Novel Anti-TCR delta variable 1 antibodies

Abstract

To provide an antibody directed to a T cell receptor of a gamma delta T cell.SOLUTION: Provided is a human isolated anti-TCR delta variable 1 (anti-V δ 1) antibody or fragment thereof that binds to an epitope of the γ δ T cell receptor (TCR) variable delta 1 (V δ 1) chain comprising one or more amino acid residues within the amino acid region of (i) 3-20 of a specific amino acid sequence, and / or (ii) 37-77 of said specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antibodies and fragments thereof directed against the T cell receptor of gamma delta T cells. [Background technology]

[0002] Growing interest in T cell immunotherapy for cancer has focused on the apparent ability of subsets of CD8+ and CD4+ alpha beta (αβ) T cells to recognize cancer cells and mediate potential host protective functions, particularly when derepressed by clinically mediated antagonism of inhibitory pathways exerted 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 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 TRGV gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV8, TRGV9, and TRGV11, as well as nonfunctional genes TRGV10, TRGV11, TRGVA, and TRGVB. The most common TRGV gene segments encode Vδ1, Vδ2, and Vδ3, with some V segments designated as both Vδ and Vα (Adams et al., 2015, Cell Immunol., 296:30-40). Human γδ T cells can be broadly classified based on their TCR chain, as particular γ and δ types are more commonly, but not exclusively, found on cells in one or more tissue types. For example, most blood-resident γδ T cells express the Vδ2 TCR (commonly Vγ9Vδ2), whereas tissue-resident γδ T cells, such as those in the skin, more frequently use the Vδ1 TCR paired with a gamma chain, e.g., often paired with Vγ4 in the gut.

[0004] To utilize γδ T cells for immunotherapy, a means is required to either expand the cells in vivo or harvest and expand them ex vivo before reinfusion. The latter approach has previously been described using the addition of exogenous cytokines; see, for example, International Publication Nos. 2017 / 072367 and 2018 / 212808. Methods for expanding a patient's own γδ T cells have been described using pharmacologically modified forms of hydroxymethyl but-2-enyl pyrophosphate (HMBPP) or clinically approved aminobisphosphonates. These approaches appear to safely treat over 250 cancer patients, although the incidence of complete remission is rare. However, there remains a need for activating agents with a demonstrated ability to expand large numbers of γδ T cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 072367 [Patent Document 2] International Publication No. 2018 / 212808 [Non-patent literature]

[0006] [Non-Patent Document 1] Adams et al.,296:30-40(2015)Cell Immunol. Summary of the Invention

[0007] According to a first aspect of the present invention there is provided an isolated human anti-TCR delta variable 1 (anti-V51) antibody or fragment thereof, comprising: (i) sequences 3 to 20 of SEQ ID NO: 1, and / or (ii) Provided is an isolated human anti-Vδ1 antibody or fragment thereof that binds to an epitope of the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) that includes one or more amino acid residues within the amino acid region of 37 to 77 of SEQ ID NO:1.

[0008] According to a further aspect of the present invention, there is provided an isolated anti-Vδ1 antibody or a fragment thereof, comprising: a CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 25; CDR2 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 and A1 to A12 (in Table 2), and / or Provided is an isolated anti-Vδ1 antibody or a fragment thereof, comprising one or more CDR1s comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38 to 61.

[0009] According to a further aspect of the present invention, there is provided an isolated anti-Vδ1 antibody or a fragment thereof comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62 to 85.

[0010] According to another aspect of the present invention, there is provided an isolated anti-Vδ1 antibody or a fragment thereof comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 86 to 97.

[0011] According to a further aspect of the invention there is provided an isolated anti-V51 antibody or fragment thereof, which has an EC50 value for down-regulation of γδ TCR upon binding of less than 0.5 μg / ml, such as less than 0.06 μg / ml.

[0012] According to a further aspect of the present invention there is provided an isolated anti-V51 antibody or fragment thereof, which has an EC50 value for γδ T cell degranulation upon binding of less than 0.05 μg / ml, for example less than 0.005 μg / ml, in particular less than 0.002 μg / ml.

[0013] According to a further aspect of the present invention there is provided an isolated anti-V51 antibody or fragment thereof, which has an EC50 value for γδ T cell killing upon binding of less than 0.5 μg / ml, such as less than 0.055 μg / ml, in particular less than 0.020 μg / ml.

[0014] According to a further aspect of the present invention, there is provided a polynucleotide sequence encoding an anti-Vδ1 antibody or a fragment thereof comprising a sequence having at least 70% sequence identity with SEQ ID NOs: 99-110.

[0015] A further aspect of the present invention provides a polynucleotide sequence encoding an anti-Vδ1 antibody consisting of any of the sequences of SEQ ID NOs: 99 to 110, or a fragment thereof.

[0016] According to a further aspect of the present invention, there are provided expression vectors comprising the VH regions of SEQ ID NOs: 99 to 110.

[0017] According to a further aspect of the present invention, there are provided expression vectors comprising the VL regions of SEQ ID NOs: 99 to 110.

[0018] According to a further aspect of the present invention there is provided a cell comprising a polynucleotide sequence or expression vector as defined herein.

[0019] According to a further aspect of the present invention there is provided a composition comprising an antibody or fragment thereof as defined herein.

[0020] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising an antibody or fragment thereof as defined herein, together with a pharmaceutically acceptable diluent or carrier.

[0021] According to a further aspect of the invention there is provided an isolated antigen comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 123 for use in generating an anti-V51 antibody or fragment thereof.

[0022] According to a further aspect of the invention there is provided a method for producing an anti-V51 antibody or fragment thereof, comprising the steps of: (i) designing a series of antigens comprising a TCR delta variable 1 (V51) amino acid sequence, wherein the CDR3 sequence of V51 is the same for all antigens in the series; (ii) exposing the first antigen designed in step (i) to an antibody library; and (iii) isolating antibodies or fragments thereof that bind to the antigen. (iv) exposing the isolated antibody or fragment thereof to the second antigen designed in step (i); (v) isolating an antibody or fragment thereof that binds to both the first and second antigens.

[0023] According to a further aspect of the present invention there is provided an antibody obtainable by the method defined herein. [Brief explanation of the drawings]

[0024] [Figure 1] ELISA detection of directly coated antigens using anti-V51 Ab (REA173, Miltenyi Biotec). Detection was only seen with antigens containing the V51 domain. The leucine zipper (LZ) format appeared to be more potent than the Fc format, consistent with cell-based flow competition assays (data not shown). [Figure 2] Polyclonal phage DELFIA data for DV1 selection. A) Heterodimer selection: heterodimeric LZ TCR format in rounds 1 and 2, with deselection for heterodimeric LZ TCR in both rounds. B) Homodimer selection: Round 1 was performed using homodimeric Fc fusion TCR with deselection for human IgG1 Fc, followed by round 2 with deselection for heterodimeric LZ TCR. Each graph contains two bars for each target, representing selections from different libraries. [Figure 3-1]IgG capture: Left) Sensorgram of the interaction of anti-L1 IgG with L1, Right) Steady-state fitting where available. All experiments were performed on a MASS-2 instrument at room temperature. Steady-state fitting with Langmuir 1:1 binding. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4] Results of TCR down-regulation assays 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] Results of T cell degranulation assays 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] Results of killing assays (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] 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]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] 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] 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] Epitope mapping data for 1141_P01_E01. Graphical representation of the epitope binding site of 1141_P01_E01 on SEQ ID NO: 1. DETAILED DESCRIPTION OF THE INVENTION

[0025] 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.

[0026] Gamma delta (γδ) T cells represent a small subset of T cells that express a distinct 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 major subtypes of human γδ T cells: those that predominate in peripheral blood and those that predominate in nonhematopoietic 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 primarily express the delta variable 2 chain (Vδ2). γδ T cells that predominate in nonhematopoietic tissues (i.e., tissue-resident) primarily express the delta variable 1 chain. Reference to "Vδ1 T cells" refers to γδ T cells that possess the Vδ1 chain, i.e., Vδ1+ cells.

[0027] References to "delta variable 1" may also be referred to as V51 or Vd1, while the nucleotides encoding the TCR chain comprising this region may be referred to as "TRDV1". Any antibody or fragment thereof that interacts with the V51 chain of a γδ TCR is an effective 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".

[0028] Further reference is made herein to other delta chains, such as the "delta variable 2" chain. These may be referred to similarly. 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, an antibody or fragment thereof that interacts with the V51 chain of a γδ TCR does not interact with other delta chains, such as V52.

[0029] Reference is also made herein to "gamma variable chains." These may also be referred to as gamma 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, an antibody or fragment thereof that interacts with the Vδ1 chain of a γδ TCR does not interact with a gamma chain such as Vγ4.

[0030] 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 IgA, IgG, IgE, IgD, and IgM types (and their subtypes). The overall structure of immunoglobulin G (IgG) antibodies, assembled from two identical heavy (H) chains and two identical light (L) chain polypeptides, is well established and highly conserved in mammals (Padlan (1994) Mol. Immunol. 31:169-217).

[0031] 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, their related domains, and domains derived therefrom 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 subdivided into regions called "complementarity-determining regions" (CDRs) and "framework regions" (FRs), interspersed with more conserved regions. The framework and complementarity-determining regions have been precisely defined (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, (1991) NIH Publication Number 91-3242). Alternative numbering conventions for CDR sequences exist, such as those shown in Chothia et al. (1989) Nature 342:877-883. In conventional antibodies, each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A conventional antibody tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains is formed, for example, by the immunoglobulin heavy and light chains interconnected by disulfide bonds and the heavy chains similarly connected. The heavy chain constant region contains three domains: CH1, CH2, and CH3. The light chain constant region is composed 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.

[0032] 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 that 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 within the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CH1 domains); (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 the VH and CH1 domains); (iv) Fv fragments (consisting of the VL and VH domains of a single arm of an antibody); (v) single-chain variable fragments, scFv, which consist of VL and VH domains connected by a synthetic linker using recombinant methods, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules; (vi) VH (an immunoglobulin chain variable domain consisting of a VH domain), (vii) VL (an immunoglobulin chain variable domain consisting of a VL domain), (viii) domain antibodies (dAbs, consisting of either VH or VL domains); (ix) a minibody (consisting of a pair of scFv fragments linked via a CH3 domain); and (x) Diabodies, which consist of a non-covalent dimer of scFv fragments consisting of the VH domain from one antibody and the VL domain from another antibody connected by a small peptide linker.

[0033] "Human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human subject administered with such a human antibody does not generate an interspecies antibody response (e.g., a HAMA response—called human anti-mouse antibody) 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 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., mouse) have been grafted onto human framework sequences. Human antibodies prepared, expressed, produced, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected 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, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences, may also be referred to as "recombinant human antibodies."

[0034] 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.

[0035] "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 other antigens. 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 more frequently, more rapidly, with longer duration, and / or with higher affinity with a particular target antigen or epitope compared to alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily, and / or with longer duration than it binds to other substances.

[0036] "Affinity" is expressed by the equilibrium constant (KD) of dissociation between an antigen and an antigen-binding polypeptide, and 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 stronger the binding strength between the antigenic determinant and the 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.

[0037] 10 -6 Any KD value less than 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 a fluorescence assay), as well as different variants known in the art.

[0038] "Avidity" is a measure of the strength of binding between an antibody or fragment thereof and an associated antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antibody, as well as the number of associated binding sites present on the antibody.

[0039] "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 either human tissues or the hematopoietic blood system or human tumors, respectively. All of the above cell types can be identified by their (i) location, or where they originate, and (ii) expression of the V51+ TCR.

[0040] A "modulatory antibody" is an antibody that, upon contact with or binding to a cell expressing the target to which the antibody binds, produces 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 towards target cells or diseased cells).

[0041] A method of "modulating" a cell or collection thereof refers to a method that triggers at least one measurable change in, or secretion from, the aforementioned cell or cells, to produce one or more "modulated cells."

[0042] 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 (including, but not limited to, cell-mediated response, humoral response, cytokine response, chemokine response) upon addition of a regulatory antibody.

[0043] "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, red blood cells, platelets, dendritic cells, phagocytes, granulocytes, innate lymphoid cells, natural killer (NK) cells, and gamma delta T cells. Typically, immune cells are classified using combinatorial cell surface molecular analysis (e.g., via flow cytometry) to identify, group, or cluster immune cells for differentiation into subpopulations. These can then be further subdivided with additional analysis. For example, CD45+ lymphocytes can be further subdivided into vδ-positive and vδ-negative populations.

[0044] A "model system" is a biological model or representation designed to aid in understanding how a pharmaceutical agent, such as an antibody or fragment thereof, may function as a pharmaceutical agent in alleviating the signs or symptoms of a disease. Such models typically include the use of diseased cells, non-diseased cells, healthy effector cells, and tissues in vitro, ex vivo, and in vivo to study and compare the performance of such pharmaceutical agents.

[0045] A "disease cell" exhibits a phenotype associated with a disease such as cancer, an infection such as a viral infection, or the progression of an inflammatory condition or disease. For example, the disease cell may be a tumor cell, an autoimmune tissue cell, or a virally infected cell. Thus, the disease cell may be defined as being neoplastic, virally infected, or inflammatory.

[0046] "Healthy cells" refer to normal cells that are not diseased. They can also be referred to as "normal" or "non-disease" cells. Non-disease cells include non-cancerous, non-infectious, or non-inflammatory cells. These cells are often used together with related diseased cells to determine the diseased cell specificity of a drug and / or to better understand the therapeutic index of a drug.

[0047] "Disease cell specificity" is a measure of how effectively an effector cell or a population thereof (e.g., a population of V51+ cells) can distinguish and kill diseased cells, such as cancer cells, while sparing non-disease or healthy cells. This potential can be measured in a model system and can involve comparing the tendency of an effector cell or a population of effector cells to selectively kill or lyse diseased cells with the potential of said effector cells to kill or lyse non-disease or healthy cells. Such disease cell specificity can inform the potential therapeutic index of a pharmaceutical.

[0048] "Enhanced disease cell specificity" describes the phenotype of an effector cell, e.g., a V51+ cell, or a 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 the fold change or percentage increase in disease cell killing specificity or selectivity.

[0049] Preferably, the antibody or fragment thereof (i.e., polypeptide) is isolated. An "isolated" polypeptide is a polypeptide that is 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 specifically bind to antigens other than Vδ1). The term "isolated" can also be used to 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, or most preferably at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure.

[0050] Preferably, the polynucleotide used in the present invention is isolated.An "isolated" polynucleotide is a polynucleotide that is removed from its original environment.For example, a naturally occurring polynucleotide is isolated when it is separated from some or all of the materials that coexist in natural system.For example, if a polynucleotide is cloned into a vector that is not part of its natural environment, or if the polynucleotide is contained in cDNA, it is considered to be isolated.

[0051] An antibody or fragment thereof may be a "functionally active variant," including naturally occurring allelic variants as well as mutants or any non-naturally occurring variants. As known in the art, allelic variants are alternative forms of (poly)peptides 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, the above-mentioned functionally active variants may still function when the framework containing the CDRs is modified, when the CDRs themselves are modified, when the above-mentioned CDRs are grafted onto alternative frameworks, or when N- or C-terminal extensions are incorporated. Furthermore, the CDRs containing the binding domains may pair with different partner chains, such as those shared with another antibody. When shared with a so-called "common" light chain or a "common" heavy chain, the above-mentioned binding domains may still function. Furthermore, the above-mentioned binding domains may function when multimerized. Furthermore, an "antibody or fragment thereof" may include functional variants in which the VH or VL or constant domains have been altered away from or towards different canonical sequences (e.g., as listed on IMGT.org) and still function.

[0052] For purposes of comparing two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0 using standard settings for polypeptide sequences (BLASTP). For purposes of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0 using standard settings for nucleotide sequences (BLASTN).

[0053] A polypeptide or polynucleotide sequence is said to be the same as, or "identical to," another polypeptide or polynucleotide sequence if the sequences share 100% sequence identity over their entire length. Residues in a sequence are numbered from left to right, i.e., from N-terminus to C-terminus for polypeptides and from 5' to 3' for polynucleotides.

[0054] A "difference" between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the second sequence compared to the first sequence. Two polypeptide sequences may contain one, two, or more such amino acid differences. An insertion, deletion, or substitution in a second sequence that is otherwise identical (100% sequence identity) to the first sequence reduces the percent sequence identity. For example, if the identical sequences are 9 amino acid residues long, a single substitution in the second sequence results in 88.9% sequence identity. If the first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share more than 66% identity (the first and second polypeptide sequences share 66.7% identity).

[0055] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence may be determined. An "addition" is the addition of one amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). A "substitution" is the replacement of one amino acid residue in the sequence of the first polypeptide with a different amino acid residue. This substitution may be conservative or non-conservative. A "deletion" is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0056] A "conservative" amino acid substitution is one in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure and is expected to have little effect on the function, activity, or other biological properties of a polypeptide. Preferably, such a conservative substitution is one in which one amino acid residue within the following group is replaced with another amino acid residue within the same group: JPEG2026009903000001.jpg157168

[0057] 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.

[0058] As used herein, the numbering of polypeptide sequences and the definitions of CDR and FR are as defined according to the Kabat system (Kabat et al., 1991, incorporated herein by reference in its entirety).The "corresponding" amino acid residue between a first polypeptide sequence and a second polypeptide sequence is the amino acid residue in the first sequence that shares the same position according to the Kabat system with the amino acid residue in the second sequence, but the amino acid residue in the second sequence may have different identity with the first sequence.Appropriately, corresponding residues share the same number (and letter) when the framework and CDR are the same length according to Kabat definition.Alignment can be achieved manually or by using known computer algorithms for sequence alignment, such as, for example, NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

[0059] The term "epitope" herein refers to the portion of a target that is specifically bound by an antibody or its fragment. An epitope can also be referred to as an "antigenic determinant." An antibody binds to "essentially the same epitope" as another antibody when both recognize the same or sterically overlapping epitopes. A commonly used method for determining whether two antibodies bind to the same or overlapping epitopes is a competitive assay, which can be configured in several different formats (e.g., well plates using radioactive or enzyme labels, or flow cytometry on antigen-expressing cells) using either labeled antigen or labeled antibody.

[0060] Epitopes found on protein targets can be defined as "linear epitopes" or "conformational epitopes." Linear epitopes are formed by contiguous sequences of amino acids 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.

[0061] The term "vector," as used herein, 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 replicating along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably 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. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), as well as bacteriophage and phagemid systems, which serve equivalent functions. The term "recombinant host cell" (or simply "host cell"), as used herein, 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 to the progeny of such a cell, for example, where such progeny are used to generate cell lines or cell banks, which are then optionally stored, provided, sold, transferred, or used to produce antibodies or fragments thereof as described herein.

[0062] 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 (such as cows), sport animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the subject is a human. In alternative embodiments, the subject is a non-human mammal, such as a mouse.

[0063] The term "sufficient amount" means an amount sufficient to produce a desired effect. The term "therapeutically effective amount" is an amount effective to alleviate the symptoms of a disease or disorder. Since prevention can be considered therapy, a therapeutically effective amount can be a "prophylactically effective amount."

[0064] A disease or disorder is "alleviated" 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, is reduced.

[0065] 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.

[0066] "Cancer," as used herein, refers to the abnormal growth or division of cells. Generally, the growth and / or lifespan of cancer cells exceeds and is uncoordinated with the growth and / or lifespan of normal cells and surrounding tissues. Cancers can be benign, premalignant, or malignant. Cancer arises 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, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous, meningioma, etc.), breast, reproductive system (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidneys, ureters, 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.).

[0067] As used herein, the term "about" as used herein means up to 10% greater than, and including, the specified value, up to 10% less than, and including, the specified value, preferably up to 5% greater than, and including, the specified value, up to 5% less than, and including, the specified value, and particularly including, the specified value. The term "between" includes the boundary values ​​specified.

[0068] Antibodies or fragments thereof Provided herein is an antibody or fragment thereof capable of specifically binding to the delta variable 1 chain (Vδ1) of the γδ T cell receptor (TCR).

[0069] 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.

[0070] The antibodies of the present invention may be of any class, e.g., IgG, IgA, IgM, IgE, IgD, or their isotypes, and may contain a kappa or lambda light chain. In one embodiment, the antibody is an IgG antibody, e.g., at least one of the isotypes IgG1, IgG2, IgG3, or IgG4. In a further embodiment, the antibody may be in a modified format (e.g., an IgG format) to confer desired properties, such as an Fc mutated to reduce effector function, extend half-life, alter ADCC, or improve hinge stability. Such modifications are well known in the art.

[0071] In one embodiment, the antibody or fragment thereof is human. Thus, the antibody or fragment thereof may be derived from a human immunoglobulin (Ig) sequence. The CDR, framework and / or constant region of the antibody (or fragment thereof) may be derived from a human Ig sequence, particularly a human IgG sequence. The CDR, framework and / or constant region may be substantially identical to a human Ig sequence, particularly a human IgG sequence. The advantage of using a human antibody is that human antibodies are less immunogenic or non-immunogenic in humans.

[0072] The antibody or fragment thereof may also be chimeric, for example, a mouse-human antibody chimera.

[0073] Alternatively, the antibody or fragment thereof is derived from a non-human species, such as a mouse. Such non-human antibodies can be modified to increase their similarity to antibody variants naturally produced in humans, such that the antibody or fragment thereof can be partially or fully humanized. Thus, in one embodiment, the antibody or fragment thereof is humanized.

[0074] The anti-V51 antibodies or fragments thereof described herein may be used to modulate delta variable 1 chain (V51) T cells.

[0075] Regulation of V51 T cells may include: - expansion of V51 T cells, e.g., by increasing the number of V51 T cells or promoting the survival of V51 T cells; - stimulation of V51 T cells, for example by increasing the potency of V51 T cells, i.e., by increasing target cell killing; - prevention of Vδ1 T cell exhaustion, e.g., by increasing Vδ1 T cell persistence, - degranulation of Vδ1 T cells, - 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, - Reducing V51 T cell numbers, for example by inhibiting V51 T cell proliferation or by inducing V51 T cell death (i.e. killing V51 T cells).

[0076] 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 upon administration of an anti-V51 antibody, or fragment thereof, as defined herein to a patient. In an alternative embodiment, V51 T cells are inhibited upon administration of an anti-V51 antibody, or fragment thereof, as defined herein to a patient. In an alternative embodiment, V51 T cells are not inhibited upon administration of an anti-V51 antibody, or fragment thereof, as defined herein to a patient.

[0077] Antibodies targeting 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, such as activation or inhibition.

[0078] In one embodiment, the epitope can be an activating epitope of a γδ T cell. An "activating" epitope can include, for example, stimulating TCR function such as cell degranulation, TCR downregulation, cytotoxicity, proliferation, mobilization, increased survival or resistance to exhaustion, intracellular signaling, cytokine or growth factor secretion, phenotypic change, or altered gene expression. For example, binding of an activating epitope can stimulate the expansion (i.e., proliferation) of γδ T cell populations, preferably Vδ1 T cell populations. Thus, these antibodies can be used to regulate γδ T cell activation and thereby regulate immune responses. Thus, in one embodiment, binding of an activating epitope downregulates a γδ TCR. In additional or alternative embodiments, binding of an activating epitope activates γδ T cell degranulation. In further additional or alternative embodiments, binding of an activating epitope activates γδ T cell killing.

[0079] Alternatively, the antibody (or fragment thereof) may have a blocking effect by preventing 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 prevents TCR binding (e.g., by steric hindrance). By blocking V51, the antibody may prevent 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.

[0080] The epitope preferably consists of at least an extracellular, soluble, hydrophilic, external or cytoplasmic part of one of the V51 chains of a γδ TCR.

[0081] 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 to 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 solely by the presence of the V51 chain. It will be appreciated, therefore, that any V51 chain comprising a γδ TCR 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 one or more amino acid residues within amino acid regions 1-24 and / or 35-90 of SEQ ID NO: 1, e.g., portions of the V51 chain that are not part of the CDR1 and / or CDR3 sequence. In one embodiment, the epitope does not include amino acid residues within amino acid region 91-105 (CDR3) of SEQ ID NO:1.

[0082] 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) does not include an epitope found in the J region of the Vδ1 chain (e.g., one of the four J regions encoded by human Delta1 chain germline: SEQ ID NO: 131 (J1*0), or 132 (J2*0), or 133 (J3*0), or 134 (J4*0)). In one embodiment, the epitope bound by the antibody (or fragment thereof) does not include an epitope found in the C region of the Vδ1 chain (e.g., SEQ ID NO: 135 (C1*0), which contains the C-terminal membrane-proximal / transmembrane region). In one embodiment, the epitope bound by the antibody (or fragment thereof) does not include an epitope found within the N-terminal leader sequence of the V51 chain (e.g., SEQ ID NO: 129). Thus, the antibody or fragment may bind only within 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).

[0083] Reference to the epitope is made in relation to the Vδ1 sequence, which is derived from the sequence described in Luoma et al. (2013) Immunity 39:1032-1042, and shown in SEQ ID NO: 1, RCSB Protein Data Bank entries: 4MNH and 3OMZ. AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGESLTRADKLIFGKGTRVTVEPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 1).

[0084] 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. Within the V region, CDR1 is defined as amino acid residues 25-34 of SEQ ID NO: 1, CDR2 is defined as amino acid residues 50-54 of SEQ ID NO: 1, and CDR3 is defined as amino acid residues 93-104 of SEQ ID NO: 1 (Xu et al., PNAS USA 108(6):2414-2419 (2011)).

[0085] Thus, according to an aspect of the present invention, there is provided an isolated antibody or fragment thereof that binds to an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR) comprising one or more amino acid residues within the following amino acid region: (i) sequences 3 to 20 of SEQ ID NO: 1, and / or (ii) 37 to 77 of SEQ ID NO: 1.

[0086] In a further embodiment, the antibody or fragment thereof further recognizes a polymorphic V region comprising an epitope of amino acid residues 1-90 of SEQ ID NO: 128. Thus, 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 when defining an epitope as described herein. The antibodies of the present invention can recognize both variants of this germline sequence. By way of example, when it is described that an antibody or fragment thereof defined herein recognizes 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.

[0087] 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 amino acids equivalently positioned 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, which encodes either alanine (A) or valine (V) at position 71 of SEQ ID NO: 1.

[0088] 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.

[0089] 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 residues 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.

[0090] It will be further understood that the above-described antibodies (or fragments thereof) need not bind to all amino acids within a defined range. Such epitopes may be referred to as linear epitopes. For example, an antibody that binds to an epitope comprising amino acid residues within amino acid region 5-20 of SEQ ID NO: 1 may bind only to one or more of the amino acid residues in the range, including, for example, only the amino acid residues at each end of the range (i.e., amino acids 5 and 20), optionally including amino acids within the range (i.e., amino acids 5, 9, 16, and 20).

[0091] 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.

[0092] In one embodiment, the epitope comprises one or more amino acid residues within the following amino acid region of SEQ ID NO:1 (or SEQ ID NO:128 as described above): (i) 3-17, (ii) 5–20; (iii) 37-53, (iv) 50-64, (v)59-72, (vi) 59-77, (vii) 62-69, and / or (viii) 62-77.

[0093] In further embodiments, the epitope comprises one or more amino acid residues within the amino acid regions 5-20 and 62-77, 50-64, 37-53 and 59-72, 59-77, or 3-17 and 62-69 of SEQ ID NO: 1. In further embodiments, the epitope consists of one or more amino acid residues within the amino acid regions 5-20 and 62-77, 50-64, 37-53 and 59-72, 59-77, or 3-17 and 62-69 of SEQ ID NO: 1.

[0094] In a further embodiment, the epitope comprises, or preferably consists of, amino acid residues 3, 5, 9, 10, 12, 16, 17, 62, 64, 68, and 69 of SEQ ID NO: 1. In a further embodiment, the epitope comprises, or preferably consists of 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 preferably consists of, amino acid residues 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77 of SEQ ID NO: 1. In a further embodiment, the epitope comprises, or preferably consists of amino acid residues 50, 53, 59, 62, and 64 of SEQ ID NO: 1. In a further embodiment, the epitope comprises, or preferably consists of amino acid residues 59, 60, 68, and 72 of SEQ ID NO: 1.

[0095] In one embodiment, the epitope comprises one or more amino acid residues within amino acid regions 5-20 and / or 62-77 of SEQ ID NO: 1. In a further embodiment, the epitope consists of one or more amino acid residues within amino acid regions 5-20 and 62-77 of SEQ ID NO: 1. In alternative further embodiments, the epitope comprises one or more amino acid residues within amino acid regions 5-20 or 62-77 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may have some 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.

[0096] 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 have some 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.

[0097] 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 have some 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.

[0098] 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 have some 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.

[0099] In one embodiment, the epitope does not include amino acid residues in the amino acid region 11-21 of SEQ ID NO: 1. In one embodiment, the epitope does not include amino acid residues in the amino acid region 21-28 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 67-82 of SEQ ID NO: 1.

[0100] 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 WO 2017 / 197347, binding of TS-1 and TS8.2 to soluble TCRs was detected when the 51 chain included the V51 J1 and V51 J2 sequences, but not binding to the V51 J3 chain, indicating that binding of TS-1 and TS8.2 involves critical residues in the deltaJ1 and deltaJ2 regions.

[0101] 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.

[0102] Various techniques for determining which epitopes bind to 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. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used. Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry (as described in Example 9). Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and 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 can retain deuterium and therefore exhibit a relatively higher mass than amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts.

[0103] Antibody sequence The isolated anti-V51 antibodies or fragments thereof of the invention may be described with reference to their CDR sequences.

[0104] According to one aspect of the invention there is provided an isolated anti-V51 antibody or fragment thereof, comprising: CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 25; CDR2 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 and A1 to A12, and / or Provided is an isolated anti-Vδ1 antibody or a fragment thereof, comprising one or more CDR1s comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38 to 61.

[0105] According to one aspect of the present invention, there is provided an isolated anti-Vδ1 antibody or fragment thereof, comprising 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.

[0106] 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: 2-25. 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: 26-37 and sequences A1-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: 38-61.

[0107] 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: 2-25. 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: 26-37 and sequences A1-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: 38-61.

[0108] According to a further aspect of the present invention, there is provided 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: 2 to 13, and / or 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. According to a further aspect of the present invention, there is provided 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: 2 to 13, 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: 14 to 25.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] According to a further aspect of the present invention, there is provided 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: 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. According to a further aspect of the present invention, there is provided 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: 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.

[0113] According to a specific aspect of the present invention, there is provided 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: 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. According to a further aspect of the present invention, there is provided 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: 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.

[0114] According to a further aspect of the present invention, there is provided 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: 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. According to a further aspect of the present invention, there is provided 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: 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.

[0115] According to a specific aspect of the present invention, there is provided 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. According to another aspect of the present invention, there is provided 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.

[0116] According to a specific aspect of the present invention, there is provided 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. According to another aspect of the present invention, there is provided 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.

[0117] According to a specific aspect of the present invention, there is provided 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. According to another aspect of the present invention, there is provided 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.

[0118] Embodiments referred to herein as "at least 80%" or "80% or greater" will be understood to include all values ​​of 80% or greater, e.g., 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity, etc. In one embodiment, an antibody or fragment of the invention comprises at least 85%, e.g., at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the designated sequence.

[0119] Instead of percent sequence identity, embodiments may 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 fragment thereof of the present invention comprises, or more preferably 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.

[0120] Suitably, residues in any of CDR1, CDR2 or CDR3 that differ from their corresponding residues in SEQ ID NOs: 2-61 and A1-A12 are conservative substitutions with respect to their corresponding residues, for example, any residue in CDR3 that differs from its corresponding residue in SEQ ID NOs: 2-25 is a conservative substitution with respect to its corresponding residue.

[0121] 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 comprising 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 sequences A1 to A12, and / or (vi) A VL region including a CDR1 having a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 50 to 61.

[0122] 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 heavy chain having a VH region including a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38 to 49.

[0123] 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 comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of sequences A1 to A12; and (iii) A light chain having a VL region including a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 50 to 61.

[0124] 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, such as 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, such as 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, such as 38, 39, 40, or 41, particularly 38, 39, or 40.

[0125] 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.

[0126] 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.

[0127] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ ID NO: 27, and a CDR1 sequence of SEQ ID NO: 39. In one embodiment, the CDR3 consists of the sequence of SEQ ID NO: 3, the CDR2 consists of the sequence of SEQ ID NO: 27, and the CDR1 consists of the sequence of SEQ ID NO: 39.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 8, a CDR2 sequence of SEQ ID NO: 32, and a CDR1 sequence of SEQ ID NO: 44. In one embodiment, the CDR3 consists of the sequence of SEQ ID NO: 8, the CDR2 consists of the sequence of SEQ ID NO: 32, and the CDR1 consists of the sequence of SEQ ID NO: 44.

[0132] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 9, a CDR2 sequence of SEQ ID NO: 33, and a CDR1 sequence of SEQ ID NO: 45. In one embodiment, the CDR3 consists of the sequence of SEQ ID NO: 9, the CDR2 consists of the sequence of SEQ ID NO: 33, and the CDR1 consists of the sequence of SEQ ID NO: 45.

[0133] 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: 47.

[0134] 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, the CDR3 consists of the sequence of SEQ ID NO: 11, the CDR2 consists of the sequence of SEQ ID NO: 35, and the CDR1 consists of the sequence of SEQ ID NO: 47.

[0135] 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: 14-25, 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-A12 (of 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.

[0136] 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 of sequence A1, and CDR1 consists of the sequence of SEQ ID NO: 50.

[0137] 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 of sequence A2, and CDR1 consists of the sequence of SEQ ID NO: 51.

[0138] 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 of sequence A3, and CDR1 consists of the sequence of SEQ ID NO: 52.

[0139] 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 of sequence A4, and CDR1 consists of the sequence of SEQ ID NO: 53.

[0140] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 18, a CDR2 comprising the sequence of sequence 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 sequence A5, and CDR1 consists of the sequence of SEQ ID NO: 54.

[0141] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of sequence 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 sequence A7, and CDR1 consists of the sequence of SEQ ID NO: 56.

[0142] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 21, a CDR2 comprising the sequence of sequence 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 sequence A8, and CDR1 consists of the sequence of SEQ ID NO: 57.

[0143] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 22, a CDR2 comprising the sequence of sequence 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 sequence A9, and CDR1 consists of the sequence of SEQ ID NO: 58.

[0144] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 23, a CDR2 comprising the sequence of sequence 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 sequence A10, and CDR1 consists of the sequence of SEQ ID NO: 59.

[0145] 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, 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, the HCDR3 consists of the sequence of SEQ ID NO: 2, the HCDR2 consists of the sequence of SEQ ID NO: 26, the HCDR1 consists of the sequence of SEQ ID NO: 38, the LCDR3 consists of the sequence of SEQ ID NO: 14, the LCDR2 consists of the sequence of sequence A1, and the LCDR1 consists of the sequence of SEQ ID NO: 50.

[0146] 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, 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, the HCDR3 consists of the sequence of SEQ ID NO: 3, the HCDR2 consists of the sequence of SEQ ID NO: 27, the HCDR1 consists of the sequence of SEQ ID NO: 39, the LCDR3 consists of the sequence of SEQ ID NO: 15, the LCDR2 consists of the sequence of sequence A2, and the LCDR1 consists of the sequence of SEQ ID NO: 51.

[0147] 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, 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, the HCDR3 consists of the sequence of SEQ ID NO: 4, the HCDR2 consists of the sequence of SEQ ID NO: 28, the HCDR1 consists of the sequence of SEQ ID NO: 40, the LCDR3 consists of the sequence of SEQ ID NO: 16, the LCDR2 consists of the sequence of sequence A3, and the LCDR1 consists of the sequence of SEQ ID NO: 52.

[0148] 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, 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, the HCDR3 consists of the sequence of SEQ ID NO: 5, the HCDR2 consists of the sequence of SEQ ID NO: 29, the HCDR1 consists of the sequence of SEQ ID NO: 41, the LCDR3 consists of the sequence of SEQ ID NO: 17, the LCDR2 consists of the sequence of sequence A4, and the LCDR1 consists of the sequence of SEQ ID NO: 53.

[0149] 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, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 18, a CDR2 comprising the sequence of sequence A5, and a CDR1 comprising the sequence of SEQ ID NO: 54. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 6, the HCDR2 consists of the sequence of SEQ ID NO: 30, the HCDR1 consists of the sequence of SEQ ID NO: 42, the LCDR3 consists of the sequence of SEQ ID NO: 18, the LCDR2 consists of the sequence of sequence A5, and the LCDR1 consists of the sequence of SEQ ID NO: 54.

[0150] 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, and 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, the HCDR3 consists of the sequence of SEQ ID NO: 7, the HCDR2 consists of the sequence of SEQ ID NO: 31, the HCDR1 consists of the sequence of SEQ ID NO: 43, the LCDR3 consists of the sequence of SEQ ID NO: 19, the LCDR2 consists of the sequence of sequence A6, and the LCDR1 consists of the sequence of SEQ ID NO: 55.

[0151] 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, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of sequence A7, and a CDR1 comprising the sequence of SEQ ID NO: 56. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 8, the HCDR2 consists of the sequence of SEQ ID NO: 32, the HCDR1 consists of the sequence of SEQ ID NO: 44, the LCDR3 consists of the sequence of SEQ ID NO: 20, the LCDR2 consists of the sequence of sequence A7, and the LCDR1 consists of the sequence of SEQ ID NO: 56.

[0152] 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, and the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 21, a CDR2 comprising the sequence of sequence A8, and a CDR1 comprising the sequence of SEQ ID NO: 57. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 9, the HCDR2 consists of the sequence of SEQ ID NO: 33, the HCDR1 consists of the sequence of SEQ ID NO: 45, the LCDR3 consists of the sequence of SEQ ID NO: 21, the LCDR2 consists of the sequence of sequence A8, and the LCDR1 consists of the sequence of SEQ ID NO: 57.

[0153] 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, and 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 sequence A9, and a CDR1 comprising the sequence of SEQ ID NO: 58. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 10, the HCDR2 consists of the sequence of SEQ ID NO: 34, the HCDR1 consists of the sequence of SEQ ID NO: 46, the LCDR3 consists of the sequence of SEQ ID NO: 22, the LCDR2 consists of the sequence of sequence A9, and the LCDR1 consists of the sequence of SEQ ID NO: 58.

[0154] 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, and 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 sequence A10, and a CDR1 comprising the sequence of SEQ ID NO: 59. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 11, the HCDR2 consists of the sequence of SEQ ID NO: 35, the HCDR1 consists of the sequence of SEQ ID NO: 47, the LCDR3 consists of the sequence of SEQ ID NO: 23, the LCDR2 consists of the sequence of sequence A10, and the LCDR1 consists of the sequence of SEQ ID NO: 59.

[0155] 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, and 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 sequence A11, and a CDR1 comprising the sequence of SEQ ID NO: 60. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 12, the HCDR2 consists of the sequence of SEQ ID NO: 36, the HCDR1 consists of the sequence of SEQ ID NO: 48, the LCDR3 consists of the sequence of SEQ ID NO: 24, the LCDR2 consists of the sequence of sequence A11, and the LCDR1 consists of the sequence of SEQ ID NO: 60.

[0156] 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, and 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 sequence A12, and a CDR1 comprising the sequence of SEQ ID NO: 61. In one embodiment, the HCDR3 consists of the sequence of SEQ ID NO: 13, the HCDR2 consists of the sequence of SEQ ID NO: 37, the HCDR1 consists of the sequence of SEQ ID NO: 49, the LCDR3 consists of the sequence of SEQ ID NO: 25, the LCDR2 consists of the sequence of sequence A12, and the LCDR1 consists of the sequence of SEQ ID NO: 61.

[0157] 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 described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P02_F07 described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P01_G06 described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1245_P01_G09 described 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 described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1251_P02_G10 described in Table 2.

[0158] Preferably, the VH and VL regions listed above each comprise four framework regions (FR1-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: 62-85. 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: 62-85. 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: 62-85. 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: 62-85.

[0159] The antibodies described herein may be defined by their complete light and / or heavy chain variable sequences. Thus, according to a further aspect of the present invention, there is provided an isolated anti-Vδ1 antibody, or a fragment thereof, comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 62 to 85. According to a further aspect of the present invention, there is provided an isolated anti-Vδ1 antibody, or a fragment thereof, consisting of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 62 to 85.

[0160] 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, such as 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, such as 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.

[0161] 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, such as 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, such as 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.

[0162] 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.

[0163] 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 one embodiment, the antibody or fragment thereof comprises 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).

[0164] 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).

[0165] 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).

[0166] 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).

[0167] 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).

[0168] 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_G06) and a VL region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0169] For fragments comprising both the VH and VL regions, these may be associated either covalently (e.g., by 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 regions linked by a linker. In one embodiment, the VH and VL regions are linked by a (e.g., synthetic) polypeptide linker. The polypeptide linker may be (Gly4Ser) nA linker may be included, where n=1 to 8, for example, 2, 3, 4, 5, or 7. The polypeptide linker may be [(Gly4Ser) n (Gly3AlaSer) m ] p It may comprise a linker, where n=1 to 8, for example, 2, 3, 4, 5, or 7, m=1 to 8, for example, 0, 1, 2, or 3, and p=1 to 8, for example, 1, 2, or 3. In a further embodiment, the linker comprises SEQ ID NO: 98. In a further embodiment, the linker consists of SEQ ID NO: 98.

[0170] 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).

[0171] 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 yet a 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).

[0172] Those skilled in the art will appreciate that scFv constructs can be designed and engineered to include N- and C-terminal modifications to aid 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., starting with QVQ or EVQ). At the C-terminus (i.e., C-terminal to the canonical VL domain sequence ending 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, including tags such as His-tags and Flag-tags, to aid 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 well understood that the N- or C-terminal sequences of the scFvs described above are optional and may be removed, modified, or substituted if alternative scFv design, translation, purification, or detection strategies are employed.

[0173] As described herein, antibodies may be in any format. In a preferred embodiment, the antibody is 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.

[0174] 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: 111-122. In a further embodiment, the antibody or fragment thereof consists of the amino acid sequence of any one of SEQ ID NOs: 111-122. In yet a further embodiment, the antibody or fragment thereof consists of 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 consists of 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 consists of 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.

[0175] In one embodiment, the antibody binds to or competes with the same or essentially the same epitope as an antibody or fragment thereof defined herein. 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 with binding thereto. For example, to determine whether a test antibody binds to the same epitope as a reference anti-V51 antibody of the present invention, 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 is likely to bind to the same epitope as the reference anti-V51 antibody of the present invention.

[0176] The present invention also encompasses anti-V51 antibodies that compete for binding to V51 with antibodies or fragments thereof defined herein, or with antibodies having the CDR sequences of any of the exemplary antibodies described herein. For example, competition assays can be performed using the antibodies of the present invention to determine which proteins, antibodies, and other antagonists compete with the antibodies of the present invention for binding to V51 and / or share epitopes. These assays are readily known to those skilled in the art and assess competition between antagonists or ligands for a limited number of binding sites on a protein (e.g., V51). The antibody (or fragment) is immobilized or insolubilized before or after competition, and the sample bound to the V51 chain is separated from the unbound sample, for example, by decantation (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 its function is altered by the binding or lack of binding of the antibody to the protein, for example, whether the antibody molecule inhibits or enhances, for example, the enzymatic activity of a label. ELISA and other functional assays may be used as known in the art and described herein.

[0177] Two antibodies bind to the same or overlapping epitopes 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.

[0178] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed loss 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 loss 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.

[0179] In some embodiments, the antibody or fragment thereof contains an altered effector function through a modification to the sugar linked to Asn 297 (EU numbering scheme). In a further modification described above, Asn 297 indicates no or reduced fucosylation (i.e., a defucosylated or non-fucosylated antibody). Fucosylation involves 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. An antibody can be modified to prevent or inhibit antibody fucosylation. Typically, glycosylation modification involves expressing the antibody or fragment thereof in a host cell with alternative glycosylation processing capabilities, either through targeted engineering or through targeted or serendipitous host or clonal selection (see, e.g., Example 13). These and other effector modifications are further described in recent reviews, for example, by Xinhua Wang et al. (2018) Protein & Cell 9:63-73 and by Pereira et al. (2018) mAbs 10(5):693-711, which are incorporated herein.

[0180] 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 ordinary skill in the art. The following examples are non-limiting.

[0181] During antibody discovery and sequence recovery from phage libraries, desired antibody variable domains can be reformatted into full-length IgGs by subcloning. To accelerate this process, variable domains are often transferred using restriction enzymes. These unique restriction sites may introduce additional / alternative amino acids, deviating from the canonical sequence (such canonical sequences may be found, for example, in the international ImMunoGeneTics [IMGT] information system; see http: / / www.imgt.org). These may be introduced as kappa or lambda light chain sequence modifications.

[0182] Kappa light chain modification The variable kappa light chain variable sequence can be cloned using restriction sites (e.g., Nhe1-Not1) during reformatting to full-length IgG. 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 during further development, such as to generate the canonical 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 included herein that contain this sequence (e.g., SEQ ID NOs: 86, 88-90, and 92-97).

[0183] Additional amino acid changes may 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 resulted in an RT A The constant domain starting with AAPS is obtained. Preferably, this sequence is VThe canonical kappa light chain constant region can be further modified during development to generate a canonical kappa light chain constant region starting with AAPS. Thus, in one embodiment, the kappa light chain containing antibody described herein comprises a constant domain described using the sequence RTV. Thus, in one embodiment, the sequence RTV of SEQ ID NOs: 111-114 and 117-122 can be used. A AAPS is sequence RT V Replaces AAPS.

[0184] Lambda light chain modification Similar to the kappa example above, lambda light chain variable domains may also be cloned by introducing restriction sites (e.g., Nhe1-Not1) during reformatting to full-length IgG. More specifically, an additional Ala-Ser sequence may be introduced at the lambda light chain N-terminus to aid cloning. Preferably, this additional AS sequence is then removed during further development, such as to generate the canonical 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 included 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 six residues; i.e., the ASSYEL sequence is removed.

[0185] 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 a GQP (from the NotI restriction site) A A constant domain beginning with AAPS is obtained. Preferably, this sequence is GQP KTo generate a canonical lambda light chain constant region starting from AAPS, it can be modified during further development.Thus, in one embodiment, the lambda light chain containing antibody described herein comprises a constant domain starting from the sequence GQPK.Thus, in one embodiment, the sequence GQP of SEQ ID NO: 115 or 116. A AAPS is the sequence GQP K Replaces AAPS.

[0186] Heavy chain modifications Typically, human variable heavy chain sequences begin with either basic glutamine (Q) or acidic glutamate (E). However, both of these sequences are known to subsequently be converted to pyroglutamate (pE), an acidic amino acid residue. The Q-to-pE conversion alters the charge on the antibody, while the E-to-pE conversion does not. Therefore, to avoid variable charge changes over time, one option is to modify the starting heavy chain sequence from Q to E in the first instance. Thus, in one embodiment, the heavy chain of an antibody described herein contains a Q to E modification at the N-terminus. In particular, the initial residues of SEQ ID NOs: 62, 64, and / or 67-71 may 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).

[0187] 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 results in a change in the charge on the antibody through the variable loss of the C-terminal lysine residue. Therefore, one option is to remove the lysine in the first instance to obtain 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.

[0188] Optional allotypic modification During antibody discovery, specific human allotypes can be used. Optionally, antibodies can be switched to different human allotypes during development. As a non-limiting example, for kappa chains, there are three human allotypes called Km1, Km1,2, and Km3, which define three Km alleles (using allotype numbering). Km1 correlates with valine 153 (IMGT V45.1) and leucine 191 (IMGT L101), Km1,2 correlates with alanine 153 (IMGT A45.1) and leucine 191 (IMGT L101), and Km3 correlates with alanine 153 (IMGT A45.1) and valine 191 (IMGT V101). Therefore, optionally, sequences can be altered from one allotype to another by standard cloning approaches. For example, the L191V (IMGT L101V) change converts the Km1,2 allotype to the Km3 allotype. For further reference to such allotypes, see Jefferis and Lefranc (2009) MAbs 1(4):332-8, which is incorporated herein by reference.

[0189] 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.

[0190] antibody binding The antibody or fragment thereof of the present invention has a solubility of 1.5×10 -7 In a preferred embodiment, the KD is less than 1.5 x 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 yet a further embodiment, 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 less than 2.0 × 10 -8 M (i.e., 20 nM). For example, according to one embodiment, the 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).

[0191] In one embodiment of the invention, 4.0×10 -8 M (i.e., less than 40 nM), 3.0 × 10 -8 M (i.e., 30 nM) or less than 2.0 × 10 -8 Antibodies or fragments thereof that bind to the Vδ1 chain of a γδ TCR with a binding affinity (KD) of less than M (i.e., 20 nM) are provided.

[0192] In one embodiment, the binding affinity of an antibody or fragment thereof is established by coating the antibody or fragment thereof directly or indirectly (e.g., by capturing with anti-human IgG Fc) onto a sensor surface (e.g., an amine high-capacity chip or equivalent), and the target bound by the antibody or fragment (i.e., the V51 chain of the γδ TCR) is flowed over the chip and binding detected. Suitably, a MASS-2 instrument (which may also be referred to as a Sierra SPR-32) is used at 25°C and 30 μL / min in PBS + 0.02% Tween 20 running buffer.

[0193] Other assays that can be used to define antibody function are described herein. For example, an antibody or fragment thereof described herein can be evaluated by measuring γδ TCR engagement, e.g., downregulation of γδ TCR upon antibody binding. Surface expression of γδ TCR (optionally displayed on the surface of cells) after application of the antibody or fragment thereof can be measured, for example, by flow cytometry. An antibody or fragment thereof described herein can also be evaluated by measuring γδ T cell degranulation. For example, expression of CD107a, a marker for cell degranulation, can be measured, for example, by flow cytometry, after application of the antibody or fragment thereof (optionally displayed on the surface of cells) to γδ T cells. An antibody or fragment thereof described herein can also be evaluated 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 cells). After incubation, the culture can be stained with a cell viability dye to distinguish between live and dead target cells. The proportion of dead cells can then be determined, for example, by flow cytometry.

[0194] As described herein, the antibody or fragment thereof used in the assay can be displayed on a surface, for example, the surface of a cell, such as a cell containing an Fc receptor. For example, the antibody or fragment thereof can be displayed on the surface of a THP-1 cell, such as a TIB-202™ cell (available from the American Type Culture Collection (ATCC)). Alternatively, the antibody or fragment thereof can be used directly in the assay.

[0195] In such functional assays, output can be measured by calculating the half maximal effective concentration, also referred to as "EC50" or "50% effective concentration." The term "IC50" refers to inhibitory concentration. Both EC50 and IC50 can be measured using methods known in the art, such as 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

[0196] The EC50 for down-regulation of γδ TCR upon antibody (or fragment) binding may be less than 0.50 μg / ml, for example, less than 0.40 μg / ml, less than 0.30 μg / ml, less than 0.20 μg / ml, less than 0.15 μg / ml, less than 0.10 μg / ml, less than 0.06 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, less than 0.05 μg / ml, less than 0.04 μg / ml, or less than 0.03 μg / ml. In particular, the above-mentioned EC50 values ​​are when the antibody is measured in IgG1 format. For example, EC50 γδ TCR down-modulation values ​​can be measured using flow cytometry (eg, as described in the assay in Example 6).

[0197] 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, less than 0.030 μg / ml, less than 0.020 μg / ml, less than 0.015 μg / ml, less than 0.010 μg / ml, or less than 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 above-mentioned EC50 values ​​are when the antibody is measured in IgG1 format. For example, γδ T cell degranulation EC50 values ​​can be measured by detecting CD107a expression (i.e., a marker of cell degranulation) using flow cytometry (e.g., as described in the assay in Example 7). In one embodiment, CD107a expression is measured using an anti-CD107a antibody, such as anti-human CD107a BV421 (clone H4A3) (BD Biosciences).

[0198] 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, less than 0.30 μg / ml, less than 0.20 μg / ml, less than 0.15 μg / ml, less than 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 above-mentioned EC50 values ​​are those when the antibody is measured in an IgG1 format. For example, an EC50 γδ T cell killing value can be measured by detecting the percentage of dead cells (e.g., using a cell viability dye) using flow cytometry after incubation of the antibody, γδ T cells, and target cells (e.g., as described in the assay in Example 8). In one embodiment, target cell death is measured using a cell viability dye, where the viability dye is Viability Dye eFluor™ 520 (ThermoFisher).

[0199] 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 such as CellTracker™ Orange CMTMR (ThermoFisher).

[0200] Immunoconjugates The antibodies or fragments thereof of the present invention may be conjugated to a therapeutic moiety such as a cytotoxin or a chemotherapeutic agent. Such conjugates may also be referred to as immunoconjugates. As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically linked to another moiety, such as a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide or protein, or therapeutic agent. The antibody may be linked to the cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide, or therapeutic agent at any position along the molecule, as long as it is capable of binding to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second, different antibody against V51. In certain embodiments, the antibody may be conjugated to an agent specific for tumor cells or virus-infected cells. The type of therapeutic moiety that may be conjugated to an anti-V51 antibody takes into account the condition to be treated and the desired therapeutic effect to be achieved. In one embodiment, the agent may be a second antibody, or a fragment thereof, that binds to a molecule other than V51.

[0201] multispecific antibodies Antibodies of the present invention may be monospecific, or they may bind to additional targets and thus be bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide, or may be specific for more than one target polypeptide. Thus, in one embodiment, the antibody or fragment thereof comprises a first binding specificity for V51 and a second binding specificity for a second target epitope.

[0202] The second binding specificity may target an antigen on the same cell as V51, or on a different cell of the same tissue type, or on a different cell of a different tissue type. In certain embodiments, the target epitopes may be on different cells, including different T cells, B cells, tumor cells, autoimmune tissue cells, or virus-infected cells. Alternatively, the target epitopes may be on the same cell.

[0203] Polynucleotides and Expression Vectors In one aspect of the present invention, there is provided a polynucleotide encoding an anti-Vδ1 antibody or fragment of the present invention. In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NOs: 99-110. In one embodiment, an expression vector comprises a VH region of SEQ ID NOs: 99-110. In another embodiment, an expression vector comprises 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, there is provided a cDNA comprising the above-described polynucleotide.

[0204] In one aspect of the present invention, a polynucleotide encoding an anti-Vδ1 antibody or fragment of the present invention is provided. In one embodiment, the polynucleotide comprises or consists of a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to SEQ ID NOs: 99-101 or 105-108. In one embodiment, an expression vector comprises a VH region of SEQ ID NOs: 99-101 or 105-108. In another embodiment, an expression vector comprises a VL region of SEQ ID NOs: 99-101 or 105-108. In a further embodiment, the polynucleotide comprises or consists of SEQ ID NOs: 99-101 or 105-108. In a further aspect, a cDNA comprising the above-described polynucleotide is provided.

[0205] 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 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, there is provided a cDNA comprising the above-mentioned polynucleotide.

[0206] In one aspect of the invention, there is provided a polynucleotide comprising or consisting of a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to any one of a portion of SEQ ID NOs: 99-110 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%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to any one of a portion of SEQ ID NOs: 99-101 or 105-108 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.

[0207] In one aspect of the invention, there is provided a polynucleotide comprising or consisting of a sequence having at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to any one of a portion of SEQ ID NOs: 99-110 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%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99% sequence identity to any one of a portion of SEQ ID NOs: 99-101 or 105-108 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.

[0208] Polynucleotides and expression vectors of the invention may also be described with reference to the encoded amino acid sequence. Thus, in one embodiment, a polynucleotide comprises or consists of a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 62-85. In one embodiment, an expression vector comprises a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 62-73. In another embodiment, an expression vector comprises a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 74-85.

[0209] 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. Accordingly, one aspect of the present invention provides an expression vector 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.

[0210] It is understood that the nucleotide sequences described herein include additional sequences encoding amino acid residues to aid in translation, purification, and detection; however, 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, the last (3'-end) 36 nucleotides of SEQ ID NOs: 99-100, 102-103, and 105-110, or the last (3'-end) 39 nucleotides of SEQ ID NOs: 101 and 104 are optional sequences. These optional sequences can be removed, modified, or substituted if alternative design, translation, purification, or detection strategies are employed.

[0211] 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.

[0212] 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, radiolytic decay mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, or a combination thereof.

[0213] 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 attached 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 collected. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity.

[0214] 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 may include promoters, enhancers, transcription terminators, a start 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 include nucleotide sequences encoding single-chain variable fragments of the invention, comprising VH and VL regions connected by a synthetic linker (encoding SEQ ID NO: 98). It will be understood that the polynucleotides or expression vectors of the invention may 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 alternatively, 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).

[0215] 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. Expression vectors can also encode a signal peptide that facilitates secretion of the antibody (or fragment thereof) from the host cell. A 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).

[0216] In one aspect of the present invention, a cell (e.g., a host cell) is provided that contains a polynucleotide or expression vector as defined herein.It will be understood that the cell may contain a first vector encoding the light chain of an antibody or its fragment, and a second vector encoding the heavy chain of an antibody or its fragment.Alternatively, both the heavy chain and the light chain are encoded on the same expression vector introduced into the cell.

[0217] In one embodiment, the polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to an antibody or fragment thereof, such that the antibody or fragment thereof is displayed on the extracellular surface of the cell.

[0218] Transformation can be by any known method for introducing polynucleotide into host cell.The method for introducing heterologous polynucleotide into mammalian cell is well known in the art, and includes dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide in liposome, biolistic injection, and direct microinjection of DNA into nucleus.In addition, nucleic acid molecule can be introduced into mammalian cell by virus vector.

[0219] 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 cell lines 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., HepG2), A549 cells, 3T3 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. A particular preferred cell line is selected by determining which cell line has a high expression level. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Antigen-binding fragments of antibodies, such as ScFv and Fv fragments, can be isolated and expressed in E. coli using methods known in the art.

[0220] 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 medium that the host cells are grown in. The antibody can be recovered from the culture medium using standard protein purification methods.

[0221] 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 Edition, Cold Spring Harbour Laboratory Press.

[0222] Monoclonal antibodies can be produced using hybridoma technology by fusing specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for the ability to grow in tissue culture and the absence of antibody chain synthesis.

[0223] Monoclonal antibodies directed against the determined antigens can be prepared, for example, by Antibodies can be obtained by: a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a determined antigen with immortal cells, preferably myeloma cells, to form hybridomas; b) culturing the formed immortalized cells (hybridomas) and recovering the cells that produce antibodies with the desired specificity.

[0224] Alternatively, the use of hybridoma cells is not necessary. Antibodies capable of binding to the target antigens described herein can be isolated from suitable antibody libraries through conventional practices, for example, using 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 a phage, more particularly a filamentous bacteriophage, DNA or cDNA sequence obtained from lymphocytes, in particular peripheral blood lymphocytes, of an animal (preferably previously immunized with the determined antigen); b) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the antibody; c) selecting antibodies by performing antigen affinity selection; and d) recovering antibodies having the desired specificity.

[0225] Pharmaceutical Compositions According to a further aspect of the present invention, there is provided a composition comprising an antibody or fragment thereof as defined herein. In such embodiments, the composition may comprise the antibody, optionally 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).

[0226] According to a further aspect of the present invention, a pharmaceutical composition is provided comprising an antibody or fragment thereof defined herein together with a pharmaceutically acceptable diluent or carrier. The antibody of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, the pharmaceutical composition comprises the antibody of the present invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents. Examples of pharmaceutically acceptable carriers include one or more of water, saline, salt, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferable to include an isotonicity agent, such as a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride, in the composition. Pharmaceutically acceptable substances, such as wetting or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, that enhance the shelf life or effectiveness of the antibody or fragment thereof, are also included.

[0227] The compositions of the present invention can be in various forms.These compositions include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.Preferred forms depend on intended administration modes and therapeutic applications.Typical preferred compositions are in the form of injectable and infusible solutions.

[0228] The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intrathecal). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

[0229] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.

[0230] It is within the scope of the present invention to use the pharmaceutical compositions of the present invention in therapeutic methods for the treatment of the diseases described herein as an adjunct to, or in conjunction with, other established therapies commonly used to treat such diseases.

[0231] In a further embodiment of the invention, the antibody, composition or pharmaceutical composition is administered sequentially, simultaneously or separately with at least one active agent.

[0232] Uses of antibodies or fragments thereof According to a further aspect of the present invention, there is provided a use of an anti-V51 antibody or fragment thereof as 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 proliferation of γδ T cells and therefore may be used in methods of expanding γδ T cells (e.g., V51 T cells).

[0233] Antibodies that bind to the V51 chain can be used to detect γδ T cells. For example, the antibodies can be labeled with a detectable label or reporter molecule, or can be used as a capture ligand to selectively detect and / or isolate V51 T cells in a sample. Labeled antibodies find use in many methods known in the art, such as immunohistochemistry and ELISA.

[0234] The detectable label or reporter molecule may be 3 H, 14 C. 32 P, 35 S or 125The fluorescent label may be a radioisotope such as I, 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) methods.

[0235] Methods for producing antibodies or fragments thereof Described herein are soluble TCRs for use in generating antibodies. Thus, according to one aspect of the invention, there is provided an isolated antigen comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 123 for use in generating an anti-V51 antibody or fragment thereof. Also provided is the use of an isolated antigen comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 123 for use in generating an anti-V51 antibody or fragment thereof. In one embodiment, the isolated antigen comprises SEQ ID NO: 123.

[0236] According to one aspect of the invention there is provided a method for producing an anti-V51 antibody or fragment thereof, comprising the steps of: (i) designing a series of antigens comprising a TCR delta variable 1 (V51) amino acid sequence, wherein the CDR3 sequence of V51 is the same for all antigens in the series; (ii) exposing the first antigen designed in step (i) to an antibody library (e.g., by phage display); (iii) isolating an antibody or fragment thereof that binds to the antigen; and (iv) exposing the isolated antibody or fragment thereof to the second antigen designed in step (i); (v) isolating an antibody or fragment thereof that binds to both the first and second antigens.

[0237] Isolating an antibody (or fragment thereof) that binds to both the first and second antigens aims to provide an antibody that recognizes sequences within the variable domain that are germline-encoded and therefore identical in all clones, thereby providing an antibody that recognizes a broader subset of γδ T cells. The set of antigens described herein may also include antigens in different formats (i.e., TCR delta variable 1 chains). Thus, the antigens described above may be synthetic / recombinant antigens. For example, the antigens may be presented as leucine zippers or Fc fusions. In one embodiment, the TCR delta variable 1 (TRDV1) amino acid sequence comprises SEQ ID NO: 123. Suitable antigen sequences may be designed using, for example, sequences listed in the IMMUNOGENETICS Information System database (http: / / www.imgt.org).

[0238] Antigens may also include additional features to aid in protein expression. For example, the recombinant TCR antigens described herein may be fused to the TCR alpha or TCR beta constant regions (see Xu et al. (2011) PNAS 108:2414-2419).

[0239] In one embodiment, the method further comprises exposing the isolated antibody or fragment thereof to a second set of antigens comprising a γδ TCR with a different delta variable chain, for example delta variable 2 (Vδ2) or delta variable 3 (Vδ3), and then deselecting antibodies or fragments thereof that also bind to the second set of antigens.

[0240] In a further embodiment, the second set of antigens comprising a γδ TCR with a different delta variable chain comprises the same CDR3 sequence as the first set of antigens, and therefore all antigens comprise the same CDR3 sequence (from Vδ1).

[0241] In one embodiment, the first and / or second series of antigens are presented as leucine zipper and / or Fc fusions.

[0242] In one embodiment, the set of antigens is in heterodimeric and / or homodimeric format.

[0243] In further embodiments, the series of antigens comprises a pair of TCR variable chains together with a target (i.e., a TCR delta variable 1 chain). In a particular embodiment, the paired TCR variable chain is a variable gamma (Vγ) chain (i.e., the antigen is in a heterodimeric format). In one embodiment, the Vδ1 chain and the Vγ chain are covalently linked by at least one disulfide bond. In a further embodiment, the Vδ1 chain and the Vγ chain are paired by a specific heterodimerization interaction (e.g., a leucine zipper). In an alternative embodiment, the Vδ1 chain and the Vγ chain comprise a single-chain in-frame fusion. In a particular embodiment, the Vδ1 chain is N-terminal to the Vγ chain. In an alternative embodiment, the Vδ1 chain is C-terminal to the Vγ chain. In a further embodiment, the single-chain in-frame fusion comprises an internal linker sequence. In one embodiment, the antigen in the first series is in a heterodimeric format comprising a Vδ1 chain and a different Vγ chain, such as Vγ2, Vγ4, or Vγ8. Selecting an antibody that binds to all formats ensures that the above-mentioned isolated antibody or fragment thereof recognizes the Vδ1 chain independently of the partner chain present in the heterodimer.

[0244] In an alternative embodiment, the paired TCR variable chain is another Vδ chain. In a further embodiment, the Vδ chain is the same as the target (i.e., the antigen is in a homodimeric format).

[0245] Example 2 provided herein describes examples of sets of antigens that may be used. It will be understood that the (first) set of antigens includes antigens in which TRDV1 (V51) is present (e.g., L1, L2, L3, F1, F2, F3 and Fc1 / 1), and the second set of antigens includes antigens in which V51 is absent (e.g., L4, F9, Fc3 / 3, Fc4 / 4, Fc8 / 8).

[0246] In a further embodiment, the set of antigens comprises a target fused in-frame to a TCR constant region (i.e., a TCR delta variable 1 chain). For example, the TCR constant region described above may be fused in-frame to the C-terminus of the V51 chain. In one embodiment, the TCR constant region may be a human TCR constant region. In one embodiment, the TCR constant region is selected from a TCR alpha or a TCR beta constant region. In another embodiment, the constant region is a TCR gamma constant region. In yet another embodiment, the set of antigens may comprise an additional, second TCR constant region, which is fused in-frame to the paired TCR variable chain. In a further embodiment, the second TCR constant region is selected from a TCR alpha or a TCR beta constant region. In a further embodiment, the constant region is a TCR gamma constant region.

[0247] It will be understood that the set of antigens described herein can be presented in either soluble or linked / fused form, or can be associated with a cell membrane. For example, for presentation purposes, the set of antigens can be fused or tethered to inorganic or organic materials (e.g., beads, plates, columns, or phage), or expressed on the cell surface.

[0248] According to various embodiments of the invention, a set of antigens comprising a TCR delta variable 1 (V51) amino acid sequence comprises a CDR3 sequence of V51 that is the same for all antigens. In one embodiment, the CDR3 sequence is derived from the CDR3 sequence of RCSB Protein Data Bank entry: 3OMZ.

[0249] According to a further aspect of the present invention there is provided an antibody obtainable by the method defined herein.

[0250] section A series of clauses defining the invention and its preferred embodiments are set forth below.

[0251] Section 1. An isolated anti-Vδ1 antibody or fragment thereof, CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 25; CDR2 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 and A1 to A12 (in Table 2), and / or An isolated anti-Vδ1 antibody or a fragment thereof, comprising one or more CDR1s comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61.

[0252] Clause 2. An isolated anti-Vδ1 antibody or fragment thereof as defined in clause 1, comprising a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2-13, such as SEQ ID NOs: 2, 3, or 4.

[0253] Clause 3. An isolated anti-Vδ1 antibody or fragment thereof as defined in clause 1 or clause 2, comprising a VH region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 26-37, such as SEQ ID NOs: 26, 27, or 28.

[0254] Clause 4. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1-3, comprising a VH region comprising a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 38-49, such as SEQ ID NOs: 38, 39, or 40.

[0255] Clause 5. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 4, comprising a VH region comprising: 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.

[0256] Clause 6. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 4, comprising a VH region comprising 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.

[0257] Clause 7. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 4, comprising a VH region comprising: 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.

[0258] Clause 8. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 4, comprising a VH region comprising: 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.

[0259] Clause 9. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1-8, comprising a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 14-25, such as SEQ ID NOs: 14, 15, or 16.

[0260] Clause 10. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1-9, comprising a VL region comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of sequences A1-A12, e.g., SEQ ID NO: A1, A2, or A3.

[0261] Clause 11. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1-10, comprising a VL region comprising a CDR1 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 50-61, such as SEQ ID NOs: 50, 51, or 52.

[0262] Clause 12. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 11, comprising a VL region comprising 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.

[0263] Clause 13. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 11, comprising a VL region comprising: 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.

[0264] Clause 14. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 11, comprising a VL region comprising 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.

[0265] Clause 15. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 11, comprising a VL region comprising 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.

[0266] Clause 16. An isolated anti-Vδ1 antibody or fragment thereof, comprising a VH region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 5, and a VL region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 12.

[0267] Clause 17. An isolated anti-Vδ1 antibody or fragment thereof, comprising a VH region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 6, and a VL region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 13.

[0268] Clause 18. An isolated anti-Vδ1 antibody or fragment thereof, comprising a VH region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 7, and a VL region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 14.

[0269] Clause 19. An isolated anti-Vδ1 antibody or fragment thereof, comprising a VH region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 8, and a VL region comprising the CDR1, CDR2 and CDR3 sequences defined in clause 15.

[0270] Section 20. An isolated anti-Vδ1 antibody or fragment thereof comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62-85.

[0271] Clause 21. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 20, comprising a VH region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62-73.

[0272] Clause 22. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 21, wherein the VH region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 62, 63, or 64.

[0273] Clause 23. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 20-22, comprising a VL region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74-85.

[0274] Clause 24. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 23, wherein the VL region comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74, 75, or 76.

[0275] Clause 25. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0276] Clause 26. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0277] Clause 27. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0278] Clause 28. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0279] Clause 29. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0280] Clause 30. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0281] Clause 31. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 24, comprising 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.

[0282] Clause 32. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 20 to 31, wherein the VH region and the VL region are linked by a linker, such as a polypeptide linker.

[0283] Item 33. The linker is (Gly4Ser) n 33. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 32, comprising the format, wherein n=1 to 8.

[0284] Clause 34. The linker is [(Gly4Ser) n (Gly3AlaSer) m ] p 34. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 32 or clause 33, comprising a linker, and wherein n, m and p=1 to 8.

[0285] Clause 35. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 32 to 34, wherein the linker comprises SEQ ID NO: 98.

[0286] Clause 36. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 35, wherein the linker consists of SEQ ID NO: 98.

[0287] Section 37. An isolated anti-Vδ1 antibody or fragment thereof comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 86-97.

[0288] Clause 38. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 37, comprising the amino acid sequence of any one of SEQ ID NOs: 86-97.

[0289] Clause 39. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 37 or clause 38, comprising SEQ ID NO: 86.

[0290] Clause 40. The isolated anti-Vδ1 antibody or fragment thereof as defined in clause 37 or clause 38, comprising SEQ ID NO: 87.

[0291] Clause 41. The isolated anti-Vδ1 antibody or fragment thereof as defined in clause 37 or clause 38, comprising SEQ ID NO: 88.

[0292] Clause 42. The isolated anti-Vδ1 antibody or fragment thereof as defined in clause 37 or clause 38, comprising SEQ ID NO: 92.

[0293] Clause 43. The isolated anti-Vδ1 antibody or fragment thereof as defined in clause 37 or clause 38, comprising SEQ ID NO: 93.

[0294] Clause 44. The isolated anti-Vδ1 antibody or fragment thereof as defined in clause 37 or clause 38, comprising SEQ ID NO: 94.

[0295] Clause 45. The isolated anti-Vδ1 antibody or fragment thereof as defined in clause 37 or clause 38, comprising SEQ ID NO: 95.

[0296] Clause 46. An isolated anti-Vδ1 antibody or fragment thereof that binds to or competes with the same or essentially the same epitope as an antibody or fragment thereof defined in any one of clauses 1 to 45.

[0297] 47. A human isolated anti-TCR delta variable 1 (anti-Vδ1) antibody or fragment thereof, (i) sequences 3 to 20 of SEQ ID NO: 1, and / or (ii) An isolated human anti-Vδ1 antibody or fragment thereof that binds to an epitope of the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) that includes one or more amino acid residues within the amino acid region of 37 to 77 of SEQ ID NO: 1.

[0298] Clause 48. The human isolated anti-Vδ1 antibody or fragment thereof defined in clause 47, wherein 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.

[0299] Clause 49. The human isolated anti-Vδ1 antibody or fragment thereof defined in clause 47 or clause 48, wherein the epitope comprises one or more amino acid residues within amino acid regions 5-20 and 62-77, 50-64, 37-53 and 59-72, 59-77, or 3-17 and 62-69 of SEQ ID NO:1.

[0300] Clause 50. The human isolated anti-Vδ1 antibody or fragment thereof defined in clause 49, wherein the epitope consists of one or more amino acid residues within amino acid regions 5-20 and 62-77, 50-64, 37-53 and 59-72, 59-77, or 3-17 and 62-69 of SEQ ID NO:1.

[0301] Clause 51. The human isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 47-50, wherein the epitope comprises one or more amino acid residues within amino acid regions 5-20 and 62-77 of SEQ ID NO:1.

[0302] Clause 52. The human isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 47-50, wherein the epitope comprises one or more amino acid residues within amino acid region 50-64 of SEQ ID NO:1.

[0303] Clause 53. The human isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 47-50, wherein the epitope comprises one or more amino acid residues within amino acid regions 37-53 and 59-77 of SEQ ID NO:1.

[0304] Clause 54. The human isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 46 to 53, wherein the epitope is an activating epitope of a gamma delta T cell.

[0305] Clause 55. An isolated human anti-Vδ1 antibody or fragment thereof as defined in clause 54, wherein binding of the activating epitope (i) downregulates a γδ TCR, (ii) activates γδ T cell degranulation, and / or (iii) activates γδ T cell killing.

[0306] Clause 56. An isolated human anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 47 to 55, wherein the antibody or fragment binds only to an epitope in the V region of the Vδ1 chain of a γδ TCR.

[0307] Clause 57. An isolated human anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 47 to 56, which does not bind to an epitope found in the CDR3 of the Vδ1 chain of a γδ TCR.

[0308] Clause 58. An isolated human anti-Vδ1 antibody or fragment thereof as defined in clause 57, which does not bind to an epitope within amino acid region 91-105 (CDR3) of SEQ ID NO:1.

[0309] Section 59. 1.5 × 10 as measured by surface plasmon resonance -7 10. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1-58, which binds to the variable delta 1 (Vδ1) chain of a gamma delta T cell receptor (TCR) with a binding affinity (KD) of less than M.

[0310] Section 60.KD is 1.3 x 10 -7 Less than M or smaller, e.g., 1.0 × 10 -7 Less than M, especially 5.0 × 10 -859. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 59, wherein the antibody or fragment thereof is less than M.

[0311] Item 61. An isolated anti-Vδ1 antibody or fragment thereof, having an EC50 value for down-regulation of γδ TCR upon binding of less than 0.5 μg / ml.

[0312] Clause 62. An isolated anti-Vδ1 antibody or fragment thereof, having an EC50 value for down-regulation of γδ TCR upon binding of less than 0.06 μg / ml.

[0313] Item 63. An isolated anti-Vδ1 antibody or fragment thereof, having an EC50 value for γδ T cell degranulation upon binding of less than 0.05 μg / ml.

[0314] Clause 64. An isolated anti-Vδ1 antibody or fragment thereof, which has an EC50 value for γδ T cell degranulation upon binding of less than 0.005 μg / ml, such as less than 0.002 μg / ml.

[0315] Clause 65. The isolated anti-Vδ1 antibody or fragment thereof defined in clause 63 or clause 64, wherein the gamma delta T cell degranulation EC50 value is measured by detecting CD107a expression.

[0316] Item 66. An isolated anti-Vδ1 antibody or fragment thereof, which has an EC50 value for γδ T cell killing upon binding of less than 0.5 μg / ml.

[0317] Clause 67. An isolated anti-Vδ1 antibody or fragment thereof, which has an EC50 value for γδ T cell killing upon binding of less than 0.055 μg / ml, such as less than 0.020 μg / ml.

[0318] Clause 68. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 61 to 67, wherein the EC50 value is measured using flow cytometry.

[0319] Clause 69. An isolated anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 68, which is an scFv, Fab, Fab', F(ab')2, Fv, variable domain (e.g., VH or VL), diabody, minibody, or full-length antibody.

[0320] Clause 70. An isolated anti-Vδ1 antibody or fragment thereof as defined in clause 69, which is an scFv, or a full length antibody, e.g., an IgG1.

[0321] Clause 71. The isolated anti-Vδ1 antibody defined in clause 70, comprising an amino acid sequence having at least 80% sequence identity to that of any one of SEQ ID NOs: 111-122.

[0322] Clause 72. The isolated anti-Vδ1 antibody defined in clause 70 or clause 71, comprising the amino acid sequence of any one of SEQ ID NOs: 111-122.

[0323] Clause 73. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 111.

[0324] Clause 74. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 112.

[0325] Clause 75. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 116.

[0326] Clause 76. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 117.

[0327] Clause 77. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 118.

[0328] Clause 78. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 119.

[0329] Clause 79. The isolated anti-Vδ1 antibody defined in clause 71 or clause 72, comprising SEQ ID NO: 120.

[0330] Clause 80. The isolated anti-Vδ1 antibody or fragment thereof defined in any one of clauses 1 to 79, which is human.

[0331] Clause 81. A polynucleotide sequence encoding an anti-Vδ1 antibody or fragment thereof as defined in any one of clauses 1 to 80.

[0332] Section 82. A polynucleotide sequence encoding an anti-Vδ1 antibody or a fragment thereof comprising a sequence having at least 70% sequence identity to SEQ ID NOs: 99-110.

[0333] Item 83. Polynucleotide sequence encoding an anti-Vδ1 antibody or a fragment thereof consisting of the sequence of SEQ ID NO: 99-110.

[0334] Clause 84. An expression vector comprising a polynucleotide sequence as defined in any one of clauses 81-83.

[0335] Item 85. An expression vector comprising a VH region of SEQ ID NO: 99-110.

[0336] Item 86. An expression vector comprising a VL region of SEQ ID NO: 99-110.

[0337] Clause 87. An expression vector comprising the VH region of clause 85 and the VL region of clause 86.

[0338] Clause 88. A cell comprising a polynucleotide sequence as defined in any one of clauses 81-83 or an expression vector as defined in any one of clauses 84-87.

[0339] Clause 89. A cell comprising a first expression vector defined in clause 85 and a second expression vector defined in clause 86.

[0340] Clause 90. A cell comprising an expression vector as defined in clause 87.

[0341] Clause 91. The cell defined in any one of clauses 88-90, wherein the polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to the antibody or fragment thereof, and wherein the antibody or fragment thereof is displayed on the extracellular surface of the cell.

[0342] Clause 92. A composition comprising an antibody or fragment thereof as defined in any one of clauses 1 to 80.

[0343] Clause 93. A pharmaceutical composition comprising an antibody or fragment thereof as defined in any one of clauses 1 to 80 together with a pharmaceutically acceptable diluent or carrier.

[0344] Item 94. An isolated antigen comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 123 for use in generating an anti-Vδ1 antibody or fragment thereof.

[0345] Item 95. A method for producing an anti-Vδ1 antibody or fragment thereof, comprising: (i) designing a series of antigens comprising a TCR delta variable 1 (TRDV1) amino acid sequence, wherein the CDR3 sequence of TRDV1 is the same for all antigens in the series; (ii) exposing the first antigen designed in step (i) to an antibody library; and (iii) isolating antibodies or fragments thereof that bind to the antigen. (iv) exposing the isolated antibody or fragment thereof to the second antigen designed in step (i); (v) isolating an antibody or fragment thereof that binds to both the first and second antigens.

[0346] Clause 96. The method defined in clause 95, further comprising exposing the isolated antibodies or fragments thereof to a second set of antigens comprising a gamma delta TCR with a different delta variable chain, for example, TCR delta variable 2 (TRDV2) or TCR delta variable 3 (TRDV3), and then deselecting antibodies or fragments thereof that also bind to the second set of antigens.

[0347] Clause 97. The method defined in clause 95 or clause 96, wherein the first and / or second set of antigens are presented as leucine zipper and / or Fc fusions.

[0348] Clause 98. The method defined in any one of clauses 95 to 97, wherein the set of antigens is in heterodimeric and / or homodimeric format.

[0349] Clause 99. An antibody obtained by the method defined in any one of clauses 95 to 98.

[0350] Other features and advantages of the present invention will be apparent from the description provided herein. However, since various changes and modifications will become 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 using the following non-limiting examples. [Example]

[0351] 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 by Schofield et al. (Genome biology 2007, 8(11):R254) and contained single-chain fragment variable (scFv) antibodies representing a library of approximately 40 billion human clones. The library was screened using the antigens, methods, selection, deselection, screening, and characterization strategies described herein.

[0352] Antigen preparation The design of the soluble yδ TCR heterodimers containing TCRα and TCRβ constant regions used in the following examples was generated according to Xu et al. (2011) PNAS 108:2414-2419. The Vγ or Vδ domain was fused in frame to a TCRα or TCRβ constant region lacking the transmembrane domain, followed by a leucine zipper sequence or Fc sequence, and a histidine tag / linker.

[0353] Expression constructs were transiently transfected (either as single or cotransfections of heterodimers) into mammalian EXPI HEK293 suspension cells. Secreted recombinant proteins were collected and purified from culture supernatants by affinity chromatography. To ensure good recovery of 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.

[0354] Verification of antigen function The specificity of antigens containing the delta variable 1 (Vδ1) chain was confirmed in a flow-based assay in competition with γδ T cells using the DELFIA immunoassay (Perkin Elmer) and REA173-Miltenyi Biotec anti-Vδ1 antibody.

[0355] Dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) To confirm antigen specificity, a DELFIA immunoassay (Nunc #437111) was performed with antigen directly coated onto the plate (3 μg / mL antigen in 50 μL PBS overnight at 4°C) and serial dilutions of the primary antibody starting at 300 nM. For detection, 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) nonfat milk powder). Development was performed with 50 μL of DELFIA enhancement solution (Perkin Elmer #4001-0010).

[0356] Affinity ranking of the antibodies of interest was performed using the DELFIA immunoassay, in which antibodies were captured via protein G coated onto a plate, and soluble biotinylated L1 (DV1-GV4) antigen was added at 5 nM in 50 μL (3M PBS). 50 μL of streptavidin-Eu (1:500 in assay buffer, Perkin Elmer) was used for detection, and the signal was developed using DELFIA enhancement solution. D1.3 hIgG1 (described in England et al. (1999) J. Immunol. 162:2129-2136) was used as a negative control.

[0357] 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 DELFIA signals exceeding 3000 fluorescent units.

[0358] Antibody preparation The selected scFvs were subcloned into an IgG1 framework using commercially available plasmids. expi293F suspension cells were transfected with the above-mentioned plasmids for antibody expression. 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 in any antibody format, as described above.

[0359] 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.

[0360] γδT cell preparation Enriched γδ T cell populations were prepared according to the methods described in WO 2016 / 198480 (i.e., blood-derived γδ T cells) or WO 2020 / 095059 (i.e., skin-derived γδ T cells). Briefly, for blood-derived γδ T cells, PBMCs were collected from the blood and magnetically depleted for αβ T cells. The αβ-depleted PBMCs were then cultured in CTS OpTmiser medium (ThermoFisher) for 7 days 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 of culture onwards, cultures were supplemented with OKT-3 (or the respective anti-Vδ1 antibody) and IL-15 every 3–4 days, and half of the medium was replaced with fresh medium every 7 days.

[0361] For skin-derived γδ T cells, skin samples were prepared by removing subcutaneous fat and creating multiple punches using a 3 mm biopsy punch. The punches were placed on a carbon matrix grid and placed in the wells of a G-REX6 (Wilson Wolf) plate. Each well was filled with complete isolation medium containing AIM-V medium (Gibco, Life Technologies), CTS Immune Serum Replacement (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"). 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 released cells are then passaged into fresh tissue culture vessels and fresh medium (e.g., AIM-V medium or TexMAX medium (Miltenyi)), further supplemented with recombinant IL-2, IL-4, IL-15, and IL-21, before harvesting. Optionally, αβ T cells also present in the culture are removed using an αβ T cell depletion kit and associated protocol, such as that provided by Miltenyi. For further references, see WO 2020 / 095059.

[0362] 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 secondary fluorescent dye-conjugated antibody against human IgG1. For negative controls, cells were prepared with a) isotype antibody alone (recombinant human IgG), b) fluorescent dye-conjugated anti-human IgG antibody alone, and c) a combination of a) and b). Control wells of completely unstained cells were also prepared and analyzed. As positive controls, purified mouse monoclonal IgG2 anti-human CD3 antibody and purified mouse monoclonal IgG1 anti-human TCR Vδ1 antibody were used at two different concentrations and stained with a fluorescent dye-conjugated goat anti-mouse secondary antibody. The assay was accepted if the mean fluorescence intensity of the low-concentration positive control in the FITC channel was at least 10-fold higher than that of the highest negative control.

[0363] SPR analysis SPR analysis was performed using a MASS-2 instrument equipped with an amine-high-capacity chip (both Sierra Sensors, Germany). 15 nM IgG was captured onto the amine-high-capacity chip via protein G (100 nM for TS8.2). L1 (DV1-GV4) antigen was sprayed onto the cells in a 1:2 serial dilution from 2000 nM to 15.625 nM using the following parameters: 180 s association, 600 s dissociation, flow rate 30 μL / min, operating 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.

[0364] Comparator antibody The antibodies of the present invention were compared to commercially available antibodies in the test assays described. JPEG2026009903000002.jpg79169

[0365] γδ TCR down-regulation and degranulation assays 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 a 2-hour incubation, flow cytometry was used to assess surface expression of the γδ TCR (to measure TCR downregulation) and CD107a expression (to measure degranulation) on γδ T cells.

[0366] Killing assay The killing activity of gamma delta T cells and the effect of test antibodies on the killing activity of gamma delta T cells were assessed by flow cytometry. A 20:1 ratio of gamma delta T cells and CellTracker™ Orange CMTMR (ThermoFisher, C2927)-labeled THP-1 cells (with or without antibody loading) were cocultured in vitro for 4 hours and then stained with Viability Dye eFluor™ 520 (ThermoFisher, 520 65-0867-14) to distinguish between viable and dead target THP-1 cells. During sample acquisition, target cells were gated for 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. The killing activity of gamma delta T cells was presented as the percentage of dead target cells.

[0367] 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.

[0368] 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 crosslinkers and subjected to multienzyme proteolysis using trypsin, chymotrypsin, Asp-N, elastase, and thermolysin. After enrichment of the crosslinked 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.

[0369] Example 2. Antigen design Gamma delta (γδ) T cells are polyclonal with CDR3 polyclonality. To avoid the situation where generated antibodies are selected against the CDR3 sequence (since the CDR3 sequence will be different for each TCR clone), antigen design involves maintaining a consistent CDR3 across different formats. This design aims to generate antibodies that recognize sequences within the variable domain, which are germline encoded and therefore the same in all clones, thus providing antibodies that recognize a broader subset of γδ T cells.

[0370] Another important aspect of the antigen preparation process was designing antigens suitable for expression as proteins. The γδ TCR is a complex protein with a heterodimer containing inter- and intra-chain disulfide bonds. Leucine zipper (LZ) and Fc formats were used to generate soluble TCR antigens for use in phage display selection. Both the LZ and Fc formats expressed well and successfully displayed TCRs, particularly heterodimeric TCRs, such as Vδ1Vγ4.

[0371] The CDR3 sequence 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.

[0372] Antigens containing the delta variable 1 chain were expressed in the LZ format as heterodimers (i.e., in combination with different gamma variable chains "L1," "L2," and "L3"), and in the Fc format either as heterodimers ("F1," "F2," and "F3") or as homodimers (i.e., in combination with another delta variable 1 chain "Fc1 / 1"). All of the antigens' delta variable 1 chains contained a 30M Z CDR3. Another series of gamma delta TCR antigens using a similar format were designed to contain different delta variable chains (e.g., delta variable 2 and delta variable 3) and were used to deselect antibodies with nonspecific or off-target binding ("L4," "F9," "Fc4 / 4," "Fc8 / 8"). These antigens also contained a 30M Z CDR3, ensuring deselection of antibodies that bind in the CDR3 region.

[0373] Antigen functional validation was performed to confirm that the designed antigens were suitable for generating anti-TRDV1 (TCR delta variable 1) antibodies. Detection was observed only when antigens containing the δ1 domain were used (Figure 1).

[0374] Example 3. Phage display Phage display selections were performed on a library of human scFvs using the heterodimeric LZ TCR format in either rounds 1 and 2, with deselection of the heterodimeric LZ TCR in both rounds. Alternatively, round 1 was performed using a homodimeric Fc-fusion TCR with deselection for human IgG1 Fc, followed by round 2 on the heterodimeric LZ TCR with deselection for the heterodimeric LZ TCR (see Table 1). [Table 1]

[0375] Selection was performed in solution phase using 100 nM biotinylated protein. Deselection was performed using 1 μM non-biotinylated protein.

[0376] The success of the phage display selection was analyzed by polyclonal phage ELISA (DELFIA). All DV1 selection outputs showed desirable binding to targets Fc1 / 1, L1, L2, L3, F1, and F3. Varying degrees of binding to non-targets L4, F9, Fc4 / 4, Fc8 / 8, and Fc were detected (see Figures 2A and B).

[0377] 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.

[0378] Unique clones were resequenced and specificity analyzed by ELISA (DELFIA). From the selection, we identified a panel of 94 unique human scFv binders that bound to TRDV1 (L1, L2, L3, F1, F2, F3) but not TRDV2 (L4).

[0379] The inclusion of affinity rankings of selected binders helped guide clone selection. Many binders showed affinities in the nanomolar range, reacting with 25-100 nM biotinylated antigen. A handful of binders showed strong reactivity with 5 nM antigen, indicating potential single-digit nanomolar affinities. Several binders showed no reactivity with 100 nM antigen, indicating affinities in the micromolar range.

[0380] For the selection of clones to proceed to IgG conversion, the aim was to include as many germline lineages and as many different CDR3s as possible, while avoiding sequence liabilities such as glycosylation, integrin binding sites, CD11c / CD18 binding sites, and unpaired cysteines. Additionally, a range of affinities was included.

[0381] Selected clones were screened for binding to the native cell surface-expressed γδ TCR using skin-derived γδ T cells obtained from different donors. Clones selected for conversion to IgG are shown in Table 2. [Table 2]

[0382] Example 5: Antibody SPR analysis IgG antibodies that passed the γδ cell binding assay were prepared, and five were selected for further functional and biophysical characterization. SPR analysis was performed to determine the equilibrium dissociation constant (K D ) was determined. Sensorgrams of the tested antibody-analyte interactions, along with steady-state fits (where available), are shown in Figure 3. No binding was detected for TS8.2, where 80 RU of IgG was captured on the chip. The results are summarized in Table 3. [Table 3]

[0383] Example 6: TCR Engagement Assay We designed several assays to characterize the functionality of selected antibodies. The first assay assessed γδ TCR engagement by measuring downregulation of the γδ TCR upon antibody binding. Selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies, which served as positive controls, or against 1252_P01_C08 (versus 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09). A commercially available anti-panγδ antibody was used as a negative control because it likely has a different mode of action, being a panγδ antibody and recognizing all γδ T cells regardless of their variable chain.

[0384] Assays were 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. EC50 values ​​are summarized in Table 4 below.

[0385] 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 activation of apoptosis. γδ T cell cytoplasmic lytic granules can be released toward target cells upon T cell activation. Therefore, target cells can be labeled with an antibody against CD107a and expression measured by flow cytometry to identify degranulated γδ T cells.

[0386] For Example 6, selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies as positive controls, or 1252_P01_C08 (against 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) as positive controls. IgG2a, IgG1, and D1.3 antibodies were used as negative controls. Assays were 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.

[0387] Example 8: Killing Assay The third assay assessed the ability of γδ T cells activated with selected antibodies to kill target cells.

[0388] For Example 6, selected antibodies were tested against commercially available anti-CD3 and anti-Vδ1 antibodies as positive controls, or against 1252_P01_C08 (against 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) as positive controls, and against anti-panγδ as a negative control. IgG2a, IgG1, and D1.3 antibodies were also used as isotype controls. Assays were performed using skin-derived γδ T cells (94% and 80% pure) obtained from two donors, and the results are shown in Figure 6.

[0389] The results from the three functional assays tested in Examples 6-8 are summarized in Table 4. [Table 4]

[0390] Example 9: Epitope Mapping To determine the epitopes of the antigen / antibody complexes with high resolution, the protein complexes were incubated with a deuterated cross-linker and subjected to multi-enzyme cleavage. After enrichment of the cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry (nLC-LTQ-Orbitrap MS). The generated data were analyzed using XQuest (version 2.0) and Stavrox (version 3.6) software.

[0391] After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis of the protein complex L1(DV1-GV4) / 1245_P01_E07 containing deuterated d0d12, nLC-orbitrap MS / MS analysis detected 13 cross-linked peptides between L1(DV1-GV4) and antibody 1245_P01_E07. The results are presented in Figure 7.

[0392] After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis of the protein complex L1(DV1-GV4) / 1252_P01_C08 containing deuterated d0d12, nLC-orbitrap MS / MS analysis detected five cross-linked peptides between L1(DV1-GV4) and antibody 1252_P01_C08. The results are presented in Figure 8.

[0393] After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis of the protein complex L1(DV1-GV4) / 1245_P02_G04 containing deuterated d0d12, nLC-orbitrap MS / MS analysis detected 20 cross-linked peptides between L1(DV1-GV4) and antibody 1245_P02_G04. The results are presented in Figure 9.

[0394] After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis of the protein complex L1(DV1-GV4) / 1251_P02_C05 containing deuterated d0d12, nLC-orbitrap MS / MS analysis detected five cross-linked peptides between L1(DV1-GV4) and antibody 1251_P02_C05. The results are presented in Figure 10.

[0395] Epitope binding with another antibody, clone ID1141_P01_E01, was also tested. After trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis of the protein complex L1(DV1-GV4) / 1141_P01_E01 containing deuterated d0d12, nLC-orbitrap MS / MS analysis detected 20 cross-linked peptides between L1(DV1-GV4) and antibody 1141_P01_E01. The results are presented in Figure 11.

[0396] A summary of the epitope mapping results is presented in Table 5. [Table 5]

Claims

1. An isolated human anti-TCR delta variable 1 (anti-Vδ1) antibody or fragment thereof, comprising: (i) a sequence selected from the group consisting of sequences 3-20 of SEQ ID NO: 1; and / or (ii) An isolated human anti-Vδ1 antibody or fragment thereof that binds to an epitope of the variable delta 1 (Vδ1) chain of a gamma delta T cell receptor (TCR) comprising one or more amino acid residues within the amino acid region of 37 to 77 of SEQ ID NO:

1.

2. 2. The human isolated anti-Vδ1 antibody or fragment thereof of claim 1, wherein 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.

3. 3. The human isolated anti-Vδ1 antibody or fragment thereof of claim 1 or claim 2, wherein the epitope comprises one or more amino acid residues within amino acid regions 5-20 and 62-77 of SEQ ID NO:

1.

4. 3. The human isolated anti-Vδ1 antibody or fragment thereof of claim 1 or claim 2, wherein the epitope comprises one or more amino acid residues within amino acid region 50 to 64 of SEQ ID NO:

1.

5. 3. The human isolated anti-Vδ1 antibody or fragment thereof of claim 1 or claim 2, wherein the epitope comprises one or more amino acid residues within amino acid regions 37-53 and 59-77 of SEQ ID NO:

1.

6. The human isolated anti-Vδ1 antibody or fragment thereof according to any one of claims 1 to 5, wherein the epitope is an activating epitope of a γδ T cell.

7. 1. An isolated anti-Vδ1 antibody or fragment thereof, comprising: CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 25; a CDR2 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26-37 and A1-A12 (of Table 2); and / or An isolated anti-Vδ1 antibody or fragment thereof, comprising one or more of its CDR1s comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38-61.

8. 8. The isolated anti-Vδ1 antibody or fragment thereof of claim 7, comprising a VH region comprising: 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.

9. 8. The isolated anti-Vδ1 antibody or fragment thereof of claim 7, comprising a VH region comprising: 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.

10. 8. The isolated anti-Vδ1 antibody or fragment thereof of claim 7, comprising a VH region comprising: 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.

11. 8. The isolated anti-Vδ1 antibody or fragment thereof of claim 7, comprising a VL region comprising: 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.

12. 8. The isolated anti-Vδ1 antibody or fragment thereof of claim 7, comprising a VL region comprising: 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.

13. 8. The isolated anti-Vδ1 antibody or fragment thereof of claim 7, comprising a VL region comprising: 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.

14. An isolated anti-Vδ1 antibody or fragment thereof, comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 62 to 85.

15. The isolated anti-Vδ1 antibody or fragment thereof of claim 14, comprising 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.

16. The isolated anti-Vδ1 antibody or fragment thereof of claim 14, comprising 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.

17. The isolated anti-Vδ1 antibody or fragment thereof of claim 14, comprising 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.

18. 18. The isolated anti-Vδ1 antibody or fragment thereof of any one of claims 7 to 17, wherein the anti-Vδ1 antibody or fragment thereof comprises a VH domain and a VL domain, and the VH domain and VL domain are linked by a linker, such as a polypeptide linker.

19. An isolated anti-Vδ1 antibody or fragment thereof comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 86 to 97.

20. 20. The isolated anti-Vδ1 antibody or fragment thereof of claim 19, comprising SEQ ID NO:

86.

21. 20. The isolated anti-Vδ1 antibody or fragment thereof of claim 19, comprising SEQ ID NO:

87.

22. 20. The isolated anti-Vδ1 antibody or fragment thereof of claim 19, comprising SEQ ID NO:

88.

23. 1.5×10 as measured by surface plasmon resonance －７ 23. The isolated anti-Vδ1 antibody or fragment thereof of any one of claims 1 to 22, which binds to the variable delta 1 (Vδ1) chain of a gamma delta T cell receptor (TCR) with a binding affinity (KD) of less than M.

24. 24. The isolated anti-Vδ1 antibody or fragment thereof of any one of claims 1 to 23, which 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.

25. 25. The isolated anti-Vδ1 antibody or fragment thereof of claim 24, which is an scFv, or a full-length antibody, e.g., IgG1.

26. 26. The isolated anti-Vδ1 antibody of claim 25, comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 111-122.

27. 27. The isolated anti-Vδ1 antibody of claim 26, comprising SEQ ID NO:

111.

28. 27. The isolated anti-Vδ1 antibody of claim 26, comprising SEQ ID NO:

112.

29. 27. The isolated anti-Vδ1 antibody of claim 26, comprising SEQ ID NO:

116.

30. 30. The isolated anti-Vδ1 antibody or fragment thereof of any one of claims 7 to 29, which is human.

31. A polynucleotide sequence encoding the anti-Vδ1 antibody or fragment thereof according to any one of claims 1 to 30.

32. A polynucleotide sequence encoding an anti-Vδ1 antibody or a fragment thereof comprising a sequence having at least 70% sequence identity with SEQ ID NOs: 99-110.

33. 33. An expression vector comprising the polynucleotide sequence of claim 31 or claim 32.

34. 34. A cell comprising the polynucleotide sequence of claim 31 or claim 32 or the expression vector of claim 33.

35. A composition comprising an antibody or fragment thereof according to any one of claims 1 to 30.

36. A pharmaceutical composition comprising an antibody or fragment thereof according to any one of claims 1 to 30 together with a pharmaceutically acceptable diluent or carrier.

37. 1. A method for producing an anti-Vδ1 antibody or fragment thereof, comprising: (i) designing a series of antigens comprising a TCR delta variable 1 (TRDV1) amino acid sequence, wherein the CDR3 sequence of the TRDV1 is the same for all antigens in the series; (ii) exposing the first antigen designed in step (i) to an antibody library; (iii) isolating antibodies or fragments thereof that bind to said antigen; (iv) exposing the isolated antibody or fragment thereof to the second antigen designed in step (i); (v) isolating an antibody or fragment thereof that binds to both the first and second antigens.

38. 38. The method of claim 37, further comprising exposing the isolated antibodies or fragments thereof to a second set of antigens comprising a γδ TCR with a different delta variable chain, e.g., TCR delta variable 2 (TRDV2) or TCR delta variable 3 (TRDV3), and then deselecting antibodies or fragments thereof that also bind to the second set of antigens.

39. 39. The method of claim 37 or claim 38, wherein the set of antigens is in heterodimeric and / or homodimeric format.

40. An antibody obtained by the method according to any one of claims 37 to 39.

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