γδ T cells are recovered by magnetic separation and / or labeling. In yet a further embodiment, γδ T cells are recovered by flow cytometry techniques, e.g., FACS. Thus, in certain embodiments, γδ T cells are recovered by specific labeling of γδ T cells. It will be understood that such recovery of γδ T cells may include physical removal of the sample from the culture, transfer to a separate culture vessel, or transfer to separate or different culture conditions.

[0115] It will be understood that such recovery of γδ T cells occurs after a period sufficient to obtain a population of γδ T cells isolated from the sample. In certain embodiments, the γδ T cells are recovered at least 1 week, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after culturing the sample. Preferably, the γδ T cells are recovered within 40 days, e.g., within 38 days, 36 days, 34 days, 32 days, 30 days, 28 days, 26 days, or 24 days. In one embodiment, the γδ T cells are recovered at least 14 days after culturing the sample. In a further embodiment, the γδ T cells are recovered between 14 and 21 days after culturing the sample.

[0116] In one embodiment, the sample is cultured in a medium that is substantially free of serum (e.g., a serum-free medium or a medium containing a serum replacement (SR)). Thus, in one embodiment, the sample is cultured in a serum-free medium. Such serum-free medium may also include serum replacement medium, in which the serum replacement is based on chemically defined components to avoid the use of human or animal-derived serum. 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)).

[0117] The culture medium may further comprise other components that can aid in the growth and expansion of γδ T 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.

[0118] The predominant γδ T cells in the blood are primarily Vδ2 T cells, whereas the predominant γδ T cells in non-hematopoietic tissues are primarily Vδ1 T cells, resulting in Vδ1 T cells comprising approximately 70-80% of the non-hematopoietic tissue-resident γδ T cell population. In one preferred embodiment, the isolated γδ T cells comprise a population of Vδ1 T cells.

[0119] (Antibody or its fragment) Provided herein are antibodies or fragments thereof capable of specifically binding to the delta variable 1 chain (Vδ1) of the γδ T cell receptor (TCR).

[0120] In one embodiment, the antibody or fragment thereof is an scFv, Fab, Fab', F(ab'), Fv, a variable domain (e.g., VH or VL), a diabody, a minibody, or a monoclonal antibody. In a further embodiment, the antibody or fragment thereof is an scFv.

[0121] The antibodies described herein can be of any class, e.g., IgG, IgA, IgM, IgE, IgD, or their isotypes, and can include a kappa or lambda light chain. In one embodiment, the antibody is an IgG antibody, e.g., at least one of the isotypes IgG1, IgG2, IgG3, or IgG4. In further embodiments, the antibody is in a format, e.g., an IgG format, with an Fc mutated to confer desired properties, e.g., reduce effector function, extend half-life, alter ADCC, or improve hinge stability. Such modifications are well known in the art.

[0122] In one embodiment, the antibody or fragment thereof is human. Thus, the antibody or fragment thereof can be derived from a human immunoglobulin (Ig) sequence. The CDR, framework, and / or constant region of the antibody (or fragment thereof) can be derived from a human Ig sequence, particularly a human IgG sequence. The CDR, framework, and / or constant region can be substantially identical to the human Ig sequence, particularly a human IgG sequence. The advantage of using a human antibody is that it is less immunogenic or non-immunogenic in humans.

[0123] The antibody or fragment thereof can also be chimeric, for example, a mouse-human antibody chimera.

[0124] Alternatively, the antibody or fragment thereof is derived from a non-human species, such as a mouse. Such a non-human antibody can be modified to increase its similarity to antibody variants naturally produced in humans; thus, the antibody or fragment thereof can be partially or fully humanized. Thus, in one embodiment, the antibody or fragment thereof is humanized.

[0125] (Antibodies targeted to epitopes) Provided herein are antibodies (or fragments thereof) that bind to an epitope on the V51 chain of a γδ TCR. Such binding may optionally have an effect on γδ TCR activity, for example, activation or inhibition.

[0126] In one embodiment, the epitope may be an activating epitope of γδ T cells. An "activating" epitope can include, for example, stimulating TCR function, such as degranulation, TCR downregulation, cytotoxicity, proliferation, recruitment, increased survival or resistance to exhaustion, intracellular signaling, cytokine or growth factor secretion, a phenotypic change, or a change in gene expression. For example, binding of an activating epitope can stimulate the expansion (i.e., proliferation) of a γδ T cell population, preferably a Vδ1+ T cell population. Thus, these antibodies can be used to regulate γδ T cell activation and thereby regulate an immune response. Thus, in one embodiment, binding of an activating epitope downregulates a γδ TCR. In a further or alternative embodiment, binding of an activating epitope activates degranulation of γδ T cells. In further additional or alternative embodiments, binding of an activating epitope activates γδ T cell killing.

[0127] Alternatively, the antibody (or fragment thereof) may have a blocking effect by interfering with the binding or interaction of another antibody or molecule. In one embodiment, the invention provides an isolated antibody or fragment thereof that blocks V51 and interferes with TCR binding (e.g., by steric hindrance). By interfering with V51, the antibody may interfere with TCR activation and / or signaling. The epitope may be an inhibitory epitope of γδ T cells. An "inhibitory" epitope may include, for example, blocking TCR function, thereby inhibiting TCR activation.

[0128] The epitope preferably consists of at least one extracellular, soluble, hydrophilic, external, or cytoplasmic portion of the V51 chain of a γδ TCR.

[0129] In particular, the epitope does not include epitopes found in the hypervariable region of the V51 chain of a γδ TCR, particularly in the CDR3 of the V51 chain. In a preferred embodiment, the epitope is within the non-variable region of the V51 chain of a γδ TCR. It will be appreciated that such binding allows for unique recognition of the V51 chain without being limited by the highly variable sequence of the TCR (particularly the CDR3). Various γδ TCR complexes that recognize MHC-like peptides or antigens can be recognized in this manner simply by the presence of the V51 chain. It will therefore be appreciated that any γδ TCR comprising a V51 chain can be recognized using an antibody or fragment thereof defined herein, regardless of the specificity of the γδ TCR. In one embodiment, the epitope comprises amino acid regions 1-24 and / or 35-90 of SEQ ID NO: 1, e.g., one or more amino acid residues within the portion of the V51 chain that is not part of the CDR1 and / or CDR3 sequence. In one embodiment, the epitope does not include amino acid residues within amino acid region 91 to 105 (CDR3) of SEQ ID NO:1.

[0130] In a manner similar to well-characterized αβ T cells, γδ T cells utilize a different set of somatically rearranged variable (V), diversity (D), joining (J), and constant (C) genes, but γδ T cells contain fewer V, D, and J segments than αβ T cells. In one embodiment, the epitope bound by the antibody (or fragment thereof) is located in the J region of the Vδ1 chain (e.g., one of 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 * In one embodiment, the epitope bound by the antibody (or fragment thereof) does not include an epitope found in the C-region of the V51 chain (e.g., one of SEQ ID NO: 135 (C1 *0)). In one embodiment, the epitope bound by the antibody (or fragment thereof) does not include an epitope found in the N-terminal leader sequence of the V51 chain (e.g., SEQ ID NO: 129). Thus, the antibody or fragment can bind only in the V region of the V51 chain (e.g., SEQ ID NO: 130). Thus, in one embodiment, the epitope consists of an epitope in the V region of the γδ TCR (e.g., amino acid residues 1-90 of SEQ ID NO: 1).

[0131] Reference to the epitope is made to SEQ ID NO:1: [ka] (2013) Immunity 39: 1032-1042, and the Vδ1 sequences from RCSB Protein Data Bank entries 4MNH and 3OMZ, shown as

[0132] SEQ ID NO: 1 represents a soluble TCR comprising a V region (also referred to as a variable domain), a D region, a J region, and a TCR constant region. The V region comprises amino acid residues 1-90, the D region comprises amino acid residues 91-104, the J region comprises amino acid residues 105-115, and the constant region comprises amino acid residues 116-209. In 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)).

[0133] Thus, in one embodiment, the isolated antibody or fragment thereof comprises the amino acid region: (i) SEQ ID NO: 1, 3 to 20; and / or (ii) 37 to 77 of SEQ ID NO: 1 It binds to an epitope on the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) that contains one or more amino acid residues within

[0134] In a further embodiment, the antibody or fragment thereof further recognizes a polymorphic V region comprising an epitope at amino acid residues 1-90 of SEQ ID NO: 128. Thus, 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, SEQ ID NO: 128) can be considered interchangeable. The antibodies of the present invention can recognize both variants of this germline sequence. By way of example, when an antibody or fragment thereof defined herein is described as recognizing an epitope comprising one or more amino acid residues within amino acid region 1-24 and / or 35-90 of SEQ ID NO: 1, this also refers to the same region of SEQ ID NO: 128; specifically, amino acid region 1-24 and / or 35-90 of SEQ ID NO: 128.

[0135] In one embodiment, the antibody or fragment thereof recognizes one or more amino acid residues within amino acid region 1-90 of SEQ ID NO: 1 and the equivalently positioned amino acids in region 1-90 of SEQ ID NO: 128. More specifically, in one embodiment, the antibody or fragment thereof defined herein recognizes a human germline epitope, wherein the germline encodes either an alanine (A) or a valine (V) at position 71 of SEQ ID NO: 1.

[0136] In one embodiment, an epitope includes one or more, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues within the described region.

[0137] In a further embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 3-20 of SEQ ID NO: 1. In an alternative embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 37-77 (e.g., amino acid region 50-54) of SEQ ID NO: 1. In yet a further embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 3-20 (e.g., 5-20 or 3-17) and one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 37-77 (e.g., 62-77 or 62-69) of SEQ ID NO: 1.

[0138] It will be further understood that the antibody (or fragment thereof) need not bind to every amino acid within a defined range. Such an epitope can be referred to as a linear epitope. For example, an antibody that binds to an epitope comprising amino acid residues within amino acid region 5-20 of SEQ ID NO: 1 can bind only to amino acid residues within that range, e.g., one or more of the amino acid residues at each end of that range (i.e., amino acids 5 and 20), optionally including the amino acids within that range (i.e., amino acids 5, 9, 16, and 20).

[0139] 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 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.

[0140] In one embodiment, the epitope is the following amino acid region of SEQ ID NO: 1 (or SEQ ID NO: 128 above): (i) 3–17; (ii) 5–20; (iii) 37–53; (iv) 50–64; (v)59-72; (vi) 59-77; (vii) 62 to 69; and / or (viii) 62-77 It contains one or more amino acid residues in

[0141] In further embodiments, the epitope comprises one or more amino acid residues within the amino acid regions 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 further embodiments, the epitope consists of one or more amino acid residues within the amino acid regions 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.

[0142] In a further embodiment, the epitope comprises or is preferred from 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 or is preferred from amino acid residues 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO: 1. In still further embodiments, the epitope comprises or is preferred from amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77 of SEQ ID NO: 1. In further embodiments, the epitope comprises or is preferred from amino acid residues 50, 53, 59, 62, and 64 of SEQ ID NO: 1. In further embodiments, the epitope comprises or is preferred from amino acid residues 59, 60, 68, and 72 of SEQ ID NO: 1.

[0143] In one embodiment, the epitope comprises one or more amino acid residues within the amino acid region 5-20 and / or 62-77 of SEQ ID NO: 1. In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region 5-20 and 62-77 of SEQ ID NO: 1. In an alternative further embodiment, the epitope comprises one or more amino acid residues within the amino acid region 5-20 or 62-77 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may comprise part or all of the sequence of 1245_P01_E07, or such an antibody or fragment thereof may be derived from 1245_P01_E07. For example, an antibody or fragment 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 an epitope.

[0144] In one embodiment, the epitope comprises one or more amino acid residues within the 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 the amino acid region 50-64 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may comprise part or all of the sequence of 1252_P01_C08, or such an antibody or fragment thereof may be derived from 1252_P01_C08. For example, an antibody or fragment 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 an epitope.

[0145] In one embodiment, the epitope comprises one or more amino acid residues within the amino acid region 37-53 and / or 59-77 of SEQ ID NO: 1. In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region 37-53 and 59-77 of SEQ ID NO: 1. In an alternative further embodiment, the epitope comprises one or more amino acid residues within the amino acid region 37-53 or 59-77 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may comprise part or all of the sequence of 1245_P02_G04, or such an antibody or fragment thereof may be derived from 1245_P02_G04. For example, an antibody or fragment 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 an epitope.

[0146] In one embodiment, the epitope comprises one or more amino acid residues within the 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 the amino acid region 59-72 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may comprise part or all of the sequence of 1251_P02_C05, or such an antibody or fragment thereof may be derived from 1251_P02_C05. For example, an antibody or fragment 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 an epitope.

[0147] In one embodiment, the epitope does not include amino acid residues in the amino acid region 11 to 21 of SEQ ID NO: 1. In one embodiment, the epitope does not include amino acid residues in the amino acid region 21 to 28 of SEQ ID NO: 1. In one embodiment, the epitope does not include amino acid residues in the amino acid region 59 and 60 of SEQ ID NO: 1. In one embodiment, the epitope does not include amino acid residues in the amino acid region 67 to 82 of SEQ ID NO: 1.

[0148] In one embodiment, the epitope is not the same epitope bound by commercially available anti-V51 antibodies, such as TS-1 or TS8.2. As described in WO2017197347, binding of TS-1 and TS8.2 to soluble TCRs was detected when the 51 chain comprised the V51 J1 and V51 J2 sequences, but not the V51 J3 chain, indicating that binding of TS-1 and TS8.2 requires key residues in the deltaJ1 and deltaJ2 regions.

[0149] References herein to "within" include the ends of the defined range. For example, "within amino acid region 5-20" refers to all amino acid residues from residue 5, inclusive, through residue 20, inclusive.

[0150] Various techniques for determining which epitopes are bound by antibodies are known in the art. Exemplary techniques include, for example, routine cross-blocking assays, alanine scanning mutation analysis, peptide blot analysis, peptide truncation analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be utilized. Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange (described in Example 9) detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding an antibody to the deuterium-labeled protein. Next, when the protein / antibody complex is transferred to water, exchangeable protons in amino acids protected by the antibody complex undergo back exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface are able to retain deuterium and therefore exhibit a relatively larger mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts.

[0151] (antibody sequence) An isolated anti-V51 antibody or fragment thereof can be described with reference to its CDR sequences.

[0152] In one embodiment, the anti-V51 antibody or fragment thereof a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 25; SEQ ID NOs: 26 to 37 and a CDR2 comprising a sequence having at least 80% sequence identity with any one of the sequences: A1 to A12; and / or CDR1 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61 : Contains one or more of the following.

[0153] In one embodiment, the isolated anti-Vδ1 antibody or fragment thereof comprises a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 25. In one embodiment, the antibody or fragment thereof comprises a CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26 to 37 and sequences: A1 to A12 (in Table 2). In one embodiment, the antibody or fragment thereof comprises a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38 to 61.

[0154] In one embodiment, the antibody or fragment thereof comprises a CDR3 comprising a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2 to 25. In one embodiment, the antibody or fragment thereof comprises a CDR2 comprising a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 26 to 37 and sequences: A1 to A12 (in Table 2). In one embodiment, the antibody or fragment thereof comprises a CDR1 comprising a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 38 to 61.

[0155] In one embodiment, the antibody or fragment thereof comprises a CDR3 consisting of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2 to 25. In one embodiment, the antibody or fragment thereof comprises a CDR2 consisting of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 26 to 37 and sequences A1 to A12 (in Table 2). In one embodiment, the antibody or fragment thereof comprises a CDR1 consisting of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 38 to 61.

[0156] In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 sequence sharing at least 80% sequence identity with any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 14 to 25. In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 consisting of a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 14 to 25.

[0157] In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14 to 25. In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 consisting of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14 to 25.

[0158] In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 14 to 25. In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 consisting of a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 14 to 25.

[0159] In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 13, and a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 25. In one embodiment, the antibody or fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 13, and a VL region comprising a CDR3 consisting of a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 25.

[0160] In one embodiment, an antibody or a fragment thereof comprises a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4, and a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16. In one embodiment, an antibody or a fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4, and a VL region comprising a CDR3 consisting of a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16.

[0161] In one embodiment, an antibody or a fragment thereof comprises a VH region comprising a CDR3 comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4, and / or a VL region comprising a CDR3 comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16. In one embodiment, an antibody or a fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4, and / or a VL region comprising a CDR3 consisting of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16.

[0162] In one embodiment, an antibody or a fragment thereof comprises a VH region comprising a CDR3 comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4, and / or a VL region comprising a CDR3 comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16. In one embodiment, an antibody or a fragment thereof comprises a VH region comprising a CDR3 consisting of a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4, and / or a VL region comprising a CDR3 consisting of a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16.

[0163] In one embodiment, an antibody or a fragment thereof, comprising a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8 to 13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20 to 25, particularly 20, 21, 22, or 23. In one embodiment, an antibody or a fragment thereof, comprising a VH region comprising a CDR3 consisting of a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8 to 13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 consisting of a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20 to 25, particularly 20, 21, 22, or 23.

[0164] In one embodiment, an antibody or a fragment thereof, comprising a VH region comprising a CDR3 comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 8 to 13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20 to 25, particularly 20, 21, 22, or 23. In one embodiment, an antibody or a fragment thereof, comprising a VH region comprising a CDR3 consisting of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 8 to 13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 consisting of a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20 to 25, particularly 20, 21, 22, or 23.

[0165] In one embodiment, an antibody or a fragment thereof, comprising a VH region comprising a CDR3 comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 8 to 13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 comprising a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 20 to 25, particularly 20, 21, 22, or 23. In one embodiment, an antibody or a fragment thereof, comprising a VH region comprising a CDR3 consisting of a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 8 to 13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 consisting of a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 20 to 25, particularly 20, 21, 22, or 23.

[0166] As used herein, embodiments referring to "at least 80%" or "80% or more" will be understood to include all values ​​of sequence identity of 80% or more, e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In one embodiment, the antibody or fragment thereof comprises at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the designated sequence.

[0167] Instead of percentage sequence identity, embodiments can also be defined using one or more amino acid changes, for example, one or more additions, substitutions, and / or deletions. In one embodiment, the sequence may contain up to five amino acid changes, for example, 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, for example, up to three amino acid substitutions, particularly up to one or two amino acid substitutions. For example, the CDR3 of the antibody or a fragment thereof preferably comprises or consists of a sequence having two or fewer substitutions, more preferably one or fewer substitutions, compared to any one of SEQ ID NOs: 2 to 25.

[0168] Suitably, any residue in CDR1, CDR2 or CDR3 that differs from its corresponding residue in SEQ ID NOs: 2 to 61 and SEQ ID NOs: A1 to A12 is a conservative substitution for its corresponding residue, for example, any residue in CDR3 that differs from its corresponding residue in SEQ ID NOs: 2 to 25 is a conservative substitution for its corresponding residue.

[0169] In one embodiment, the antibody or fragment thereof (i) a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 13; (ii) a VH region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26 to 37; (iii) a VH region comprising a CDR1 having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38 to 49; (iv) a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 25; (v) a VL region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of the sequences: A1 to A12; and / or (vi) a VL region comprising a CDR1 having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 50 to 61; Contains:

[0170] In one embodiment, the antibody or fragment thereof (i) a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 13; (ii) a VH region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26 to 37; and (iii) a VH region comprising a CDR1 having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38 to 49; :

[0171] In one embodiment, the antibody or fragment thereof (i) a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 25; (ii) a VL region including a CDR2 having a sequence having at least 80% sequence identity with any one of the sequences: A1 to A12; and (iii) a VL region comprising a CDR1 having a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 50 to 61 :

[0172] In one embodiment, the antibody or fragment thereof comprises (or consists of) a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 3, 4, 5, or 6, e.g., 2, 3, 4, or 5, particularly 2, 3, or 4. In one embodiment, the antibody or fragment thereof comprises (or consists of) a VH region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26, 27, 28, 29, or 30, e.g., 26, 27, 28, or 29, particularly 26, 27, or 28. In one embodiment, the antibody or fragment thereof comprises (or consists of) a VH region comprising a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38, 39, 40, 41, or 42, e.g., 38, 39, 40, or 41, particularly 38, 39, or 40.

[0173] In one embodiment, the antibody or fragment thereof comprises (or consists of) a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 8, 9, 10, or 11. In one embodiment, the antibody or fragment thereof comprises (or consists of) a VH region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 32, 33, 34, or 35. In one embodiment, the antibody or fragment thereof comprises (or consists of) a VH region comprising a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 44, 45, 46, or 47.

[0174] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 2, a CDR2 comprising the sequence of SEQ ID NO: 26, and a CDR1 comprising 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.

[0175] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 3, a CDR2 comprising the sequence of SEQ ID NO: 27, and a CDR1 comprising 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.

[0176] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 4, a CDR2 comprising the sequence of SEQ ID NO: 28, and a CDR1 comprising 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.

[0177] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 5, a CDR2 comprising the sequence of SEQ ID NO: 29, and a CDR1 comprising 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.

[0178] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 6, a CDR2 comprising the sequence of SEQ ID NO: 30, and a CDR1 comprising 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.

[0179] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 8, a CDR2 comprising the sequence of SEQ ID NO: 32, and a CDR1 comprising 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.

[0180] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 9, a CDR2 comprising the sequence of SEQ ID NO: 33, and a CDR1 comprising 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.

[0181] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 10, a CDR2 sequence of SEQ ID NO: 34, and a CDR1 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.

[0182] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 11, a CDR2 sequence of SEQ ID NO: 35, and a CDR1 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.

[0183] In one embodiment, the antibody or fragment thereof comprises (or consists of) a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14 to 25, e.g., SEQ ID NOs: 14, 15, 16, 17, or 18, e.g., 14, 15, 16, or 17, particularly 14, 15, or 16. In one embodiment, the antibody or fragment thereof comprises (or consists of) a VL region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of sequences: A1 to A12 (in Table 2), e.g., sequence: A1, A2, A3, A4, or A5, e.g., A1, A2, A3, or A4, particularly A1, A2, or A3. In one embodiment, the antibody or fragment thereof comprises (or consists of) a VL region comprising a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 50 to 61, e.g., SEQ ID NOs: 50, 51, 52, 53, or 54, e.g., 50, 51, 52, or 53, particularly 50, 51, or 52.

[0184] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 14, a CDR2 comprising the sequence of sequence: A1, and a CDR1 comprising 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: A1, and CDR1 consists of the sequence of SEQ ID NO: 50.

[0185] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 15, a CDR2 comprising the sequence of sequence: A2, and a CDR1 comprising 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: A2, and CDR1 consists of the sequence of SEQ ID NO: 51.

[0186] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 16, a CDR2 comprising the sequence of sequence: A3, and a CDR1 comprising 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: A3, and CDR1 consists of the sequence of SEQ ID NO: 52.

[0187] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 17, a CDR2 comprising the sequence of sequence: A4, and a CDR1 comprising 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: A4, and CDR1 consists of the sequence of SEQ ID NO: 53.

[0188] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 18, a CDR2 comprising the sequence of A5, and a CDR1 comprising 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 A5, and CDR1 consists of the sequence of SEQ ID NO: 54.

[0189] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of A7, and a CDR1 comprising 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 A7, and CDR1 consists of the sequence of SEQ ID NO: 56.

[0190] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 21, a CDR2 comprising the sequence of A8, and a CDR1 comprising 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 A8, and CDR1 consists of the sequence of SEQ ID NO: 57.

[0191] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 22, a CDR2 comprising the sequence of A9, and a CDR1 comprising 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 A9, and CDR1 consists of the sequence of SEQ ID NO: 58.

[0192] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 23, a CDR2 comprising the sequence of A10, and a CDR1 comprising 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 A10, and CDR1 consists of the sequence of SEQ ID NO: 59.

[0193] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 2, a CDR2 comprising the sequence of SEQ ID NO: 26, a CDR1 comprising the sequence of SEQ ID NO: 38, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 14, a CDR2 comprising the sequence of sequence: A1, and a CDR1 comprising the sequence of SEQ ID NO: 50. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 2, HCDR2 consists of the sequence of SEQ ID NO: 26, HCDR1 consists of the sequence of SEQ ID NO: 38, LCDR3 consists of the sequence of SEQ ID NO: 14, LCDR2 consists of the sequence: A1, and LCDR1 consists of the sequence of SEQ ID NO: 50.

[0194] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 3, a CDR2 comprising the sequence of SEQ ID NO: 27, a CDR1 comprising the sequence of SEQ ID NO: 39, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 15, a CDR2 comprising the sequence of sequence: A2, and a CDR1 comprising the sequence of SEQ ID NO: 51. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 3, HCDR2 consists of the sequence of SEQ ID NO: 27, HCDR1 consists of the sequence of SEQ ID NO: 39, LCDR3 consists of the sequence of SEQ ID NO: 15, LCDR2 consists of the sequence: A2, and LCDR1 consists of the sequence of SEQ ID NO: 51.

[0195] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 4, a CDR2 comprising the sequence of SEQ ID NO: 28, a CDR1 comprising the sequence of SEQ ID NO: 40, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 16, a CDR2 comprising the sequence of sequence: A3, and a CDR1 comprising the sequence of SEQ ID NO: 52. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 4, HCDR2 consists of the sequence of SEQ ID NO: 28, HCDR1 consists of the sequence of SEQ ID NO: 40, LCDR3 consists of the sequence of SEQ ID NO: 16, LCDR2 consists of the sequence: A3, and LCDR1 consists of the sequence of SEQ ID NO: 52.

[0196] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 5, a CDR2 comprising the sequence of SEQ ID NO: 29, a CDR1 comprising the sequence of SEQ ID NO: 41, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 17, a CDR2 comprising the sequence of sequence: A4, and a CDR1 comprising the sequence of SEQ ID NO: 53. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 5, HCDR2 consists of the sequence of SEQ ID NO: 29, HCDR1 consists of the sequence of SEQ ID NO: 41, LCDR3 consists of the sequence of SEQ ID NO: 17, LCDR2 consists of the sequence: A4, and LCDR1 consists of the sequence of SEQ ID NO: 53.

[0197] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 6, a CDR2 comprising the sequence of SEQ ID NO: 30, a CDR1 comprising the sequence of SEQ ID NO: 42, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 18, a CDR2 comprising the sequence of the sequence: A5, and a CDR1 comprising the sequence of SEQ ID NO: 54. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 6, HCDR2 consists of the sequence of SEQ ID NO: 30, HCDR1 consists of the sequence of SEQ ID NO: 42, LCDR3 consists of the sequence of SEQ ID NO: 18, LCDR2 consists of the sequence: A5, and LCDR1 consists of the sequence of SEQ ID NO: 54.

[0198] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 7, a CDR2 comprising the sequence of SEQ ID NO: 31, a CDR1 comprising the sequence of SEQ ID NO: 43, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 19, a CDR2 comprising the sequence of sequence: A6, and a CDR1 comprising the sequence of SEQ ID NO: 55. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 7, HCDR2 consists of the sequence of SEQ ID NO: 31, HCDR1 consists of the sequence of SEQ ID NO: 43, LCDR3 consists of the sequence of SEQ ID NO: 19, LCDR2 consists of the sequence: A6, and LCDR1 consists of the sequence of SEQ ID NO: 55.

[0199] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 8, a CDR2 comprising the sequence of SEQ ID NO: 32, a CDR1 comprising the sequence of SEQ ID NO: 44, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of A7, and a CDR1 comprising the sequence of SEQ ID NO: 56. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 8, HCDR2 consists of the sequence of SEQ ID NO: 32, HCDR1 consists of the sequence of SEQ ID NO: 44, LCDR3 consists of the sequence of SEQ ID NO: 20, LCDR2 consists of the sequence of A7, and LCDR1 consists of the sequence of SEQ ID NO: 56.

[0200] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 9, a CDR2 comprising the sequence of SEQ ID NO: 33, a CDR1 comprising the sequence of SEQ ID NO: 45, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 21, a CDR2 comprising the sequence of A8, and a CDR1 comprising the sequence of SEQ ID NO: 57. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 9, HCDR2 consists of the sequence of SEQ ID NO: 33, HCDR1 consists of the sequence of SEQ ID NO: 45, LCDR3 consists of the sequence of SEQ ID NO: 21, LCDR2 consists of the sequence of A8, and LCDR1 consists of the sequence of SEQ ID NO: 57.

[0201] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 10, a CDR2 comprising the sequence of SEQ ID NO: 34, a CDR1 comprising the sequence of SEQ ID NO: 46, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 22, a CDR2 comprising the sequence of A9, and a CDR1 comprising the sequence of SEQ ID NO: 58. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 10, HCDR2 consists of the sequence of SEQ ID NO: 34, HCDR1 consists of the sequence of SEQ ID NO: 46, LCDR3 consists of the sequence of SEQ ID NO: 22, LCDR2 consists of the sequence of A9, and LCDR1 consists of the sequence of SEQ ID NO: 58.

[0202] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 11, a CDR2 comprising the sequence of SEQ ID NO: 35, a CDR1 comprising the sequence of SEQ ID NO: 47, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 23, a CDR2 comprising the sequence of A10, and a CDR1 comprising the sequence of SEQ ID NO: 59. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 11, HCDR2 consists of the sequence of SEQ ID NO: 35, HCDR1 consists of the sequence of SEQ ID NO: 47, LCDR3 consists of the sequence of SEQ ID NO: 23, LCDR2 consists of the sequence of A10, and LCDR1 consists of the sequence of SEQ ID NO: 59.

[0203] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 12, a CDR2 comprising the sequence of SEQ ID NO: 36, a CDR1 comprising the sequence of SEQ ID NO: 48, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 24, a CDR2 comprising the sequence of the sequence: A11, and a CDR1 comprising the sequence of SEQ ID NO: 60. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 12, HCDR2 consists of the sequence of SEQ ID NO: 36, HCDR1 consists of the sequence of SEQ ID NO: 48, LCDR3 consists of the sequence of SEQ ID NO: 24, LCDR2 consists of the sequence: A11, and LCDR1 consists of the sequence of SEQ ID NO: 60.

[0204] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 13, a CDR2 comprising the sequence of SEQ ID NO: 37, a CDR1 comprising the sequence of SEQ ID NO: 49, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 25, a CDR2 comprising the sequence of A12, and a CDR1 comprising the sequence of SEQ ID NO: 61. In one embodiment, HCDR3 consists of the sequence of SEQ ID NO: 13, HCDR2 consists of the sequence of SEQ ID NO: 37, HCDR1 consists of the sequence of SEQ ID NO: 49, LCDR3 consists of the sequence of SEQ ID NO: 25, LCDR2 consists of the sequence of A12, and LCDR1 consists of the sequence of SEQ ID NO: 61.

[0205] In one embodiment, the antibody or fragment thereof comprises one or more CDR sequences set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 listed 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_F07 listed 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_G06 listed 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_G09 listed in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1138_P01_B09 listed in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (eg, all) of the CDR sequences of clone 1251_P02_G10 listed in Table 2.

[0206] Preferably, the VH and VL regions listed above each comprise four framework regions (FR1 to FR4). In one embodiment, the antibody or fragment thereof comprises a framework region (e.g., FR1, FR2, FR3, and / or FR4) comprising a sequence having at least 80% sequence identity to a framework region in any one of SEQ ID NOs: 62 to 85. In one embodiment, the antibody or fragment thereof comprises a framework region (e.g., FR1, FR2, FR3, and / or FR4) comprising a sequence having at least 90%, e.g., at least 95%, 97%, or 99% sequence identity to a framework region in any one of SEQ ID NOs: 62 to 85. In one embodiment, the antibody or fragment thereof comprises a framework region (e.g., FR1, FR2, FR3, and / or FR4) comprising a sequence in any one of SEQ ID NOs: 62 to 85. In one embodiment, the antibody or fragment thereof comprises a framework region (e.g., FR1, FR2, FR3, and / or FR4) consisting of a sequence in any one of SEQ ID NOs: 62 to 85.

[0207] The antibodies described herein can be defined by their complete light and / or heavy chain variable sequences. In one embodiment, the antibody or fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 85. In one embodiment, the antibody or fragment thereof consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 85.

[0208] In one embodiment, the antibody or fragment thereof comprises a VH region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 73. In one embodiment, the antibody or fragment thereof comprises a VH region consisting of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 73. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62, 63, 64, 65, or 66, e.g., 62, 63, 64, or 65, particularly 62, 63, or 64. In a further embodiment, the VH region consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62, 63, 64, 65, or 66, e.g., 62, 63, 64, or 65, particularly 62, 63, or 64. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 68, 69, 70, 71, 72, or 73, such as 68, 69, 70, or 71. In a further embodiment, the VH region consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 68, 69, 70, 71, 72, or 73, such as 68, 69, 70, or 71.

[0209] In one embodiment, the antibody or fragment thereof comprises a VL region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74 to 85. In one embodiment, the antibody or fragment thereof comprises a VL region consisting of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74 to 85. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74, 75, 76, 77, or 78, e.g., 74, 75, 76, or 77, particularly 74, 75, or 76. In a further embodiment, the VL region consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74, 75, 76, 77, or 78, e.g., 74, 75, 76, or 77, particularly 74, 75, or 76. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 80, 81, 82, 83, 84, or 85, e.g., 80, 81, 82, or 83. In a further embodiment, the VL region consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 80, 81, 82, 83, 84, or 85, e.g., 80, 81, 82, or 83.

[0210] In a further embodiment, the antibody or fragment thereof comprises a VH region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 73, and a VL region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74 to 85. In a further embodiment, the antibody or fragment thereof comprises a VH region consisting of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 73, and a VL region consisting of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74 to 85.

[0211] In one embodiment, the antibody or fragment thereof comprises a VH region comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09).

[0212] 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 an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09).

[0213] In one embodiment, the antibody or fragment thereof comprises a VL region comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises a VL region comprising the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0214] 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 an alternative embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0215] In one embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 63 (1252_P01_C08) and a VL region comprising the amino acid sequence of SEQ ID NO: 75 (1252_P01_C08). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07) and a VL region comprising the amino acid sequence of SEQ ID NO: 74 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04) and a VL region comprising the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04) and a VL region comprising the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07) and a VL region comprising the amino acid sequence of SEQ ID NO: 81 (1245_P02_F07). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06) and a VL region comprising the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 71 (1245_P01_G06) and a VL region comprising the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0216] 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) and 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 VH region consisting of the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07) and 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 VH region consisting of the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04) and 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 VH region consisting of the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04) and 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 VH region consisting of the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07) and a VL region consisting of the amino acid sequence of SEQ ID NO: 81 (1245_P02_F07). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06) and a VL region consisting of the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09) and a VL region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0217] For fragments comprising both the VH and VL domains, these may be associated either covalently (e.g., via a disulfide bond or a linker) or non-covalently. The antibody fragments described herein may include scFvs, i.e., fragments comprising the VH and VL domains connected by a linker. In one embodiment, the VH and VL domains are connected by a (e.g., synthetic) polypeptide linker. The polypeptide linker may be (Gly4Ser) nThe polypeptide linker may include a linker (where n=1 to 8, e.g., 2, 3, 4, 5, or 7). n (Gly3AlaSer) m ] p In a further embodiment, the linker comprises SEQ ID NO: 98. In a further embodiment, the linker consists of SEQ ID NO: 98.

[0218] In one embodiment, the antibody or fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 86-97. In a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 86-97. In yet a further 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 an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0219] In one embodiment, the antibody or fragment thereof consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 86-97. In a further embodiment, the antibody or fragment thereof consists of the amino acid sequence of any one of SEQ ID NOs: 86-97. In a still further 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 an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0220] It will be understood by those skilled in the art that scFv constructs can be designed and generated with N- and C-terminal modifications to aid in translation, purification, and detection. For example, at the N-terminus of the scFv sequence, additional methionine and / or alanine amino acid residues can be included before the canonical VH sequence (e.g., beginning with QVQ or EVQ). At the C-terminus (i.e., C-terminal to the canonical VL domain sequence according to the IMGT definition), additional sequences can be included, such as (i) a partial sequence of a constant domain and / or (ii) an additional synthetic sequence containing a tag, such as a His-tag or Flag-tag, to aid in purification and detection. In one embodiment, SEQ ID NO: 124 is added to the C-terminus of any one of SEQ ID NOs: 86, 88-90, 92-97. In one embodiment, SEQ ID NO: 125 is added to the C-terminus of any one of SEQ ID NOs: 86, 88-90, 92-97. In one embodiment, SEQ ID NO: 126 is added to the C-terminus of any one of SEQ ID NOs: 87 or 91. In one embodiment, SEQ ID NO: 127 is added to the C-terminus of any one of SEQ ID NOs: 87 or 91. It is appreciated that the N- or C-terminal sequences of the scFv are optional and can be removed, modified, or replaced if alternative scFv design, translation, purification, or detection strategies are employed.

[0221] As described herein, the antibody can be in any format. In a preferred embodiment, the antibody is in an IgG1 format. Thus, in one embodiment, the antibody or fragment thereof comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111-122. In a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 111-122. In yet a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NOs: 111-116, e.g., SEQ ID NOs: 111-113 and 116. In yet a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NOs: 117-122, e.g., SEQ ID NOs: 117-120. In yet a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NOs: 111, 112, 116-120, e.g., SEQ ID NOs: 111, 112, or 116, or SEQ ID NOs: 117-120.

[0222] In one embodiment, the antibody or fragment thereof consists of an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111 to 122. In a further embodiment, the antibody or fragment thereof consists of the amino acid sequence of any one of SEQ ID NOs: 111 to 122. In an even further embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NOs: 111 to 116, for example, SEQ ID NOs: 111 to 113 and 116. In an even further embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NOs: 117 to 122, for example, SEQ ID NOs: 117 to 120. In an even further embodiment, the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NOs: 111, 112, 116 to 120, for example, SEQ ID NO: 111, 112 or 116, or SEQ ID NOs: 117 to 120.

[0223] In one embodiment, the antibody binds to the same or essentially the same epitope as an antibody or fragment thereof defined herein, or competes with said antibody or fragment thereof. Using routine methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference anti-V51 antibody, or competes for binding with the reference anti-V51 antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-V51 antibody, the reference antibody is bound to a V51 protein or peptide under saturating conditions. The ability of the test antibody to bind to the V51 chain is then evaluated. If the test antibody can bind to V51 after saturation binding with the reference anti-V51 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-V51 antibody. On the other hand, if the test antibody cannot bind to the V51 chain after saturation binding with the reference anti-V51 antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-V51 antibody.

[0224] The present invention also encompasses anti-V51 antibodies that compete for V51 binding with antibodies or fragments thereof defined herein, or with antibodies having the CDR sequences of any of the exemplary antibodies described herein. 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 V51 chain and / or share epitopes. These assays are readily apparent 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., V51. The antibody (or fragment thereof) is immobilized or insolubilized before or after competition, and the sample bound to the V51 chain is separated from the unbound sample, e.g., by decanting (if the antibody was pre-insolubilized) or centrifugation (if the antibody was precipitated after the competition reaction). Competitive binding can also be determined by whether a function is altered by the binding or lack of binding of the antibody to the protein, e.g., whether the antibody molecule inhibits or enhances, for example, an enzymatic activity of a target. ELISAs and other functional assays known in the art and described herein can be used.

[0225] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to a target antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the target antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0226] Additional routine experiments (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of 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.

[0227] In some embodiments, the antibody or fragment thereof contains a modified effector function by modifying the sugar attached to Asn 297 (Kabat numbering system). In further such modifications, Asn 297 is not fucosylated or exhibits reduced fucosylation (i.e., a defucosylated antibody or afucosylated antibody). Fucosylation includes the addition of the sugar fucose to a molecule, for example, the attachment of fucose to N-glycans, O-glycans, and glycolipids. Thus, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. Antibodies can be modified to prevent or inhibit antibody fucosylation. Typically, glycosylation modification involves expressing the antibody or fragment thereof in a host cell that contains alternative glycosylation processing capabilities, either by targeted engineering or by targeted or fortuitous host or clonal selection. These and other effector modifications are further discussed in recent reviews, e.g., by Xinhua Wang et al. (2018) Protein & Cell 9: 63-73 and Pereira et al. (2018) mAbs 10(5): 693-711, which are incorporated by reference.

[0228] (Antibody sequence modification) Antibodies and fragments thereof can be modified using known methods. The sequence modifications to the antibody molecules described herein can be readily incorporated by one of skill in the art. The following examples are non-limiting.

[0229] Upon antibody discovery and sequence recovery from phage libraries, desired antibody variable domains can be reformatted by subcloning into full-length IgGs. To accelerate this process, variable domains are often transferred using restriction enzymes. These unique restriction sites can introduce additional / alternative amino acids away from the standard sequence (such standard sequences can be found, for example, in the International ImMunoGeneTics [IMGT] information system, see http: / / www.imgt.org). These can be introduced as kappa or lambda light chain sequence modifications.

[0230] (Kappa light chain modification) The variable kappa light chain variable sequence can be cloned into a full-length IgG using restriction sites (e.g., Nhe1-Not1) upon reformatting. More specifically, an additional Ala-Ser sequence was introduced at the kappa light chain N-terminus to aid in cloning. Preferably, this additional AS sequence is then removed in further development to generate the standard N-terminal sequence. Thus, in one embodiment, the kappa light chain containing antibodies described herein do not contain an AS sequence at their N-terminus, i.e., SEQ ID NOs: 74, 76-78, and 80-85 do not contain the initial AS sequence. In a further embodiment, SEQ ID NOs: 74 and 76-78 do not contain the initial AS sequence. It will be understood that this embodiment also applies to other sequences contained herein that contain this sequence (e.g., SEQ ID NOs: 86, 88-90, and 92-97).

[0231] Additional amino acid changes can be made to aid in cloning. For example, for the antibodies described herein, a valine to alanine change was introduced at the kappa light chain variable domain / constant domain boundary to aid in cloning. This resulted in a kappa constant domain modification. Specifically, this [ka] (from the NotI restriction site) is obtained. Preferably, this sequence is modified in further development to [ka] A standard kappa light chain constant region beginning with the sequence RTV can be generated. Thus, in one embodiment, the kappa light chain containing antibodies described herein contain a constant domain beginning with the sequence RTV. Thus, in one embodiment, the sequences of SEQ ID NOs: 111-114 and 117-122 can be generated. [ka] is an array [ka] has been replaced with

[0232] (lambda light chain modification) Similar to the kappa example above, lambda light chain variable domains can also be cloned into full-length IgGs by introducing restriction sites (e.g., Nhe1-Not1) during reformatting. More specifically, an additional Ala-Ser sequence can be introduced at the lambda light chain N-terminus to aid cloning. Preferably, this additional AS sequence is then removed in further development to generate the standard N-terminal sequence. Thus, in one embodiment, the lambda light chain containing antibodies described herein do not contain an AS sequence at their N-terminus, i.e., SEQ ID NOs: 75 and 79 do not contain the initial AS sequence. It will be understood that this embodiment also applies to other sequences contained herein that contain this sequence (e.g., SEQ ID NOs: 87, 91, 115, and 116). In one embodiment, SEQ ID NO: 75 does not contain the first 6 residues, i.e., [ka] The sequence has been removed.

[0233] As another example, for the antibodies described herein, a lysine to alanine sequence change was introduced at the lambda light chain variable domain / constant domain boundary to aid in cloning. This resulted in a lambda constant domain modification. Specifically, this resulted in: [ka] (from the NotI restriction site) was obtained. Preferably, this sequence is [ka] In further development, the lambda light chain constant region can be modified to generate a standard lambda light chain constant region beginning with the sequence [ka] Thus, in one embodiment, the sequence of SEQ ID NO: 115 or 116 [ka] is an array [ka] has been replaced with

[0234] (heavy chain modification) Typically, human variable heavy chain sequences begin with either a basic glutamine (Q) or an acidic glutamic acid (E). However, both such sequences are known to subsequently convert to the acidic amino acid residue pyroglutamic acid (pE). The Q to pE conversion results in a charge change of the antibody, whereas the E to pE conversion does not. Therefore, to avoid variable charge changes over time, one option is to first modify the starting heavy chain sequence from Q to E. Thus, in one embodiment, the heavy chain of the antibody described herein contains a Q to E modification at the N-terminus. In particular, the first residue of SEQ ID NOs: 62, 64, and / or 67-71 can be modified from Q to E. It will be understood that this embodiment also applies to other sequences contained herein that contain this sequence (e.g., SEQ ID NOs: 86, 88, 91-97, and 111, 112, 115, 117-120).

[0235] Furthermore, the C-terminus of the IgG1 constant domain ends with PGK. However, the terminal basic lysine (K) is often subsequently cleaved during expression (e.g., in CHO cells). This, in turn, results in a change in the charge of the antibody due to the variable loss of the C-terminal lysine residue. Therefore, one option is to first remove the lysine, resulting in a uniform and consistent heavy chain C-terminal sequence ending with PG. Thus, in one embodiment, the heavy chain of the antibody described herein has the terminal K removed from its C-terminus. In particular, the antibody of the present invention may comprise any one of SEQ ID NOs: 111-122 in which the terminal lysine residue has been removed.

[0236] (any allotype modification) Specific human allotypes can be utilized during antibody discovery. Optionally, antibodies can be converted to different human allotypes during development. As a non-limiting example, for kappa chains, there are three human allotypes designated Km1, Km1,2, and Km3, which define three Km alleles (using allotype numbering): Km1 is related to valine 153 (IMGT V45.1) and leucine 191 (IMGT L101); Km1,2 is related to alanine 153 (IMGT A45.1) and leucine 191 (IMGT L101); and Km3 is related to alanine 153 (IMGT A45.1) and valine 191 (IMGT V101). Optionally, sequences can therefore be modified from one allotype to another by standard cloning techniques. For example, the L191V (IMGT L101V) change converts the Km1,2 allotype to the Km3 allotype. For further discussion of such allotypes, see Jefferis and Lefranc (2009) MAbs 1(4):332-8, incorporated herein by reference.

[0237] Thus, in one embodiment, the antibodies described herein contain amino acid substitutions derived from different human allotypes of the same gene. In a further embodiment, the antibodies contain a L191V (IMGT L101V) substitution in the kappa chain to convert the c-domain from the km1,2 to km3 allotype.

[0238] (antibody binding) The antibody or fragment thereof has a molecular weight of 1.5 x 10 as measured by surface plasmon resonance. -7 In a preferred embodiment, the KD is less than 1.5×10 -7 In a further embodiment, the KD is less than 1.3 x 10 -7 M (i.e., 130 nM) or less, e.g., 1.0 x 10 -7In still further embodiments, the KD is 5.0 x 10 -8 M (i.e., less than 50 nM), e.g., 4.0 x 10 -8 M (i.e., less than 40 nM), 3.0 × 10 -8 M (i.e., 30 nM), or 2.0 × 10 -8 M (i.e., less than 20 nM). For example, according to one embodiment, the ion concentration is less than 1.5×10 as measured by surface plasmon resonance. -7 Human anti-V51 antibodies are provided that bind to the V51 chain of a γδ TCR with a binding affinity (KD) of less than M (i.e., 150 nM).

[0239] In one embodiment, the antibody or fragment thereof has a denaturation coefficient of 4.0 x 10 as measured by surface plasmon resonance. -8 M (i.e., less than 40 nM), 3.0 × 10 -8 M (i.e., 30 nM) or less than 2.0 × 10 -8 It binds to the Vδ1 chain of the γδ TCR with a binding affinity (KD) of less than M (i.e., 20 nM).

[0240] In one embodiment, the binding affinity of an antibody or fragment thereof is determined by coating the antibody or fragment thereof directly or indirectly (e.g., by capture with anti-human IgG Fc) onto the surface of a sensor (e.g., an amine high-capacity chip or equivalent), where the target bound by the antibody or fragment thereof (i.e., the V51 chain of the γδ TCR) is flowed over the chip to detect binding. Suitably, a MASS-2 instrument (which may also be referred to as a Sierra SPR-32) is used at 30 μl / min at 25° C. in PBS+0.02% Tween 20 running buffer.

[0241] Described herein are other assays that can be used to define antibody function. For example, the antibodies or fragments thereof described herein can be assessed by γδ TCR engagement, e.g., by measuring downregulation of γδ TCR upon antibody binding. Surface expression of γδ TCR after application of the antibody or fragment thereof (optionally, displayed on the surface of a cell) can be measured, e.g., by flow cytometry. The antibodies or fragments thereof described herein can also be assessed by measuring γδ T cell degranulation. For example, expression of CD107a, a marker of cell degranulation, can be measured, e.g., by flow cytometry, after application of the antibody or fragment thereof (optionally, displayed on the surface of a cell) to γδ T cells. The antibodies or fragments thereof described herein can also be assessed by measuring γδ T cell killing activity (to test whether the antibody has an effect on the killing activity of γδ T cells). For example, target cells can be incubated with γδ T cells in the presence of the antibody or fragment thereof (optionally, displayed on the surface of a cell). After incubation, the cultures can be stained with a cell viability dye to distinguish between live and dead target cells, and the proportion of dead cells can then be measured, for example, by flow cytometry.

[0242] As described herein, the antibodies or fragments thereof used in the assays can be displayed on a surface, e.g., the surface of a cell, such as a cell containing an Fc receptor. For example, the antibodies or fragments thereof can be displayed on the surface of THP-1 cells, e.g., TIB-202™ cells (available from the American Type Culture Collection (ATCC)). Alternatively, the antibodies or fragments thereof can be used directly in the assays.

[0243] In such functional assays, output can be measured by calculating the half-maximal concentration, also referred to as "EC50" or "50 percent effective concentration." The term "IC50" refers to inhibitory concentration. Both EC50 and IC50 can be measured using methods known in the art, for example, flow cytometry. For the avoidance of doubt, EC50 values ​​in this application are provided using IgG1 formatted antibodies. Such values ​​can be readily converted based on the molecular weight of the antibody format for equivalent values, as follows: (μg / ml) / (MW in kDa) = μM

[0244] The EC50 for down-regulation of γδ TCR upon antibody (or fragment) binding may be less than 0.50 μg / ml, for example, 0.40 μg / ml, 0.30 μg / ml, 0.20 μg / ml, 0.15 μg / ml, 0.10 μg / ml, or less than 0.05 μg / ml. In a preferred embodiment, the EC50 for down-regulation of γδ TCR upon antibody (or fragment) binding is less than 0.10 μg / ml. In particular, the EC50 for down-regulation of γδ TCR upon antibody (or fragment) binding may be less than 0.06 μg / ml, for example, 0.05 μg / ml, 0.04 μg / ml, or less than 0.03 μg / ml. In particular, the EC50 value is when the antibody is measured in IgG1 format. For example, the EC50 value for γδ TCR down-regulation can be measured using flow cytometry (e.g., as described in the assay of Example 6).

[0245] The EC50 for γδ T cell degranulation upon antibody (or fragment) binding may 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. In particular, the EC50 for γδ T cell degranulation upon antibody (or fragment) binding may be less than 0.005 μg / ml, for example, less than 0.002 μg / ml. In a preferred embodiment, the EC50 for γδ T cell degranulation upon antibody (or fragment) binding is less than 0.007 μg / ml. In particular, the EC50 value is when the antibody is measured in an IgG1 format. For example, the EC50 value for γδ T cell degranulation can be measured by measuring CD107a expression (i.e., a marker of cell degranulation) using flow cytometry (e.g., as described in the assay of Example 7). In one embodiment, CD107a expression is measured using an anti-CD107a antibody, e.g., anti-human CD107a BV421 (clone H4A3) (BD Biosciences).

[0246] The EC50 for γδ T cell killing upon antibody (or fragment) binding may 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 less than 0.07 μg / ml. In a preferred embodiment, the EC50 for γδ T cell killing upon antibody (or fragment) binding is less than 0.10 μg / ml. In particular, the EC50 for γδ T cell killing upon antibody (or fragment) binding may 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. In particular, the EC50 value is when the antibody is measured in an IgG1 format. For example, the EC50 value for γδ T cell killing can be measured by detecting the proportion of dead cells (i.e., using a cell viability dye) using flow cytometry (e.g., as described in the assay in Example 8) after incubation of the antibody, γδ T cells, and target cells. In one embodiment, target cell death is measured using the cell viability dye Viability Dye eFluor™ 520 (ThermoFisher).

[0247] In the assays described in these embodiments, the antibody or fragment thereof can be displayed on the surface of cells, such as THP-1 cells, e.g., TIB-202™ (ATCC). The THP-1 cells are optionally labeled with a dye, e.g., CellTracker™ Orange CMTMR (ThermoFisher).

[0248] Antibodies (or fragments) can be obtained and engineered using, for example, the techniques disclosed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012), 4th ed., Cold Spring Harbour Laboratory Press.

[0249] Monoclonal antibodies can be produced using hybridoma technology by fusing specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to grow in tissue culture and for their lack of antibody chain synthesis.

[0250] Monoclonal antibodies against a given antigen can be, for example, a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a predetermined antigen with immortal cells, preferably myeloma cells, to form hybridomas; b) Culturing the formed immortalized cells (hybridomas) and recovering cells that produce antibodies with the desired specificity. : can be obtained by

[0251] Alternatively, the use of hybridoma cells is not required. Antibodies capable of binding to the target antigens described herein can be routinely isolated from suitable antibody libraries using, for example, phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Thus, monoclonal antibodies can be isolated, for example, from a) cloning into a vector, in particular into a phage, more particularly into a filamentous bacteriophage, a DNA or cDNA sequence obtained from lymphocytes, in particular peripheral blood lymphocytes, of an animal (preferably one that has been previously immunized with a predetermined antigen); b) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the antibody; c) selecting the antibody by subjecting it to antigen-affinity selection d) recovering antibodies with the desired specificity : can be obtained by a process including

[0252] (Pharmaceutical composition) According to a further aspect of the present invention, there is provided a composition comprising a V51 T cell population obtained by the method defined herein. In one embodiment, the V51 T cell population is an expanded V51 T cell population. In such embodiments, the composition may optionally comprise cells in combination with other excipients. Also included are compositions comprising one or more additional active agents (e.g., active agents suitable for treating the diseases referred to herein).

[0253] Pharmaceutical compositions may comprise the V51 T cells described herein, particularly expanded V51 T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include 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. Cryoprotective solutions that may be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions may be formulated, for example, for intravenous administration.

[0254] In one embodiment, the pharmaceutical composition is substantially free of endotoxin or mycoplasma contaminants, eg, has no detectable levels of endotoxin or mycoplasma contaminants.

[0255] The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal). In a preferred embodiment, the composition is administered by intravenous infusion or injection. In another preferred embodiment, the composition is administered by intramuscular or subcutaneous injection.

[0256] It is within the scope of the present invention to use the pharmaceutical compositions of the present invention in therapeutic regimens for the treatment of the diseases described herein, as an adjunct to, or in conjunction with, other established therapies commonly used in the treatment of such diseases.

[0257] In a further embodiment of the invention, the cell population, composition or pharmaceutical composition is administered sequentially, simultaneously or separately with at least one active agent.

[0258] (Therapeutic method using cell populations) According to a further aspect of the present invention, there is provided a cell population obtained by the method defined herein for use as a medicament. According to a further aspect of the present invention, there is provided an expanded cell population defined herein for use as a medicament. References herein to a cell population "for use" as a medicament or in therapy are limited to administration of the cell population to a subject. Such use does not include direct administration of an antibody or fragment thereof to a patient, i.e., in this case, the antibody is used as a therapeutic agent.

[0259] In one embodiment, the cell population is for use in the treatment of cancer, an infectious disease, or an inflammatory disease. In a further embodiment, the cell population is for use in the treatment of cancer.

[0260] In one embodiment, the cell population for use as a medicament comprises more than 50% V51 T cells, such as more than 60%, more than 70%, more than 80%, more than 90%, more than 95% or more than 99% V51 T cells. In a further embodiment, the cell population for use as a medicament consists of V51 T cells.

[0261] In one embodiment, the cell population for use as a medicament comprises less than 10% αβ T cells, for example, less than 8%, 7%, 6%, 5%, 4%, or 3% αβ T cells. In one embodiment, the cell population for use as a medicament comprises less than 10% V52 T cells, for example, less than 8%, 7%, 6%, 5%, 4%, or 3% V52 T cells. In one embodiment, the cell population for use as a medicament comprises less than 50% NK cells, for example, less than 40%, 30%, 20%, 10%, or 5% NK cells. In one embodiment, less than 50% of the cells present in the cell population for use as a medicament express CD56, for example, less than 40%, 30%, 20%, 10%, or 5% express CD56.

[0262] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising the cell population as defined herein for use as a medicament. In one embodiment, the pharmaceutical composition comprising the cell population is for use in the treatment of cancer, an infectious disease, or an inflammatory disease. In a further embodiment, the pharmaceutical composition comprising the cell population is for use in the treatment of cancer.

[0263] According to a further aspect of the present invention there is provided a method of modulating an immune response in a subject in need thereof, the method comprising administering a therapeutically effective amount of the cell population defined herein.

[0264] According to a further aspect of the present invention there is provided a method of treating cancer, an infectious disease or an inflammatory disease in a subject in need thereof, comprising administering a therapeutically effective amount of a cell population as defined herein, or alternatively a therapeutically effective amount of a pharmaceutical composition comprising said cell population.

[0265] According to a further aspect of the present invention there is provided the use of a cell population as defined herein for the manufacture of a medicament, for example in the treatment of cancer, an infectious disease or an inflammatory disease.

[0266] (Adoptive T cell therapy) Gamma delta T cells obtained by the expansion methods of the present invention can be used as a medicine, for example, for adoptive T cell therapy, which involves transplanting γδ T cells into a patient. The therapy can be autologous, i.e., γδ T cells can be transplanted back into the same patient from which they were obtained, or allogeneic, i.e., γδ T cells from one person can be transplanted into a different patient. In cases involving allogeneic transplantation, the γδ T cells can be substantially free of αβ T cells. For example, αβ T cells can be removed from the γδ T cell population after expansion using any suitable means known in the art (e.g., by negative selection, e.g., using magnetic beads). The treatment method can include providing a tissue sample (e.g., a non-hematopoietic tissue sample) obtained from a donor individual; culturing γδ T cells obtained from the sample as described herein to produce an expanded population; and administering the population of γδ T cells to a recipient individual.

[0267] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor) or a virally infected patient (e.g., a CMV- or HIV-infected patient). Optionally, the patient has a solid tumor and / or is undergoing treatment for a solid tumor. Because tissue-resident V51 T cells typically reside in non-hematopoietic tissues, they are also more likely to home to and be retained within the tumor mass than their systemic, blood-resident counterparts, and adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-associated immunopathologies.

[0268] Because γδ T cells are MHC-unrestricted, they do not recognize the host into which they are transplanted as foreign, meaning that γδ T cells are unlikely to cause graft-versus-host disease, which means that γδ T cells can be used "off the shelf" and transplanted into any recipient, for example, for allogeneic adoptive T cell therapy.

[0269] The γδ T cells obtained by the methods described herein express NKG2D and can respond to NKG2D ligands (e.g., MICA) that are strongly associated with malignant tumors. The γδ T cells can also express a cytotoxic profile in the absence of any activation and are therefore likely to be effective in killing tumor cells. For example, the γδ T cells can express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin in the absence of any activation. IL-17A may not be expressed.

[0270] In some embodiments, a method for treating an individual having a tumor may comprise providing a sample of the tumor obtained from a donor individual, culturing γδ T cells obtained from said sample, and administering the population of γδ T cells to the individual having the tumor. In further embodiments, a method for treating an individual having a tumor in a non-hematopoietic tissue may comprise providing a sample of said non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells obtained from said sample, and administering the population of γδ T cells to the individual having the tumor.

[0271] Optionally, a therapeutically effective amount of γδ T cells obtained by any of the above methods can be administered to a subject in a therapeutically effective amount (e.g., for the treatment of cancer, e.g., for the treatment of a solid tumor). Optionally, a therapeutically effective amount of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) can be administered in a therapeutically effective amount of 10×10 per dose. 12 Fewer than 9 x 10 cells (e.g., 9 x 10 per dose) 12 Less than 8 x 10 cells per dose 12 Less than 7 x 10 cells 12 Less than 6 x 10 cells per dose 12 Less than 5 x 10 cells per dose 12 Less than 4 x 10 cells per dose 12 Less than 3 x 10 cells per dose 12 Less than 2 x 10 cells per dose12 Less than 1 x 10 cells per dose 12 Less than 9 x 10 cells per dose 11 Less than 8 x 10 cells per dose 11 Less than 7 x 10 cells per dose 11 Less than 6 x 10 cells per dose 11 Less than 5 x 10 cells per dose 11 Less than 4 x 10 cells per dose 11 Less than 3 x 10 cells per dose 11 Less than 2 x 10 cells per dose 11 Less than 1 x 10 cells per dose 11 Less than 9 x 10 cells per dose 10 Less than 7.5 x 10 cells per dose 10 Less than 5 x 10 cells per dose 10 Less than 2.5 x 10 cells per dose 10 Less than 1 x 10 cells per dose 10 Less than 7.5 x 10 cells per dose 9 Less than 5 x 10 cells per dose 9 Less than 2.5 x 10 cells per dose 9 Less than 1 x 10 cells per dose 9 Less than 7.5 x 10 cells per dose 8 Less than 5 x 10 cells per dose 8 Less than 2.5 x 10 cells per dose 8 Less than 1 x 10 cells per dose 8 Less than 7.5 x 10 cells per dose 7 Less than 5 x 10 cells per dose 7 Less than 2.5 x 10 cells per dose 7 Less than 1 x 10 cells per dose 7 Less than 7.5 x 10 cells per dose 6 Less than 5 x 10 cells per dose 6 Less than 2.5 x 10 cells per dose 6 Less than 1 x 10 cells per dose 6 Less than 7.5 x 10 cells per dose 5 Less than 5 x 10 cells per dose 5Less than 2.5 x 10 cells per dose 5 Less than 1 x 10 cells, or 1 x 10 per dose 5 (less than 100 cells).

[0272] In some embodiments, a therapeutically effective amount of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) is 10×10 12 Fewer than 9 x 10 cells (e.g., during the course of treatment) 12 Less than 8 x 10 cells 12 Less than 7 x 10 cells 12 Less than 6 x 10 cells 12 Less than 5 x 10 cells 12 Less than 4 x 10 cells 12 Less than 3 x 10 cells 12 Less than 2 x 10 cells 12 Less than 1 x 10 cells 12 Less than 9 x 10 cells 11 Less than 8 x 10 cells 11 Less than 7 x 10 cells 11 Less than 6 x 10 cells 11 Less than 5 x 10 cells 11 Less than 4 x 10 cells 11 Less than 3 x 10 cells 11 Less than 2 x 10 cells 11 Less than 1 x 10 cells 11 Less than 9 x 10 cells 10 Less than 7.5 x 10 cells 10 Less than 5 x 10 cells 10 Less than 2.5 x 10 cells 10 Less than 1 x 10 cells 10 Less than 7.5 x 10 cells 9 Less than 5 x 10 cells 9 Less than 2.5 x 10 cells 9 Less than 1 x 10 cells 9 Less than 7.5 x 10 cells 8 Less than 5 x 10 cells 8 Less than 2.5 x 10 cells 8 Less than 1 x 10 cells 8 Less than 7.5 x 10 cells 7 Less than 5 x 10 cells7 Less than 2.5 x 10 cells 7 Less than 1 x 10 cells 7 Less than 7.5 x 10 cells 6 Less than 5 x 10 cells 6 Less than 2.5 x 10 cells 6 Less than 1 x 10 cells 6 Less than 7.5 x 10 cells 5 Less than 5 x 10 cells 5 Less than 2.5 x 10 cells 5 Less than 1 x 10 cells, or 1 x 10 5 (less than 100 cells).

[0273] In some embodiments, the dose of γδ T cells described herein (e.g., skin-derived γδ T cells and / or Vδ1 T cells) is about 1×10 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , or 5 x 10 8 In some embodiments, the dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) comprises up to about 1 x 10 cells / kg. 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , or 5 x 10 8 In some embodiments, the dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) comprises about 1.1 x 10 cells / kg. 6 ~1.8×10 7In some embodiments, the dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) comprises about 1×10 cells / kg. 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 In some embodiments, the dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) comprises at least about 1 x 10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 In some embodiments, the dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) comprises up to about 1 x 10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 Contains cells.

[0274] In one embodiment, the subject receives 10 mg of 10 ... 4 ~10 6 In one embodiment, the subject receives an initial administration of a population of γδ T cells (e.g., 10 per kg body weight of the subject). 4 ~10 6 γδ T cells, e.g., 10 per kg body weight of the subject 4 ~10 5an initial administration of γδ T cells), and one or more (e.g., 2, 3, 4, or 5) subsequent administrations of γδ T cells (e.g., 10 per kg body weight of the subject) 4 ~10 6 γδ T cells, e.g., 10 per kg body weight of the subject 4 ~10 5 In one embodiment, the subject receives a total of about 10 γδ T cells per kg body weight of the subject over the course of at least three administrations of the population of γδ T cells. In one embodiment, the one or more subsequent administrations are administered less than 15 days, e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, since the previous administration, e.g., less than 4, 3, or 2 days since the previous administration. In one embodiment, the subject receives a total of about 10 γδ T cells per kg body weight of the subject over the course of at least three administrations of the population of γδ T cells. 6 γδ T cells, for example, a subject may receive 1×10 5 Initial dose of γδ T cells, 3 × 10 5 a second administration of 6 x 10 γδ T cells, and 5 and a third administration of γδ T cells, for example, where each administration is administered less than 4, 3, or 2 days after the previous administration.

[0275] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an anti-angiogenic agent, or a combination of two or more of these agents. The additional therapeutic agent can be administered simultaneously with, before, or after the administration of the γδ T cells. The additional therapeutic agent can be an immunotherapeutic agent that can act on targets within the subject's body (e.g., the subject's own immune system) and / or on the transplanted γδ T cells.

[0276] Administration of the compositions can be carried out in any convenient manner. The compositions described herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally, e.g., by intradermal or subcutaneous injection. Compositions of γδ T cells can be injected directly into a tumor, lymph node, or site of infection.

[0277] (genetic modification) The γδ T cells obtained by the methods of the present invention can also be genetically modified for enhanced therapeutic properties, e.g., chimeric antigen receptor T cell (CAR-T) therapy. This involves the generation of modified T cell receptors (TCRs) to reprogram T cells with new specificities, e.g., monoclonal antibody specificities. The modified TCRs can generate T cells specific to malignant cells and therefore useful for cancer immunotherapy. For example, T cells can recognize cancer cells expressing tumor antigens, e.g., tumor-associated antigens, that are not expressed by normal somatic cells derived from the target tissue. Thus, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0278] Other uses of antibodies or fragments thereof According to a further aspect of the invention, there is provided a use of an anti-V51 antibody or fragment thereof described herein for studying antigen recognition, activation, signaling or function of γδ T cells (particularly V51 T cells). As described herein, the antibodies have been shown to be active in assays that can be used to examine γδ T cell function. Such antibodies may also be useful for inducing γδ T cell proliferation and therefore can be used in methods of expanding γδ T cells (e.g., V51 T cells).

[0279] Antibodies that bind to the V51 chain can be used to detect γδ T cells (i.e., as a label). Preferably, antibodies used as labels do not stimulate cell proliferation, so that target V51 T cells are unaffected upon antibody binding. For example, the antibodies can be labeled with a detectable label or reporter molecule, or used as a capture ligand, to selectively detect and / or isolate V51 T cells in a sample. Labeled antibodies are used in many methods known in the art, such as immunohistochemistry and ELISA.

[0280] Detectable labels or reporter molecules can be radioisotopes, e.g., 3 H, 14C. 32 P, 35 S, or 125 The fluorescent label can be a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine, or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Fluorescent labels applied to antibodies of the invention can then be used in fluorescence-activated cell sorting (FACS) techniques.

[0281] Polynucleotides and Expression Vectors Also provided is a polynucleotide encoding the anti-V51 antibody or fragment of the present invention. In one embodiment, the anti-V51 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 to SEQ ID NOs: 99-110. In one embodiment, the anti-V51 antibody or fragment is encoded by an expression vector comprising a VH region of SEQ ID NOs: 99-110. In another embodiment, the anti-V51 antibody or fragment is encoded by an expression vector comprising a VL region of SEQ ID NOs: 99-110. In a further embodiment, the polynucleotide comprises or consists of SEQ ID NOs: 99-110. In a further aspect, a cDNA comprising the polynucleotide is provided.

[0282] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to SEQ ID NOs: 99 to 110. In one embodiment, the expression vector comprises a VH region of SEQ ID NOs: 99 to 110. In another embodiment, the expression vector comprises a VL region of SEQ ID NOs: 99 to 110. In a further embodiment, the polynucleotide comprises or consists of SEQ ID NOs: 99 to 110. In a further aspect, a cDNA comprising the polynucleotide is provided.

[0283] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to SEQ ID NO: 99-101 or 105-108. In one embodiment, the expression vector comprises a VH region of SEQ ID NO: 99-101 or 105-108. In another embodiment, the expression vector comprises a VL region of SEQ ID NO: 99-101 or 105-108. In a further embodiment, the polynucleotide comprises or consists of SEQ ID NO: 99-101 or 105-108. In a further aspect, a cDNA comprising the polynucleotide is provided.

[0284] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to SEQ ID NOs: 99-101. In one embodiment, the expression vector comprises a VH region of SEQ ID NOs: 99-101. In another embodiment, the expression vector comprises a VL region of SEQ ID NOs: 99-101. In a further embodiment, the polynucleotide comprises or consists of SEQ ID NOs: 99-101. In a further aspect, a cDNA comprising the polynucleotide is provided.

[0285] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to any one of the portions of SEQ ID NOs: 99 to 110 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%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to any one of the portions of SEQ ID NOs: 99 to 101 or 105 to 108 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%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to any one of the portions of SEQ ID NOs: 99-101 encoding CDR1, CDR2 and / or CDR3 of the encoded immunoglobulin chain variable domain.

[0286] In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to any one of the portions of SEQ ID NOs: 99 to 110 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%, for example at least 80%, for example at least 90%, for example at least 95%, for example at least 99% sequence identity to any one of the portions of SEQ ID NOs: 99 to 101 or 105 to 108 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%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to any one of the portions of SEQ ID NOs: 99-101 encoding FR1, FR2, FR3, and / or FR4 of the encoded immunoglobulin chain variable domain.

[0287] Polynucleotides and expression vectors of the invention can also be described in terms of the encoded amino acid sequence. Thus, in one embodiment, the polynucleotide comprises or consists of a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 62 to 85. In one embodiment, the expression vector comprises a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 62 to 73. In another embodiment, the expression vector comprises a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 74 to 85.

[0288] To express an antibody or fragment thereof, polynucleotides encoding the partial- or full-length light and heavy chains described herein are inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. Thus, in one aspect of the present invention, an expression vector is provided comprising a polynucleotide sequence defined herein. In one embodiment, the expression vector comprises a VH region of SEQ ID NO: 99-110, e.g., 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, e.g., SEQ ID NO: 99, 100, 101, 105, 106, 107, or 108.

[0289] The nucleotide sequences described herein include additional sequences encoding amino acid residues to aid in translation, purification, and detection, although it will be understood that alternative sequences may be used depending on the expression system used. For example, the first (5'-end) 9 nucleotides of SEQ ID NOs: 99-110 and the last (3'-end) 36 nucleotides of SEQ ID NOs: 99-100, 102-103, 105-110, or the last (3'-end) 39 nucleotides of SEQ ID NOs: 101 and 104 are optional sequences. These optional sequences may be removed, modified, or replaced if alternative design, translation, purification, or detection strategies are employed.

[0290] Mutations can be made to the DNA or cDNA encoding a polypeptide that are silent with respect to the amino acid sequence of the polypeptide but provide preferred codons for translation in a particular host. For example, preferred codons for translation of nucleic acids in E. coli and S. cerevisiae, as well as mammals, particularly humans, are known.

[0291] Mutations in polypeptides can be achieved, for example, by substitutions, additions, or deletions to the nucleic acid encoding the polypeptide. Substitutions, additions, or deletions to the nucleic acid encoding the polypeptide can be introduced by a number of methods, including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, artificial gene synthesis, gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination of these methods. Modifications, additions, or deletions to nucleic acids can also be introduced by methods including recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, or a combination thereof.

[0292] In particular, artificial gene synthesis can be used. Genes encoding the polypeptides of the present invention can be synthetically produced, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, building blocks are sequentially coupled to a growing oligonucleotide chain in the order required by the product sequence. Once chain assembly is complete, the product is released from the solid phase into solution, deprotected, and recovered. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity.

[0293] Expression vectors include, for example, plasmids, retroviruses, cosmids, yeast artificial chromosomes (YACs), and Epstein-Barr virus (EBV)-derived episomes. A polynucleotide is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the polynucleotide. Expression and / or control sequences can include promoters, enhancers, transcription terminators, an initiation codon (i.e., ATG) 5' to the coding sequence, splicing signals for introns, and stop codons. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. SEQ ID NOs: 99-110 comprise nucleotide sequences encoding single-chain variable fragments of the present invention, comprising a VH region and a VL region connected by a synthetic linker (e.g., encoding SEQ ID NO: 98). It will be understood that a polynucleotide or expression vector of the present invention can comprise a VH region, a VL region, or both (optionally including a linker). Thus, polynucleotides encoding the VH and VL regions can be inserted into separate vectors, or sequences encoding both regions are inserted into the same expression vector. Polynucleotides are inserted into expression vectors by standard methods (e.g., ligation of complementary restriction sites on the polynucleotide and vector, or blunt-end ligation if no restriction sites are present).

[0294] A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered to allow for the easy insertion and expression of any VH or VL sequence, as described herein. The expression vector may also encode a signal peptide that facilitates secretion of the antibody (or fragment thereof) from the host cell. The polynucleotide can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0295] The host cell may contain a first vector encoding the light chain of the antibody or fragment thereof and a second vector encoding the heavy chain of the antibody or fragment thereof. Alternatively, both the heavy and light chains are encoded on the same expression vector that is introduced into the host cell. In one embodiment, the polynucleotide or expression vector encodes a membrane-binding or transmembrane domain fused to the antibody or fragment thereof, wherein the antibody or fragment thereof is displayed on the extracellular surface of the host cell.

[0296] Transformation can be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammals are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, biolistic injection, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammals by viral vectors.

[0297] Mammalian cell lines available as expression hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, e.g., 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.

[0298] The antibody is produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium that the host cells are grown in. The antibody can be recovered from the culture medium using standard protein purification methods.

[0299] The antibodies (or fragments) of the present invention can be obtained and engineered using, for example, the techniques disclosed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012), 4th ed., Cold Spring Harbour Laboratory Press.

[0300] Monoclonal antibodies can be produced using hybridoma technology by fusing specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to grow in tissue culture and for their lack of antibody chain synthesis.

[0301] Monoclonal antibodies against a given antigen can be, for example, a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a predetermined antigen with immortal cells, preferably myeloma cells, to form hybridomas; b) Culturing the formed immortalized cells (hybridomas) and recovering cells that produce antibodies with the desired specificity. : can be obtained by

[0302] Alternatively, the use of hybridoma cells is not required. Antibodies capable of binding to the target antigens described herein can be routinely isolated from suitable antibody libraries using, for example, phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Thus, monoclonal antibodies can be isolated, for example, from a) cloning into a vector, in particular into a phage, more particularly into a filamentous bacteriophage, a DNA or cDNA sequence obtained from lymphocytes, in particular peripheral blood lymphocytes, of an animal (preferably one that has been previously immunized with a predetermined antigen); b) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the antibody; c) selecting the antibody by subjecting it to antigen-affinity selection d) recovering antibodies with the desired specificity : can be obtained by a process including

[0303] It will be understood that all embodiments described herein may be applied to all aspects of the present invention.

[0304] Other features and advantages of the present invention will be apparent from the description provided herein. However, since various changes and modifications will be apparent to those skilled in the art, it should be understood that the description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. The present invention will now be described by way of the following non-limiting examples: [Example]

[0305] (Example) Example 1. Materials and Methods (Human antibody discovery) Human phage display was used to generate the human anti-human variable V51+ domain antibodies described herein. The library was constructed as described in Schofield et al. (Genome biology 2007, 8(11): R254) and contained a single-chain fragment variable (scFv) display library of ~40 billion human clones. The library was screened using the antigens, methods, selection, deselection, screening, and characterization strategies described herein.

[0306] (antigen preparation) The soluble γδ TCR heterodimers containing TCR α and TCR β constant regions used in the Examples below were designed according to Xu et al. (2011) PNAS 108: 2414-2419. The Vγ or Vδ domains were fused in-frame to a TCR α or TCR β constant region lacking the transmembrane domain, followed by a leucine zipper sequence or Fc sequence, and a histidine tag / linker.

[0307] The expression constructs were transiently transfected into mammalian EXPI HEK293 suspension cells (either as single transfections or cotransfections of heterodimers). Secreted recombinant proteins were recovered and purified from the culture supernatant by affinity chromatography. To ensure good recovery of the monomeric antigen, samples were further purified using preparative size-exclusion chromatography (SEC). Purified antigens were analyzed for purity by SDS-PAGE and for aggregation state by analytical SEC.

[0308] (Functional validation of antigen) The specificity of antigens containing the delta variable 1 (Vδ1) chain was confirmed by a DELFIA immunoassay (Perkin Elmer) and a flow-based assay using REA173—an anti-Vδ1 antibody from Miltenyi Biotec—to compete with γδ T cells.

[0309] (Dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)) To confirm the specificity of the antigen, a DELFIA immunoassay was performed using antigen coated directly onto the plate (3 μg / mL antigen in 50 μL PBS, overnight at 4°C (Nunc #437111)) and serial dilutions of the primary antibody starting from 300 nM. For detection of DELFIA Eu-N1, anti-human IgG (Perkin Elmer # 1244-330) was used as the secondary antibody at a 1 / 500 dilution in 50 μL of 3% MPBS (PBS + 3% (w / V) skim milk powder). Color development was with 50 μL of DELFIA enhancement solution (Perkin Elmer #4001-0010).

[0310] Affinity ranking of the antibodies of interest was performed using a DELFIA immunoassay in which the antibody was captured by protein G coated on a plate and soluble biotinylated L1 (DV1-GV4) antigen was added at 5 nM in 50 μL (3M PBS). For detection, 50 μL of streptavidin-Eu (1:500 in assay buffer, Perkin Elmer) was used, and the signal was developed with DELFIA enhancement solution. D1.3 hIgG1 (described in England et al. (1999) J. Immunol. 162: 2129-2136) was used as a negative control.

[0311] The output of the phage display selection was subcloned into the scFv expression vector pSANG10 (Martin et al. (2006) BMC Biotechnol. 6: 46). Soluble scFvs were expressed and screened for binding to directly immobilized targets by DELFIA. Hits were defined as a DELFIA signal greater than 3000 fluorescent units.

[0312] (Antibody preparation) The selected scFvs were subcloned into an IgG1 framework using commercially available plasmids. For antibody expression, the plasmids were transfected into expi293F suspension cells. For convenience, unless otherwise stated, the antibodies characterized in these examples refer to IgG1-formatted antibodies selected from phage display as scFvs. However, the antibodies of the present invention may be of any of the antibody formats discussed above.

[0313] (Antibody purification) IgG antibodies were batch purified from the supernatant using Protein A chromatography. The concentrated Protein A eluate was then purified using size-exclusion chromatography (SEC). The quality of the purified IgG was analyzed using ELISA, SDS-PAGE, and SEC-HPLC.

[0314] (γδ T cell preparation) Enriched γδ T cell populations were prepared according to the methods described in WO2016 / 198480 (i.e., blood-derived γδ T cells) or WO2020 / 095059 (i.e., skin-derived γδ T cells). Briefly, for blood-derived γδ T cells, PBMCs were obtained from blood and subjected to magnetic depletion of αβ T cells. The αβ-depleted PBMCs were then cultured for 7 days in CTS OpTmiser medium (ThermoFisher) in the presence of OKT-3 (or the respective 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 respective anti-Vδ1 antibody), IL-21, and IL-15 for an additional 4 days. On day 11 of culture, the medium was supplemented with OKT-3 (or the respective anti-Vδ1 antibody) and IL-15 for an additional 3 days. On day 14 of culture, half of the medium was replaced with fresh complete OpTmiser and supplemented with OKT-3 (or the respective anti-Vδ1 antibody), IL-15, and IFN-γ. From day 17 onwards, cultures were supplemented with OKT-3 (or the respective anti-Vδ1 antibody) and IL-15 every 3–4 days; half of the medium was replaced with fresh medium every 7 days.

[0315] For skin-derived γδ T cells, skin samples were prepared by removing subcutaneous fat and multiple punches were made using a 3 mm biopsy punch. The punches were placed on a carbon matrix grid and placed into the wells of a G-REX6 (Wilson Wolf) plate. Each well was filled with complete isolation medium containing AIM-V medium (Gibco, Life Technologies), serum replacement from CTS Immune (Life Technologies), IL-2, and IL-15. For the first 7 days of culture, complete isolation medium containing amphotericin B (Life Technologies) was used ("+AMP"). The medium was changed every 7 days by gently aspirating the upper medium and replacing it with 2x complete isolation medium (without AMP), without disturbing the cells at the bottom of the plate or bioreactor. After more than three weeks in culture, the resulting transmigrated cells are then passaged into new tissue culture vessels and fresh medium (e.g., AIM-V medium or TexMAX medium (Miltenyi)) plus recombinant IL-2, IL-4, IL-15, and IL-21, followed by harvesting. Optionally, αβ T cells also present in the culture are then removed using an αβ T cell depletion kit and associated protocol, such as that provided by Miltenyi. For further reference, see WO2020 / 095059.

[0316] (γδ T cell binding assay) Antibody binding to γδ T cells was tested by incubating a fixed concentration of purified antibody with 250,000 γδ T cells. This incubation was performed under blocking conditions to prevent nonspecific antibody binding via Fc receptors. Detection was performed by adding a fluorochrome-conjugated secondary antibody against human IgG1. For negative controls, cells were prepared with a) isotype antibody alone (recombinant human IgG), b) fluorochrome-conjugated anti-human IgG antibody alone, and c) a combination of a) and b). Control wells of 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 fluorochrome-conjugated goat anti-mouse secondary antibody. The assay was accepted if the mean fluorescence intensity of the lower concentration positive control in the FITC channel was at least 10-fold higher than that of the highest negative control.

[0317] (SPR analysis) SPR analysis was performed using a MASS-2 instrument equipped with an amine high-capacity chip (both from Sierra Sensors, Germany). 15 nM IgG was captured onto the amine high-capacity chip (100 nM for the TS8.2) via protein G. L1 (DV1-GV4) antigen was flowed over the cells in a 1:2 dilution series from 2000 nM to 15.625 nM with the following parameters: 180 s association, 600 s dissociation, flow rate 30 μL / min, running buffer PBS + 0.02% Tween 20. All experiments were performed at room temperature on the MASS-2 instrument. Steady-state fitting was determined according to Langmuir 1:1 binding using the software Sierra Analyzer 3.2.

[0318] (comparator antibody) The antibodies were compared to commercially available antibodies in the test assays described. [Table 2]

[0319] γδ TCR Down-Regulation and Degranulation Assay THP-1 (TIB-202™, ATCC) target cells, either unloaded or loaded with test antibodies, were labeled with CellTracker™ Orange CMTMR (ThermoFisher, C2927) and incubated with γδ T cells at a 2:1 ratio in the presence of a CD107a antibody (anti-human CD107a BV421 (clone H4A3) BD Biosciences 562623). After 2 hours of incubation, surface expression of γδ TCR (to measure TCR down-regulation) and CD107a expression (to measure degranulation) on γδ T cells were assessed using flow cytometry.

[0320] Killing assay The effect of test antibodies on gamma delta T cell killing activity and γδ T cell killing activity was assessed by flow cytometry. After 4 hours of in vitro coculture, γδ T cells and CellTracker™ Orange CMTMR (ThermoFisher, C2927)-labeled THP-1 cells (antibody-loaded or unloaded) at a 20:1 ratio were stained with Viability Dye eFluor™ 520 (ThermoFisher, 520 65-0867-14) to distinguish between live and dead target THP-1 cells. Upon sample acquisition, target cells were gated on CellTracker™ Orange CMTMR positivity and examined for cell death based on viability dye uptake. CMTMR and eFluor™ 520 double-positive cells were recognized as dead target cells. γδ T cell killing activity was expressed as the percentage of dead target cells.

[0321] (epitope mapping) 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 for protein integrity and aggregation levels using high-mass MALDI.

[0322] 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 deuterated cross-linkers and subjected to multienzyme proteolysis using trypsin, chymotrypsin, Asp-N, elastase, and thermolysin. 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.

[0323] SYTOX-Flow Killing Assay The SYTOX assay allows for quantification of T cell-mediated cytolysis of target cells using flow cytometry. Dead / dying cells are detected by a dead cell dye (SYTOX® AADvanced™, Life Technologies, S10274), which penetrates only cells with impaired plasma membranes but cannot cross the intact membranes of healthy cells. NALM-6 target cells were labeled with a CTV dye (Cell Trace Violet™, Life Technologies, C34557), which allowed them to be distinguished from unlabeled effector T cells. Dead / dying target cells were identified by double staining with the dead cell dye and a cell tracking dye.

[0324] After 16 hours of in vitro co-culture of effector and CTV-labeled target cells at the indicated effector-to-target ratios (E:T, 1:1, or 10:1), cells were stained with SYTOX® AADvanced™ and acquired on a FACSLyric™ (BD). Killing results are shown as % target cell reduction, calculated by considering the number of live target cells in the test sample (sample count) relative to the number of live target cells in control wells where no effector cells were added (maximum count):

number

[0325] Example 2. Antigen design Gamma delta (γδ) T cells are polyclonal with respect to CDR3 polyclonality. To avoid situations in which generated antibodies are selected against CDR3 sequences (which vary between TCR clones), antigen design involved maintaining a consistent CDR3 across the various formats. This design aimed to generate antibodies that recognize sequences within the variable domains that are germline-encoded and therefore identical in all clones, thereby providing antibodies that recognize a broader subset of γδ T cells.

[0326] Another important aspect of the antigen preparation process was designing antigens suitable for expression as proteins. The γδ TCR is a complex protein comprising a heterodimer with intra- and inter-chain disulfide bonds. Leucine zipper (LZ) and Fc formats were used to generate soluble TCR antigens to be used in phage display selection. Both the LZ and Fc formats were well expressed and displayed TCRs well, particularly heterodimeric TCRs, e.g., Vδ1Vγ4.

[0327] The CDR3 sequence obtained from a public database entry for γδ TCR was found to be well expressed as a protein (RCSB Protein Data Bank entry: 3OMZ) and was therefore selected for antigen preparation.

[0328] Antigens containing the delta variable 1 chain were expressed either as heterodimers in the LZ format (i.e., combined with different gamma variable chains—"L1," "L2," "L3") and as heterodimers in the Fc format ("F1," "F2," "F3") or as homodimers (i.e., combined with another delta variable 1 chain—"Fc1 / 1"). The delta variable 1 chains of the antigens all contained a 30M Z CDR3. Another series of γδ TCR antigens using a similar format were designed with different delta variable chains (e.g., delta variable 2 and delta variable 3) and used to deselect antibodies with nonspecific or off-target binding ("L4," "F9," "Fc4 / 4," "Fc8 / 8"). These antigens were also designed to contain a 30M Z CDR3 to ensure that antibodies binding in the CDR3 region were also deselected.

[0329] To confirm that the designed antigens were suitable for generating anti-TRDV1 (TCR delta variable 1) antibodies, we performed antigen function validation. Detection was observed only for antigens containing the delta 1 domain (Figure 1).

[0330] Example 3. Phage display Phage display selections were performed against a library of human scFvs using either the heterodimeric LZ TCR format in rounds 1 and 2, with deselection on the heterodimeric LZ TCR in both rounds, or round 1 was performed using a homodimeric Fc-fusion TCR with deselection on a human IgG1 Fc, followed by round 2 with the heterodimeric LZ TCR and deselection on the heterodimeric LZ TCR (see Table 1). Table 1. Summary of phage display selection [Table 3] bt = biotin

[0331] Selection was performed in solution phase using 100 nM biotinylated protein, and deselection was performed using 1 μM non-biotinylated protein.

[0332] The success of the phage display selection was analyzed by polyclonal phage ELISA (DELFIA). All outputs of the DV1 selection showed desirable binding to the targets Fc 1 / 1, L1, L2, L3, F1, and F3. Varying degrees of binding to the non-targets L4, F9, Fc 4 / 4, Fc 8 / 8, and Fc were detected (see Figures 2A and B).

[0333] Example 4. Antibody Selection The hits obtained in Example 3 were sequenced (using standard methods known in the art). 130 unique clones were identified, which displayed unique combinations of VH and VL CDR3s. Of these 130 unique clones, 125 displayed unique VH CDR3s and 109 displayed unique VL CDR3s.

[0334] Unique clones were rearranged and specificity was analyzed by ELISA (DELFIA).A panel of 94 unique human scFv binders that bound to TRDV1 (L1, L2, L3, F1, F2, F3) but not to TRDV2 (L4) was identified from the selection.

[0335] Affinity rankings of selected binders were included to help guide clone selection. Many binders showed affinities in the nanomolar range, reacting with 25–100 nM biotinylated antigen. A few binders showed strong reactivity with 5 nM antigen, indicating potential single-digit nanomolar affinities. Some binders showed no reactivity with 100 nM antigen, indicating affinities in the micromolar range.

[0336] To advance clone selection to IgG conversion, the goal was to include as many germline lineages and as many different CDR3s as possible, while avoiding sequence preferences such as glycosylation, integrin binding sites, CD11c / CD18 binding sites, and unpaired cysteines. Additionally, a variety of affinities were included.

[0337] Selected clones were screened for binding to native cell surface-expressed γδ TCR using skin-derived γδ T cells obtained from various donors. Clones selected for conversion to IgG are shown in Table 2. Table 2. DV1 binders for IgG conversion [Table 4]

[0338] Example 5: Antibody SPR analysis The prepared IgG antibodies were subjected to a γδ cell binding assay, and the five best binders were selected for further functional and biophysical characterization. The equilibrium dissociation constants (K D SPR analysis was performed to determine the IgG binding activity. Sensorgrams of the test antibody-analyte interactions, along with steady-state fits (where available), are shown in Figure 3. For TS8.2, no binding was detected and 80 RU of IgG was captured on the chip. The results are summarized in Table 3. Table 3. IgG capture results [Table 5] * The binding of 1252_P02_C05 did not reach saturation, therefore the data were extrapolated.

[0339] Example 6: TCR engagement assay We designed several assays to be used for functional characterization of selected antibodies. The first assay assessed γδ TCR engagement by measuring downregulation of γδ TCR upon antibody binding. Selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies used as positive controls, or against 1252_P01_C08 as a positive control (for 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09). A commercially available anti-pan-γδ antibody was used as a negative control because it is a pan-γδ antibody that recognizes all γδ T cells regardless of variable chain and therefore likely has a different mode of action.

[0340] This assay was performed using skin-derived γδ T cells obtained from three different donor samples (samples with purities of 94%, 80%, and 57%). The results are shown in Figure 4. The EC50 values ​​are summarized in Table 4 below.

[0341] Example 7: T cell degranulation assay The second assay assessed γδ T cell degranulation. γδ T cells are thought to mediate target cell killing through perforin-granzyme-mediated apoptosis activation. Lytic granules within the cytoplasm of γδ T cells can be released toward target cells upon T cell activation. Therefore, labeling of target cells with an antibody against CD107a and measurement of expression by flow cytometry can be used to identify degranulated γδ T cells.

[0342] For Example 6, selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies as positive controls or 1252_P01_C08 as positive controls (for 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09). IgG2a, IgG1, and D1.3 antibodies were used as negative controls. The assay was performed using skin-derived γδ T cells obtained from three different donor samples (samples with purities of 94%, 80%, and 57%). Results are shown in Figure 5. EC50 values ​​are summarized in Table 4 below.

[0343] Example 8: Killing Assay The third assay assessed the ability of γδ T cells activated with selected antibodies to kill target cells.

[0344] For Example 6, selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies as positive controls or against 1252_P01_C08 as a positive control (for 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) and anti-pan-γδ as a negative control. IgG2a, IgG1, and D1.3 antibodies were also used as isotype controls. The assay was performed using skin-derived γδ T cells (94% and 80% pure) obtained from two donors. The results are shown in Figure 6.

[0345] The results of the three functional assays tested in Examples 6-8 are summarized in Table 4. Table 4. Summary of results obtained from functional assays [Table 6] N / D: Could not determine; N / D * : Unable to determine, titration curve did not reach plateau; N / D ** : Killing profile was reduced and EC50 was not determined.

[0346] Example 9: Epitope Mapping To determine the epitopes of the antigen / antibody complexes at high resolution, the protein complexes were incubated with deuterated cross-linkers and subjected to multiple enzymatic cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-LTQ-Orbitrap MS). The generated data were analyzed using XQuest (version 2.0) and Stavrox (version 3.6) software.

[0347] After proteolysis of the protein complex L1(DV1-GV4) / 1245_P01_E07 with deuterated d0d12, using trypsin, chymotrypsin, Asp-N, elastase, and thermolysin, 13 cross-linked peptides between L1(DV1-GV4) and antibody 1245_P01_E07 were detected by nLC-orbitrap MS / MS analysis. The results are shown in Figure 7.

[0348] After proteolysis of the protein complex L1(DV1-GV4) / 1252_P01_C08 with deuterated d0d12, five cross-linked peptides between L1(DV1-GV4) and antibody 1252_P01_C08 were detected by nLC-orbitrap MS / MS analysis. The results are shown in Figure 8.

[0349] After proteolysis of the protein complex L1(DV1-GV4) / 1245_P02_G04 with deuterated d0d12, trypsin, chymotrypsin, Asp-N, elastase, and thermolysin, 20 cross-linked peptides between L1(DV1-GV4) and antibody 1245_P02_G04 were detected by nLC-orbitrap MS / MS analysis. The results are shown in Figure 9.

[0350] After proteolysis of the protein complex L1(DV1-GV4) / 1251_P02_C05 with deuterated d0d12, five cross-linked peptides between L1(DV1-GV4) and antibody 1251_P02_C05 were detected by nLC-orbitrap MS / MS analysis. The results are shown in Figure 10.

[0351] Epitope binding by 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 using trypsin, chymotrypsin, Asp-N, elastase, and thermolysin, nLC-orbitrap MS / MS analysis detected 20 cross-linked peptides between L1(DV1-GV4) and antibody 1141_P01_E01. The results are shown in Figure 11.

[0352] A summary of the epitope mapping results is shown in Table 5. Table 5. Results of epitope mapping of antigen / antibody complexes [Table 7]

[0353] Example 10: Expansion of V51 T cells The expansion of isolated γδ T cells was examined in the presence of selected antibodies and comparator antibodies, which were selected from the following: OKT3 anti-CD3 antibody as a positive control, no antibody as a negative control, or an IgG1 antibody as an isotype control. Commercially available anti-Vδ1 antibodies, TS-1 and TS8.2, were also tested for comparison.

[0354] Test 1: Initial studies were performed by seeding 70,000 cells / well with the complete Optimizer and cytokines described in "γδ T Cell Preparation" for Blood-Derived γδ T Cells in Example 1. Selected 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 that allow for antibody binding / immobilization to plastic.

[0355] Cells were harvested on days 7, 14, and 18, and total cell counts were determined using a cell counter (NC250, ChemoMetec). Results are shown in Figure 12. Cell viability of V51 T cells was also measured in each harvest, and it was shown that all antibodies maintained cell viability throughout the experiment (data not shown). On day 18, the percentage, cell count, and fold change of V51 T cells were also analyzed. Results are shown in Figure 13.

[0356] As can be seen in Figure 12, the total number of cells produced in culture with the antibodies steadily increased throughout the culture and was comparable to or greater than that of commercially available anti-V51 antibodies. At day 18, the percentage of V51-positive cells in the presence of the 1245_P02_G04 ("G04"), 1245_P01_E07 ("E07"), 1245_P01_B07 ("B07"), and 1252_P01_C08 ("C08") antibodies at the highest concentrations tested was greater than in cultures in the presence of OKT3, TS-1, or TS8.2 control antibodies (see Figure 13A).

[0357] Test 2: Subsequent experiments were performed on isolated cells in culture vessels containing cytokines as described in Example 1, "γδ T Cell Preparation." Compared to experiment 1, a different culture vessel was used, whose surface does not promote antibody binding / immobilization. Selected antibodies and comparator antibodies were tested at various concentrations ranging from 42 pg / ml to 42 ng / ml. In experiment 2, results were obtained from experiments performed in triplicate.

[0358] Cells were harvested on days 7, 11, 14, and 17, and total cell counts were determined using the same cell counter as before. The results are shown in Figure 14. On day 17, the percentage of V51 T cells, cell count, and fold change were also analyzed. The results are shown in Figure 15.

[0359] The cellular composition, including non-V51 cells, was also determined in Experiment 2. Cells were harvested on day 17 and analyzed for surface expression of V51, V52, and αβ TCR by flow cytometry. The proportion of each cell type in each culture is shown graphically in Figure 16, and the percentage values ​​are provided in Table 6. Table 6. Cellular composition at day 17 - percentage of viable cells in each subset [Table 8]

[0360] As can be seen from these results, the proportion of V51-positive cells is greater in cultures in the presence of B07, C08, E07, and G04 compared to the OKT3, TS-1, or TS8.2 controls. Therefore, the tested antibodies generate and expand V51-positive cells more efficiently than commercially available antibodies, even when present at low concentrations in culture.

[0361] To confirm the presence of CD27-expressing (i.e., CD27+) natural killer (NK) cells and V51 T cells, cells from day 17 of experiment 2 were also analyzed for additional cell markers, including CD3-CD56+. The results are summarized in Table 7. Table 7. Cellular composition on day 17 - percentage of NK and CD27+ cells [Table 9] SEM: Standard error of the mean

[0362] Example 11: Functionality of V51 T cells V51 T cells expanded in the presence of selected antibodies retained a polyclonal repertoire of CDR3 regions and were also tested for functionality using a SYTOX-flow killing assay. Results are shown for cells obtained at day 14 in experiment 1 using cells at an effector to target (E:T) ratio of 10:1 (Figure 17A) and for cells obtained at day 17 (post-freeze-thaw) in experiment 2 using cells at E:T ratios of 1:1 and 10:1 (Figure 17B).

[0363] As can be seen in Figure 17, V51-positive cells expanded in the presence of all antibodies efficiently lysed target cells, demonstrating that the cells were functional even after freezing and thawing.

[0364] Example 12: Functionality of cells after storage The functionality of cells after the freezing and subsequent thawing preservation process was also examined. A portion of the cells was removed from the culture and frozen on day 17 of Experiment 2. The cells were then thawed and further expanded in culture with IL-15. Figure 18 shows the total cell counts 7 days after freezing and thawing for cultures that had been contacted with B07, C08, E07, G04, or OKT-3 antibodies before freezing. All cultures demonstrated the ability to proliferate after storage. Cultures were continued for up to 42 days, and total cell counts were monitored during this period (results are shown in Figure 19). Total cell numbers were maintained or increased in cultures pre-exposed to selected antibodies.

[0365] Example 13: Anti-V51 antibodies conferred immune cell regulation and proliferation in TILs Studies were initiated to investigate the modulation and proliferation of human tumor-infiltrating lymphocytes (TILs) conferred by anti-Vδ1 antibodies. For these studies, human renal cell carcinoma (RCC) tumor biopsies were shipped fresh and processed immediately upon receipt. Specifically, tissue was minced into ~2 mm 2Up to 1 g of tissue was placed in each Miltenyi C-tube with 4.7 mL of RPMI and the enzymes from the Miltenyi Tumor Dissociation Kit at the manufacturer's recommended concentrations, except for Enzyme R, which was used at a 0.2x concentration to prevent cleavage of relevant cell surface molecules. The C-tube was placed on a gentleMACS™ Octo Dissociator equipped with a heater. Program 37C_h_TDK_1 for soft tissue tumor dissociation 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 the enzyme activity. The cells were washed twice with RPMI / 10% FBS and resuspended for counting. The resulting cells were then seeded at 2.5 x 10e6 per well into TC wells (24-well G-REX, Wilson Wolf). The cells were then incubated with or without cytokines and with or without antibodies for 18 days. The antibodies included in the study are summarized in Figure 20. These include OKT3 (up to 50 ng / ml) and 1252_P01_C08 (up to 500 ng / ml), also known herein as "C08." Where included, bolus additions of these antibodies were added on days 0, 7, 11, and 14. During the incubation, the medium was replaced with fresh medium on days 11 and 14. Flow cytometry analysis was performed on days 0 and 18 to determine fold changes in lymphocyte phenotype and cell number. Cells were gated first on live CD45+ cells and then as indicated. In groups that included recombinant cytokines, these were added as follows: 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 V51 cells after 18 days of culture in the presence of C08 or OKT3 with and without cytokine supplementation (CK) where indicated.These results demonstrate a significant fold increase in TIL V51 cells with application of either C08 or comparator OKT3 antibody in the presence of cytokines compared to antibody or cytokine alone. Figure 20(B) shows the increase in total V51 cell numbers at harvest. These results demonstrate a significant increase in TIL V51 cell numbers after culture with C08 or comparator OKT3 antibody in the presence of cytokines compared to antibody or cytokine alone. Figure 20(C) shows an example of the gating strategy used in flow cytometry analysis of cells. From the live CD45+ cell population, cells were gated on lymphocytes based on their forward and side scatter characteristics (not shown), and then γδ T cells were separated from αβ T cells by staining for the T cell receptor. Finally, the percentage of V51 cells within the total γδ T cell population was determined. Example data at day 18 are shown for the two conditions indicated (+ / -1252_P01_C08): 64.3% of cells were CD45+, and of these CD45% cells, 53.1% were γδ+, and of these γδ cells, 89.7% were Vδ1+. Figure 20(D) shows the cell surface phenotype profile of TIL Vδ1+ cells at harvest. Higher levels of CD69 were observed after culture with the C08 antibody. Figure 20(E) shows the analysis of the TIL γδ-negative CD8-positive lymphocyte fraction within the live CD45-positive gate at harvest. Taken together, the combined results highlight the regulatory effect conferred by the anti-Vδ1 antibodies of the invention described herein on the TIL population. The present application provides the following aspects of the invention. (Aspect 1) 1. An ex vivo method for modulating V51 T cells, comprising administering to a subject the amino acid region: (i) SEQ ID NO: 1, 3 to 20; and / or (ii) 37 to 77 of SEQ ID NO: 1 the ex vivo method comprising administering to a cell population comprising V51 T cells a human anti-TCR delta variable 1 (anti-V51) antibody or fragment thereof that binds to an epitope of the variable delta 1 (V51) chain of the gamma delta T cell receptor (TCR) comprising one or more amino acid residues within (Aspect 2) 2. The method of aspect 1, wherein the epitope comprises one or more amino acid residues within the amino acid regions: 5 to 20 and 62 to 77; 50 to 64; 37 to 53 and 59 to 72; 59 to 77; or 3 to 17 and 62 to 69 of SEQ ID NO:1. (Aspect 3) Aspect 3. The method of aspect 1 or aspect 2, wherein the epitope is an activating epitope of a γδ T cell. (Aspect 4) The method according to any one of embodiments 1 to 3, wherein the antibody binds only to an epitope in the V region of the Vδ1 chain of γδ TCR. (Aspect 5) The method of any one of embodiments 1 to 4, wherein the antibody does not bind to an epitope found in CDR3 of the Vδ1 chain of γδ TCR. (Aspect 6) 1. An ex vivo method of modulating V51 T cells, comprising: a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 25; CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26 to 37 and sequences A1 to A12 (in Table 2); and / or CDR1 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61; The ex vivo method comprises administering an anti-V51 antibody or a fragment thereof comprising one or more of: to a cell population comprising V51 T cells. (Aspect 7) The method of aspect 6, wherein the antibody or fragment thereof comprises a VH region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 73, for example, SEQ ID NOs: 63, 62, or 64. (Aspect 8) The method of aspect 6, wherein the antibody or fragment thereof comprises a VL region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74 to 85, for example, SEQ ID NOs: 75, 74, or 76. (Aspect 9) The method according to any one of aspects 6 to 8, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 86 to 97, for example, SEQ ID NO: 87, 86, or 88. (Aspect 10) A method according to any one of aspects 6 to 9, wherein said antibody or fragment thereof binds to the same or essentially the same epitope as the antibody or fragment thereof according to any one of aspects 6 to 9, or competes with the antibody or fragment thereof according to any one of aspects 6 to 9. (Aspect 11) The antibody or fragment thereof has a surface plasmon resonance (SPR) content of 1.5×10 -7 11. The method of any one of embodiments 1 to 10, wherein the antibody binds to the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) with a binding affinity (KD) of less than M. (Aspect 12) The method of any one of Aspects 1 to 11, wherein the antibody or fragment thereof is an scFv, Fab, Fab', F(ab')2, Fv, a variable domain (e.g., VH or VL), a diabody, a minibody, or a full-length antibody. (Aspect 13) The method of any one of embodiments 1 to 12, wherein said modulation comprises expansion of Vδ1 T cells. (Aspect 14) 14. The method of embodiment 13, wherein the method provides a population of expanded V51 T cells that contains more than about 85% V51 T cells, such as more than about 90% V51 T cells. (Aspect 15) 15. The method of any one of embodiments 1 to 14, wherein said method comprises culturing said cell population for at least 5 days. (Aspect 16) 16. The method of any one of embodiments 1 to 15, wherein said method comprises culturing the cell population in the presence of at least one cytokine. (Aspect 17) 17. The method of embodiment 16, 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 a mixture thereof. (Aspect 18) 18. The method of any one of embodiments 1 to 17, wherein said method comprises culturing the cell population in the presence of IL-2, IL-9, and / or IL-15. (Aspect 19) The method of any one of embodiments 1 to 18, wherein said method comprises culturing the cell population in the presence of IL-21. (Aspect 20) 20. The method of any one of embodiments 1 to 19, wherein said method comprises culturing the cell population in the presence of IL-4. (Aspect 21) 18. The method of any one of embodiments 1 to 17, wherein the method comprises culturing the cell population in a first culture medium comprising IL-4, and subsequently culturing the cell population in a second culture medium comprising IL-15. (Aspect 22) 22. The method of embodiment 21, wherein the first culture medium lacks IL-15, IL-2, and / or IL-7. (Aspect 23) 22. The method of embodiment 21, wherein the second culture medium lacks IL-4. (Aspect 24) 24. The method of any one of embodiments 21 to 23, wherein the first or second culture medium, or both culture media, comprise one or more additional cytokines. (Aspect 25) 25. The method of embodiment 24, wherein the additional cytokine is selected from the group consisting of: IL-21, IFN-γ, and IL-1β. (Aspect 26) The method of any one of aspects 15 to 25, wherein the cell population is not in direct contact with stromal and / or epithelial cells during culture. (Aspect 27) 27. The method of embodiment 26, wherein the cell population is not in direct contact with fibroblasts during culture. (Aspect 28) The method according to any one of aspects 15 to 27, wherein the cell population is not in direct contact with tumor cells and / or feeder cells during culture. (Aspect 29) 29. The method of any one of embodiments 1 to 28, wherein said method comprises culturing said cell population in serum-free medium. (Aspect 30) The method of any one of aspects 1 to 29, wherein the cell population is enriched for T cells prior to administration of the antibody or fragment thereof. (Aspect 31) The method of any one of aspects 1 to 30, wherein the cell population is enriched for γδ T cells prior to administration of the antibody or fragment thereof. (Aspect 32) The method of any one of aspects 1 to 31, wherein the cell population is depleted of αβ T cells or NK cells prior to administration of the antibody or fragment thereof. (Aspect 33) 33. The method of any one of aspects 1 to 32, wherein said cell population is obtained from a hematopoietic sample or a fraction thereof. (Aspect 34) 34. The method of embodiment 33, wherein said hematopoietic sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, lymph node tissue, or a fraction thereof. (Aspect 35) 35. The method of embodiment 33 or embodiment 34, wherein said hematopoietic sample consists of low density mononuclear cells (LDMCs) or peripheral blood mononuclear cells (PBMCs). (Aspect 36) 33. The method of any one of aspects 1-32, wherein said cell population is obtained from a non-hematopoietic tissue sample, such as skin, colon, intestine, mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina, or other skin membrane, mucosa, or serous membrane sample. (Aspect 37) 37. The method of embodiment 36, wherein said cell population is obtained from the non-hematopoietic tissue sample by culturing the non-hematopoietic tissue sample on a synthetic scaffold configured to promote cell migration from the non-hematopoietic tissue sample. (Aspect 38) The method of any one of embodiments 1 to 37, wherein the cell population is obtained from a cancer tissue sample. (Aspect 39) The method of any one of embodiments 1 to 38, wherein the cell population is obtained from human or non-human animal tissue. (Aspect 40) The method of any one of aspects 1 to 39, wherein the cell population is isolated from a sample prior to administering the anti-Vδ1 antibody or fragment thereof. (Aspect 41) A Vδ1 T cell population obtained by the ex vivo method according to any one of embodiments 1 to 40. (Aspect 42) A composition comprising the V51 T cell population according to embodiment 41. (Aspect 43) 42. A pharmaceutical composition comprising the V51 T cell population according to embodiment 41. (Aspect 44) 44. A pharmaceutical composition according to embodiment 43, for use as a medicament. (Aspect 45) 44. The pharmaceutical composition according to embodiment 43, for use in the treatment of cancer, an infectious disease, or an inflammatory disease. (Aspect 46) 44. A method for treating cancer, an infectious disease or an inflammatory disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the population of V51 T cells according to aspect 41 or the pharmaceutical composition according to aspect 43.

Claims

1. 1. An ex vivo method of modulating V51 T cells, comprising: modulation of Vδ1 T cells includes expansion of Vδ1 T cells, stimulation of Vδ1 T cells, prevention of Vδ1 T cell exhaustion, immunosuppression of Vδ1 T cells, reduction of Vδ1 T cell numbers, Vδ1 T cell activation, or Vδ1 T cell inhibition; The method comprises: a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 38, a CDR2 comprising the sequence of SEQ ID NO: 26, and a CDR3 comprising the sequence of SEQ ID NO: 2; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 50, a CDR2 comprising the sequence of VAS, and a CDR3 comprising the sequence of SEQ ID NO: 14; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 39, a CDR2 comprising the sequence of SEQ ID NO: 27, and a CDR3 comprising the sequence of SEQ ID NO: 3; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 51, a CDR2 comprising the sequence of YDS, and a CDR3 comprising the sequence of SEQ ID NO: 15; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 40, a CDR2 comprising the sequence of SEQ ID NO: 28, and a CDR3 comprising the sequence of SEQ ID NO: 4; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 52, a CDR2 comprising the sequence of DAS, and a CDR3 comprising the sequence of SEQ ID NO: 16; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 41, a CDR2 comprising the sequence of SEQ ID NO: 29, and a CDR3 comprising the sequence of SEQ ID NO: 5; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 53, a CDR2 comprising the sequence of AAS, and a CDR3 comprising the sequence of SEQ ID NO: 17; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 42, a CDR2 comprising the sequence of SEQ ID NO: 30, and a CDR3 comprising the sequence of SEQ ID NO: 6; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 54, a CDR2 comprising the sequence of DAS, and a CDR3 comprising the sequence of SEQ ID NO: 18; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 43, a CDR2 comprising the sequence of SEQ ID NO: 31, and a CDR3 comprising the sequence of SEQ ID NO: 7; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 55, a CDR2 comprising the sequence of EVS, and a CDR3 comprising the sequence of SEQ ID NO: 19; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 44, a CDR2 comprising the sequence of SEQ ID NO: 32, and a CDR3 comprising the sequence of SEQ ID NO: 8; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 56, a CDR2 comprising the sequence of DAS, and a CDR3 comprising the sequence of SEQ ID NO: 20; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 45, a CDR2 comprising the sequence of SEQ ID NO: 33, and a CDR3 comprising the sequence of SEQ ID NO: 9; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 57, a CDR2 comprising the sequence of DAS, and a CDR3 comprising the sequence of SEQ ID NO: 21; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 46, a CDR2 comprising the sequence of SEQ ID NO: 34, and a CDR3 comprising the sequence of SEQ ID NO: 10; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 58, a CDR2 comprising the sequence of AAS, and a CDR3 comprising the sequence of SEQ ID NO: 22; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 47, a CDR2 comprising the sequence of SEQ ID NO: 35, and a CDR3 comprising the sequence of SEQ ID NO: 11; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 59, a CDR2 comprising the sequence of DAS, and a CDR3 comprising the sequence of SEQ ID NO: 23; a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 48, a CDR2 comprising the sequence of SEQ ID NO: 36, and a CDR3 comprising the sequence of SEQ ID NO: 12; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 60, a CDR2 comprising the sequence of DAS, and a CDR3 comprising the sequence of SEQ ID NO: 24; or a VH region comprising a CDR1 comprising the sequence of SEQ ID NO: 49, a CDR2 comprising the sequence of SEQ ID NO: 37, and a CDR3 comprising the sequence of SEQ ID NO: 13; and a VL region comprising a CDR1 comprising the sequence of SEQ ID NO: 61, a CDR2 comprising the sequence of AAS, and a CDR3 comprising the sequence of SEQ ID NO:

25. The ex vivo method comprises administering an anti-Vδ1 antibody or a fragment thereof comprising the antibody to a cell population comprising Vδ1 T cells.

2. The antibody or fragment thereof a VH region comprising the amino acid sequence of SEQ ID NO: 62 and a VL region comprising the amino acid sequence of SEQ ID NO: 74; a VH region comprising the amino acid sequence of SEQ ID NO: 63 and a VL region comprising the amino acid sequence of SEQ ID NO: 75; a VH region comprising the amino acid sequence of SEQ ID NO: 64 and a VL region comprising the amino acid sequence of SEQ ID NO: 76; a VH region comprising the amino acid sequence of SEQ ID NO: 68 and a VL region comprising the amino acid sequence of SEQ ID NO: 80; a VH region comprising the amino acid sequence of SEQ ID NO: 69 and a VL region comprising the amino acid sequence of SEQ ID NO: 81; a VH region comprising the amino acid sequence of SEQ ID NO: 70 and a VL region comprising the amino acid sequence of SEQ ID NO: 82; or a VH region comprising the amino acid sequence of SEQ ID NO: 71 and a VL region comprising the amino acid sequence of SEQ ID NO: 83; 2. The method of claim 1, comprising:

3. The method of claim 1 or 2, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 86 to 97.

4. The method of claim 3, wherein the antibody or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 86 to 88.

5. The antibody or fragment thereof has a surface plasmon resonance (SPR) content of 1.5×10 -7 5. The method of any one of claims 1 to 4, wherein the antibody binds to the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) with a binding affinity (KD) of less than M.

6. The method of any one of claims 1 to 5, wherein the antibody or fragment thereof is an scFv, Fab, Fab', F(ab')2, Fv, variable domain, diabody, minibody, or full-length antibody.

7. 7. The method of any one of claims 1 to 6, wherein the method provides an expanded population of V51 T cells containing more than 85% V51 T cells.

8. 8. The method of claim 7, wherein the method provides an expanded population of V51 T cells containing more than 90% V51 T cells.

9. 9. The method of any one of claims 1 to 8, wherein said method comprises culturing said cell population for at least 5 days.

10. 10. The method of any one of claims 1 to 9, wherein said method comprises culturing said cell population in the presence of at least one cytokine.

11. 11. The method of claim 10, 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 a mixture thereof.

12. The method of any one of claims 1 to 11, wherein the method comprises culturing the cell population in the presence of IL-2, IL-9, and / or IL-15.

13. The method of any one of claims 1 to 12, wherein said method comprises culturing said cell population in the presence of IL-21.

14. The method of any one of claims 1 to 13, wherein said method comprises culturing said cell population in the presence of IL-4.

15. 12. The method of any one of claims 1 to 11, wherein the method comprises culturing the cell population in a first culture medium comprising IL-4, and thereafter culturing the cell population in a second culture medium comprising IL-15.

16. 16. The method of claim 15, wherein the first culture medium lacks IL-15, IL-2, and / or IL-7.

17. 16. The method of claim 15, wherein the second culture medium lacks IL-4.

18. 18. The method of any one of claims 15 to 17, wherein the first or second culture medium, or both culture media, comprise one or more additional cytokines.

19. 19. The method of claim 18, wherein the additional cytokine is selected from the group consisting of: IL-21, IFN-γ, and IL-1β.

20. 20. The method of any one of claims 9 to 19, wherein the cell population is not in direct contact with stromal and / or epithelial cells during culture.

21. 21. The method of claim 20, wherein the cell population is not in direct contact with fibroblasts during culture.

22. 22. The method of any one of claims 9 to 21, wherein the cell population is not in direct contact with tumor cells and / or feeder cells during culture.

23. 23. The method of any one of claims 1 to 22, wherein said method comprises culturing said cell population in serum-free medium.

24. 24. The method of any one of claims 1 to 23, wherein the cell population is enriched for T cells prior to administration of the antibody or fragment thereof.

25. The method of any one of claims 1 to 24, wherein the cell population is enriched for γδ T cells prior to administration of the antibody or fragment thereof.

26. 26. The method of any one of claims 1 to 25, wherein the cell population is depleted of αβ T cells or NK cells prior to administration of the antibody or fragment thereof.

27. The method of any one of claims 1 to 26, wherein the cell population is obtained from a hematopoietic sample or a fraction thereof.

28. 28. The method of claim 27, wherein the hematopoietic sample is selected from peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, lymph node tissue, or a fraction thereof.

29. 29. The method of claim 27 or 28, wherein the hematopoietic sample consists of low density mononuclear cells (LDMCs) or peripheral blood mononuclear cells (PBMCs).

30. The method of any one of claims 1 to 26, wherein the cell population is obtained from a non-hematopoietic tissue sample.

31. 31. The method of claim 30, wherein the cell population is obtained from skin, colon, intestine, mammary gland, lung, prostate, liver, spleen, pancreas, uterus, vagina, or other skin, mucosal, or serosal sample.

32. 31. The method of claim 30, wherein the cell population is obtained from the non-hematopoietic tissue sample by culturing the non-hematopoietic tissue sample on a synthetic scaffold configured to promote cell migration from the non-hematopoietic tissue sample.

33. The method of any one of claims 1 to 32, wherein the cell population is obtained from a cancer tissue sample.

34. The method of any one of claims 1 to 33, wherein the cell population is obtained from human or non-human animal tissue.

35. The method of any one of claims 1 to 34, wherein the cell population is isolated from a sample prior to administering the anti-Vδ1 antibody or fragment thereof.

36. A Vδ1 T cell population with enhanced degranulation and killing activity conferred by the ex vivo method of any one of claims 1 to 35.

37. A composition comprising the Vδ1 T cell population of claim 36.

38. A pharmaceutical composition comprising the V51 T cell population of claim 36.

39. 39. The pharmaceutical composition of claim 38 for use as a medicament.

40. 39. The pharmaceutical composition of claim 38 for use in the treatment of cancer, an infectious disease, or an inflammatory disease.

41. 37. The V51 T cell population or V51 T cell population of claim 36 for the manufacture of a medicament for treating cancer, an infectious disease, or an inflammatory disease in a subject in need thereof. Use of the pharmaceutical composition of claim 38.

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