Ex vivo γδ t cell populations

Abstract

To provide a method for expanding γδ T cells.SOLUTION: An ex vivo method for modulating Vδ1 T cells is provided, comprising administering, to a cell population including Vδ1 T cells, a human anti-TCR delta variable 1 (anti-Vδ1) antibody or a fragment thereof that binds to an epitope of a variable delta 1 (Vδ1) chain of a γδ T-cell receptor (TCR) comprising an amino acid sequence consisting of a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] BACKGROUND OF THE INVENTION The growing interest in T cell immunotherapy for cancer is fueled by T cell receptors that recognize cancer cells and specifically target PD-1, CTLA-4, and Disinhibition is achieved by clinically mediated antagonism of inhibitory pathways mediated by steroids and other receptors. When inhibited, CD8+ and CD4+ alphabeta (α However, αβ T cells are not immune to the effects of M It is HC-restricted, which can lead to graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) have a distinctive definitive γδ T cell receptor ( This corresponds to a subset of T cells that express a TCR (T cell receptor), which consists of one gamma (γ) chain and one deoxyribonucleic acid (DMA) chain. T cells are composed of α (δ) chains, each of which undergoes chain rearrangement but is limited compared to αβ T cells. The major TGRV gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV6, TRGV7, TRGV8, TRGV9, TRGV10, TRGV11, TRGV12, TRGV13, TRGV14, TRGV15, TRGV16, TRGV17, TRGV18, TRGV19, TRGV20, TRGV21, TRGV2 V3, TRGV4, TRGV5, TRGV8, TRGV9, and TRGV11, as well as the non-functional genes TRGV10 and TRGV11 , TRGVA, and TRGVB. The most frequent TRDV gene segments are Vδ1, Vδ2, and In addition to Vδ3, several V segments are encoded that have both Vδ and Vα designations (Adam s et al., 296:30-40 (2015) Cell Immunol.). Specific γ and δ types are not exclusively It is found in cells, but more commonly in one or more tissue types, and is therefore associated with human γδ T cells. Cells can be broadly classified based on their TCR chains. For example, most blood-resident Neoplastic γδ T cells express a Vδ2 TCR, generally Vγ9Vδ2, which is similar to tissue-resident γδ T cells. T cells, e.g., are less common among tissue-resident γδ T cells in the skin, In the gut, γδ T cells are paired with gamma chains, e.g., often Vγ4. The Vδ1 TCRs formed by the α-Vδ1 TCR are more frequently used.

[0004] To utilize γδ T cells for immunotherapy, the cells can be expanded in situ or Either harvest them and expand them ex vivo before reinjecting them. The latter approach, using the addition of exogenous cytokines, has been described previously. See, for example, WO2017 / 072367 and WO2018 / 212808. A method to expand δ T cells is to use a pharmacologically modified form of hydroxy-methylbut-2-enylene. The study was conducted using bisphosphonates such as hydroxybenzoate (HMBPP) or clinically approved aminobisphosphonates. These approaches have resulted in over 250 cancer patients achieving the rare condition known as complete remission. However, the treatment appears to be safe, with the development of a resolution of the problem. There remains a need for activators with a proven ability to Summary of the Invention

[0005] (Summary of the Invention) According to a first aspect of the present invention, there is provided an ex vivo method for modulating V51 T cells, comprising administering to the patient an amino acid sequence comprising: region: (i) SEQ ID NO: 1, 3 to 20; and / or (ii) 37 to 77 of SEQ ID NO: 1 An epitope of the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) containing one or more amino acid residues within A human anti-TCR delta variable 1 (anti-Vδ1) antibody or a fragment thereof that binds to the polypeptide is administered to cells containing Vδ1 T cells. Ex vivo methods are provided that include administering to a population of cells.

[0006] According to a further aspect of the invention there is provided an ex vivo method for modulating V51 T cells comprising the steps of: a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 25; At least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 and sequences A1 to A12 (in Table 2) and / or a CDR2 comprising a sequence having the formula: CDR1 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61 and administering an anti-Vδ1 antibody or a fragment thereof comprising one or more of: Ex vivo methods are provided, including administering to a subject the cells.

[0007] According to a further aspect of the present invention, a method for treating a pulmonary arthritis, comprising administering to a subject a pulmonary arthritis or pulmonary edema, the method being obtained by the ex vivo method as defined herein. A population of V51 T cells is provided.

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

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

[0010] According to a further aspect of the present invention, a method for treating cancer, infectious diseases, or a method of treating an inflammatory disease, comprising administering to a subject a V51 T cell population or a medicament as defined herein. A method is provided that includes administering a therapeutically effective amount of the composition. [Brief explanation of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: ELISA detection of directly coated antigens with anti-Vδ1 Ab (REA173, Miltenyi Biotec). Detection was only seen with antigens containing the Vδ1 domain. The leucine zipper (LZ) format appears to be more potent than the Fc format, which is consistent with cell-based flow competition assays (data not shown). [Figure 2] Figure 2: Polyclonal phage DELFIA data for DV1 selection. A) Heterodimer selection: heterodimeric LZ TCR format in rounds 1 and 2, deselected with heterodimeric LZ TCR in both rounds. B) Homodimer selection: Round 1 is performed with homodimeric Fc fusion TCR and deselected with human IgG1 Fc, followed by round 2 with heterodimeric LZ TCR and deselected with heterodimeric LZ TCR. To represent selection from different libraries, each graph contains two bars for each target. [Figure 3] Figure 3: IgG capture: Left) Sensorgram of the interaction of anti-L1 IgG with L1, Right) Steady-state fit (where possible). All experiments were performed at room temperature on a MASS-2 instrument. Steady-state fitting with Langmuir 1:1 binding. [Figure 4]Figure 4: Results of TCR down-modulation assay for clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07, and 1251_P02_C05 (A) or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09, 1251_P02_G10, and 1252_P01_C08 (B). [Figure 5] Figure 5: Results of T cell degranulation assay for clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07, and 1251_P02_C05 (A) or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09, and 1251_P02_G10 (B). [Figure 6] Figure 6: Results of killing assay (THP-1 flow-based assay) for clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07, and 1251_P02_C05 (A) or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09, and 1251_P02_G10 (B). [Figure 7] Figure 7: Epitope mapping data for 1245_P01_E07. Graphical representation of the epitope binding site of 1245_P01_E07 on SEQ ID NO: 1. [Figure 8] Figure 8: Epitope mapping data for 1252_P01_C08. Graphical representation of the epitope binding site of 1252_P01_C08 on SEQ ID NO: 1. [Figure 9] Figure 9: Epitope mapping data for 1245_P02_G04. Graphical representation of the epitope binding site of 1245_P02_G04 on SEQ ID NO: 1. [Figure 10]Figure 10: Epitope mapping data for 1251_P02_C05. Graphical representation of the epitope binding site of 1251_P02_C05 on SEQ ID NO: 1. [Figure 11] Figure 11: Epitope mapping data for 1141_P01_E01. Graphical representation of the epitope binding site of 1141_P01_E01 on SEQ ID NO: 1. [Figure 12] Figure 12: Total cell counts for experiment 1 of Example 10. Samples were cultured with various concentrations of anti-V51 antibodies described herein and compared to samples cultured with a comparator antibody or control. Graphs show total cell counts at (A) day 7, (B) day 14, and (C) day 18. [Figure 13] Figure 13: Analysis of V51 T cells in experiment 1 of example 10. Graphs show (A) percentage of V51 T cells, (B) V51 T cell count, and (C) V51 fold change in day 18 samples. [Figure 14] Figure 14: Total cell counts for experiment 2 of Example 10. Samples were cultured with various concentrations of anti-V51 antibodies described herein and compared to samples cultured with a comparator antibody or control. Graphs show total cell counts at (A) day 7, (B) day 11, (C) day 14, and (D) day 17. [Figure 15] Figure 15: Analysis of V51 T cells in experiment 2 of Example 10. Graphs show (A) percentage of V51 T cells, (B) V51 T cell count, and (C) V51 fold change in day 17 samples. [Figure 16] Figure 16: Cell composition analysis. The cell types present in the samples (including non-V51 cells) were determined on day 17 of experiment 2. Cells were harvested and analyzed by flow cytometry for surface expression of V51, V52, and αβ TCR. Percentage values ​​are also provided in Table 6. [Figure 17]Figure 17: SYTOX-flow killing assay results. Cell functionality was tested using the SYTOX-flow killing assay. Results are presented for (A) experiment 1 on day 14 using cells at an effector-to-target (E:T) ratio of 10:1, and (B) experiment 2 on day 17 (post-freeze-thaw) using cells at E:T ratios of 1:1 and 10:1. [Figure 18] Figure 18: Total cell counts after freeze-thaw. The graph shows total cell counts 7 days after freeze-thawing for cultures that were contacted with B07, C08, E07, G04, or OKT-3 antibodies before freezing. [Figure 19] Figure 19: Monitoring cell expansion. Total cell counts were monitored for cells cultured after freeze-thawing up to 42 days. [Figure 20] Figure 20: Anti-V51 antibodies conferred modulation and proliferation of tumor infiltrating lymphocytes (TIL) in human tumors. Study on renal cell carcinoma (RCC) + / - antibodies. A) Fold increase in TIL V51+ cells. B) Total number of TIL V51+ cells. C) Example of gating strategy. D) Comparative cell surface phenotypic profile of TIL V51+ cells. E) Analysis of TIL V51-negative gated fraction. DETAILED DESCRIPTION OF THE INVENTION

[0012] (Detailed Description of the Invention) (definition) Unless otherwise defined, all technical and scientific terms used herein are intended to be used by the present invention. As used herein, the terms "term" and "term" have the meaning commonly understood by one skilled in the art to which they pertain. Where used herein, the following terms have the meanings ascribed to them below.

[0013] Gamma delta (γδ) T cells express a distinctive definitive T cell receptor (TCR) on their surface. This TCR represents a small subset of T cells that express one gamma (γ) chain and one delta (δ) chain. Each chain consists of a variable (V) region, a constant (C) region, a transmembrane region, and a cellular The V region contains the antigen-binding site. There are two main subtypes of human γδ T cells. Subtypes: There are those that are predominant in peripheral blood and those that are predominant in non-hematopoietic tissues. Subtypes of HIV can be defined by the type of δ and / or γ present on the cell. For example, gamma delta T cells, which are predominant in peripheral blood, primarily express the delta variable 2 chain (Vdelta2). The γδ T cells that predominate in non-hematopoietic tissues (i.e., are tissue-resident) are primarily delta-variable. Reference to "Vδ1 T cells" refers to γδ T cells that have a Vδ1 chain. That is, Vδ1 + Refers to T cells.

[0014] Reference to "delta variable 1" can be referred to as Vδ1 or Vd1, while the The nucleotides encoding the TCR chain having the V of the γδ TCR can be referred to as "TRDV1." Any antibody or fragment thereof that interacts with the δ1 chain is effectively an antibody or fragment thereof that binds to Vδ1. It is an "anti-TCR delta variable 1 antibody or a fragment thereof" or an "anti-Vδ1 antibody or a fragment thereof" It can be called a "piece."

[0015] Further reference may be made herein to other delta chains, such as the "delta variable 2" chain. These may be referred to in a similar manner, e.g., the delta variable 2 chain is referred to as Vδ2. The nucleotides encoding the TCR chain containing this region can be referred to as "TRDV2". In a preferred embodiment, an antibody that interacts with the V51 chain of the γδ TCR Neither Vδ1 nor its fragments interact with other delta chains, such as Vδ2.

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

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

[0018] A conventional antibody or immunoglobulin (Ig) consists of four polypeptide chains: two heavy (H) chains and two light (L) chains. A protein containing heavy (L) chains. Each chain is divided into a constant region and a variable domain. A heavy (H) chain variable domain is abbreviated herein as VH, and a light (L) chain variable domain is abbreviated herein as VL. These domains, their related domains, and domains derived from them, These may be referred to herein as immunoglobulin chain variable domains. VH and VL domains (VH The VL region (also referred to as the VL region) is separated by more conserved regions called "framework regions" ("FR"). These can be further divided into regions called "complementarity determining regions" ("CDRs"), which are interspersed with regions that The framework and complementarity determining regions are precisely defined (Kabat et al., Contribution: Sequences of Proteins of Immunological Interest terest), 5th edition, US Department of Health and Human Services (1991) NIH Publicati Other numbering conventions for CDR sequences are found in, for example, Chothia et al. (1989) Nature 342: 877-883. In conventional antibodies, each VH and VL is , three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, It consists of two heavy immunoglobulin chains and two A conventional antibody tetramer of light immunoglobulin chains, for example, is interconnected by disulfide bonds. The heavy and light immunoglobulin chains are connected in the same manner, and the heavy chains are connected in the same manner. The heavy chain constant region contains three domains, CH1, CH2, and CH3. The constant region consists of one domain, CL. The heavy chain variable domain and the light chain variable domain The constant domain of an antibody is the binding domain that interacts with an antigen. The constant domain of an antibody usually various cells (e.g., effector cells) and the first component of the classical complement system (C1q). Mediates the binding of antibodies to major tissues or factors.

[0019] As used herein, a fragment of an antibody (which may be referred to as an "antibody fragment," "immunoglobulin fragment," An "antigen-binding fragment," or alternatively referred to as an "antigen-binding polypeptide," is a fragment that binds specifically to a target. a portion of an antibody (or a construct containing said portion) that heterologously binds to the γδ T cell receptor; Delta variable 1 (Vδ1) chain (e.g., one or more immunoglobulin chains that are not full-length but are specific to the target) Examples of binding fragments encompassed by the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain); (ii) F(ab')2 fragment (consisting of two Fab fragments linked by a disulfide bridge at the hinge region) bivalent fragments); (iii) Fd fragment (consisting of a VH domain and a CH1 domain); (iv) Fv fragment (consisting of the VL and VH domains of a single arm of an antibody); (v) Single-chain variable fragments, scFv (which are monovalent fragments formed by pairing VL and VH domains using recombinant methods) The proteins are joined by a synthetic linker that allows them to be made into a single protein chain that forms a molecule. consisting of a VL domain and a VH domain connected together; (vi) VH (immunoglobulin chain variable domain consisting of a VH domain); (vii) VL (immunoglobulin chain variable domain consisting of a VL domain); (viii) domain antibodies (dAbs consisting of either a VH domain or a VL domain); (ix) a minibody (consisting of a pair of scFv fragments linked via a CH3 domain); and (x) Diabodies (VH domains from one antibody are linked to another antibody via a small peptide linker) consisting of a non-covalent dimer of scFv fragments consisting of VL domains derived from Examples include:

[0020] A "human antibody" is an antibody having variable and constant regions derived from human germline immunoglobulin sequences. A human subject administered the human antibody will have a primary amino acid sequence that is identical to that of the antibody. Generate a cross-species antibody response to the acid (e.g., a HAMA response - called human anti-mouse antibody). The human antibody may, for example, have CDRs, particularly CDR3, that are aligned to human germline immunoglobulin sequences. Amino acid residues not encoded by the However, However, this term also applies to CDR sequences derived from the germline of another mammalian species, e.g., a mouse, that are similar to those of a human. It is not intended to include antibodies that have been grafted onto framework sequences. Human antibodies prepared, expressed, produced, or isolated by, for example, recombinant human antibodies introduced into a host cell. Antibodies expressed using expression vectors, recombinant combinatorial human antibody libraries antibodies isolated from animals transgenic for human immunoglobulin genes ( For example, antibodies isolated from mice (e.g., mice) or human immunoglobulin gene sequences can be cloned into other DNA sequences. prepared, expressed, produced, or isolated by any other means involving splicing of sequences. Such antibodies can also be referred to as "recombinant human antibodies."

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

[0022] "Specificity" refers to the number of different types of antigens or antigens that a particular antibody or fragment thereof can bind. refers to the number of antigenic determinants. The specificity of an antibody is its ability to recognize a particular antigen as a unique molecular entity. The ability of an antibody to bind to an antigen or epitope and distinguish it from another. An antibody that "antigens" a molecule that binds to another React with a specific target more frequently, rapidly, for a longer period of time, and / or with greater affinity An antibody is said to exhibit "specific binding" when it reacts with the target antigen or epitope. It binds rapidly and / or for a longer period of time with greater affinity or avidity than it binds to other substances. When the antibody binds to the target antigen or epitope, it "specifically binds" to the target antigen or epitope.

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

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

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

[0026] "Human tissue Vδ1+ cells" and "Hematopoietic and blood Vδ1+ cells" and "Tumor-infiltrating lymphocytes (TIL)" "Vδ1+ cells" are those contained in human tissues or the hematopoietic system or human tumors, respectively, or These cell types are defined as V51+ cells derived from these. All of these cell types are defined as their (i) location or where they are located. and (ii) by their expression of the Vδ1+ TCR. .

[0027] A "modulatory antibody" can be used to contact or bind to cells that express the target to which the antibody binds, but is not limited to such antibodies. When bound, the cell cycle and / or cell number and / or cell viability and / or one or more Cell surface markers and / or one or more secreted molecules (e.g., cytokines, chemokines, lymphokines, etc.) secretion of lectins, icotrienes, etc., and / or function (e.g., on target cells or diseased cells) The antibodies confer a measurable change, including a measurable change in cellular cytotoxicity.

[0028] A method of "modulating" a cell or population thereof includes the step of: to induce at least one measurable change in the secretion of It refers to a method of doing something.

[0029] An "immune response" is defined as the activation of at least one cell of the immune system or one measurable changes (including but not limited to) in one cell type, one endocrine pathway, or one exocrine pathway These include cell-mediated responses, humoral responses, cytokine responses, and chemokine responses.

[0030] "Immune cells" include, but are not limited to, CD34+ cells, B cells, and 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, Defined as cells of the immune system, including natural killer (NK) cells and gamma delta T cells. Combinatorial cell tables for identifying, grouping, or clustering immune cells Immune cell subpopulations can be identified by surface molecular analysis (e.g., flow cytometry). Further analysis can then subdivide these further. For example, CD45+ lymphocytes can be further subdivided into vδ-positive and vδ-negative populations. This can be done.

[0031] A "model system" is a system in which a drug, such as an antibody or fragment thereof, can be used to assess the efficacy of the drug in ameliorating a sign or symptom of a disease. Biological models designed to help understand how drugs can function as therapeutic agents Such models are typically in vitro, ex vivo, and in vivo models. In vivo diseased cells, non-diseased cells, healthy cells, effector cells, and tissues, etc., as well as the It involves the use of drugs whose performance is studied and compared.

[0032] "Disease cells" include cells that are caused by cancer, an infection, e.g., a viral infection, or an inflammatory disease or inflammation. For example, diseased cells may be tumor cells, autologous cells, or other cells that display phenotypes associated with disease progression, such as inflammatory diseases. The diseased cells may be immune tissue cells or virus-infected cells. It can be defined as viral infectious or inflammatory.

[0033] "Healthy cells" refer to normal cells that are not diseased. This refers to "normal" or "non-disease" cells. Non-diseased cells include non-cancerous or non-infectious or non-inflammatory cells. The cells determine the disease cell specificity imparted by the drug and / or the therapeutic effect of the drug. It is often used in conjunction with associated disease cells to better understand the therapeutic index.

[0034] "Disease cell specificity" refers to the specificity of effector cells or populations thereof (e.g., a population of Vδ1+ cells, etc.). ) how effectively can diseased cells, such as cancer cells, be identified while sparing non-diseased or healthy cells? This potential can be measured in model systems. The effector cell or population of effector cells selectively kills or lyses diseased cells. The tendency of the effector cells to kill or lyse non-diseased or healthy cells is compared to the potential of the effector cells to kill or lyse non-diseased or healthy cells. The disease cell specificity can inform the potential therapeutic index of the drug. can be done.

[0035] "Enhanced disease cell specificity" refers to, for example, increasing the ability of a compound to specifically kill disease cells. and the like. This enhancement can be expressed as a fold change in disease cell killing specificity or selectivity, or percentage increase.

[0036] Preferably, the antibodies or fragments thereof (i.e., polypeptides) of the present invention are isolated. An "isolated" polypeptide is one that has been removed from its original environment. The term "isolated" is used to mean substantially free from other antibodies having different antigen specificities. can refer to an antibody that does not specifically bind to Vδ1 or a fragment thereof (e.g., an isolated antibody that specifically binds to Vδ1 or a fragment thereof). (Antibodies obtained are substantially free of antibodies that bind to antigens other than Vδ1.) The term is used to describe an isolated antibody that is therapeutic when formulated as the active ingredient of a pharmaceutical composition. or at least 70-80% (w / w) pure, more preferably or at least 80-90% (w / w) pure, even more preferably 90-95% pure; and most preferably Preferably, it is at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure. It can also refer to a preparation.

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

[0038] Antibodies or fragments thereof may be used in combination with naturally occurring allelic variants, as well as mutants or any other naturally occurring variants. The term "functionally active variant" includes variants that do not exist in nature. As is known in the art, allelic variants do not inherently alter the biological function of a polypeptide. Another form of the invention is characterized by one or more amino acid substitutions, deletions, or additions that do not affect the As a non-limiting example, the functionally active variant is a CDR-containing polypeptide. When the framework surrounding the CDR is modified, when the CDR itself is modified, when the CDR is replaced by another framework, still remain when grafted onto a framework or when N- or C-terminal extensions are incorporated. Furthermore, the CDR-containing binding domains can function in different partner chains, e.g. For example, it can pair with a chain shared with another antibody. When shared with a "common" heavy chain, the binding domain can still function. Furthermore, the binding domains can function when multimerized. "Fragments thereof" include fragments of the VH or VL or constant domains (e.g., as listed on IMGT.org). modified to move away from or towards the standard sequence and still function It may also include functional variants that

[0039] To compare two closely related polypeptide sequences, a first polypeptide sequence and a second polypeptide sequence are used. The "% sequence identity" between a second polypeptide sequence is calculated using standard matching of polypeptide sequences (BLASTP). This can be calculated using NCBI BLAST v2.0 with the appropriate settings. To compare polynucleotide sequences, a first nucleotide sequence and a second nucleotide sequence are The "% sequence identity" between sequences was calculated using NCBI BLAST using standard settings for nucleotide sequences. This can be calculated using BLASTN v2.0.

[0040] Polypeptide or polynucleotide sequences are those that are 100% identical in sequence over their entire length. A polypeptide or polynucleotide sequence is considered to be the same as or "identical" to another polypeptide or polynucleotide sequence if they share the same identity. The residues in the sequence are listed from left to right, i.e., from the N-terminus to the C-terminus of the polypeptide. and ; numbered from the 5' end to the 3' end of the polynucleotide.

[0041] A "difference" between sequences is a single amino acid at a position in the second sequence compared to the first sequence. The two polypeptide sequences may be one, two, or more residue insertions, deletions, or substitutions. The first sequence may contain as many such amino acid differences as possible. An insertion, deletion, or substitution in a second sequence that is identical (100% sequence identity) to a For example, if the identical sequences are 9 amino acid residues long, The single substitution in the sequence results in a sequence identity of 88.9%. If the peptide sequences are 9 amino acid residues long and share 6 identical residues, the first and The two polypeptide sequences share greater than 66% identity (first and second polypeptides The sequences share 66.7% identity).

[0042] Alternatively, for purposes of comparing a first reference polypeptide sequence to a second comparison polypeptide sequence, the additions, substitutions, and / or deletions made to the first sequence to produce the second sequence. An "addition" is the addition of one amino acid to the sequence of the first polypeptide. "Substitution" is the addition of a nucleotide sequence ... "Substitution" refers to the substitution of one amino acid residue in a first polypeptide sequence with one different amino acid residue. A "deletion" is a substitution. The substitution may be conservative or non-conservative. Deletion of one amino acid residue from the sequence of the polypeptide (at any end of the first polypeptide) (including deletions at the ends).

[0043] A "conservative" amino acid substitution is one in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure. and have little effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions are preferably those within the following group: One amino acid in a group is replaced by another amino acid residue from the same group is the substitution: [Table 1]

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

[0045] As used herein, the numbering of polypeptide sequences and the definitions of CDRs and FRs are as follows: as defined according to the .at system (which is incorporated herein by reference in its entirety). (Kabat et al., 1991). The "corresponding" amino acid residues occupy the same positions as the amino acid residues in the second sequence according to the Kabat system. The first sequence shares the amino acid residues with the second sequence, while the amino acid residues in the second sequence may differ in content from those in the first sequence. The preferred corresponding residues are those in the framework and CDR sequences of Kabat If they are the same length by definition, they will share the same number (and letter). The alignment can be done manually or using known algorithms for sequence alignment, e.g., using standard settings. Using a computer algorithm, e.g., NCBI BLAST v2.0 (BLASTP or BLASTN) This can be achieved by:

[0046] References herein to an "epitope" refer to an antigen specific to an antigen that is specifically bound by an antibody or fragment thereof. An epitope refers to the portion of a target that is targeted by an antigen. An epitope can also be called an "antigenic determinant." An antibody is considered "essentially identical" to another antibody if they recognize the same or sterically overlapping epitopes. Two antibodies bind to the same or overlapping epitopes. Commonly used methods to determine whether a labeled antigen or a labeled antibody using several different formats (e.g., using radioactive or enzyme labels) well plate for detecting antigen-expressing cells, or flow cytometry for detecting antigen-expressing cells. This is a competitive assay that can

[0047] Epitopes found on protein targets can be classified as "linear epitopes" or "conformational epitopes." A linear epitope can be defined as a sequence of consecutive amino acids in a protein antigen. Conformational epitopes are formed by sequences of amino acids that are discontinuous in the protein sequence. When a protein folds, it is made up of amino acids that come together to form its three-dimensional structure. It is formed from acids.

[0048] As used herein, the term "vector" refers to a molecule that can transport another nucleic acid to which it has been linked. A vector is intended to refer to a nucleic acid molecule capable of delivering a gene. "Pla" refers to a circular double-stranded DNA loop into which different DNA segments can be ligated. Another type of vector is a "smid." A viral vector that can be ligated into a vector. can replicate autonomously in the host cell into which it is introduced (e.g., a cell line containing a bacterial origin of replication). bacterial vectors and episomal mammalian and yeast vectors). Other vectors (e.g., non- Episomal mammalian vectors) are vectors that, when introduced into a host cell, integrate into the host cell genome. can be integrated into the host genome and thereby replicated along with it. Such vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are called plasmids. Plasmids are most often used in the form of vectors. In this specification, the terms "plasmid" and "vector" can be used interchangeably. However, the present invention is not limited to such other forms of expression vectors, e.g., those having equivalent functions. Viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adenosine-111) that serve as vectors for the and may also include bacteriophage and phagemid systems. As used herein, the term "recombinant host cell" (or simply, "host cell") refers to a The term is intended to refer to a cell into which a recombinant expression vector has been introduced. The term refers not only to the particular subject cell but also to the progeny of such a cell, as may be later referred to herein, for example. Optionally store, provide, sell, transport, or When used to generate a cell line or cell bank that is utilized, it is intended to refer to the progeny as well. will be done.

[0049] References to a "subject," "patient," or "individual" refer to a subject to be treated, in particular , refers to a mammalian subject. Mammalian subjects include humans, non-human primates, domestic animals (e.g., cattle), Sport animals or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, or In some embodiments, the subject is a human. In embodiments, the subject is a non-human mammal, for example, a mouse.

[0050] The term "sufficient amount" means an amount sufficient to produce a desired effect. The term "amount" refers to an amount that is effective to ameliorate the symptoms of a disease or disorder. A therapeutically effective amount can also be a "prophylactically effective amount."

[0051] As used herein, the term "about" means any quantity that is less than the specified quantity. Values ​​up to 10% (inclusive) greater than the specified value and up to 10% (inclusive) greater than the specified value (including) smaller values, preferably up to 5% (including 5%) larger than the specified value and A value up to 5% (inclusive) less than the specified value, specifically including the specified value. The term contains the specified range of values.

[0052] The severity of the signs or symptoms of the disease or disorder, the extent to which such signs or symptoms are targeted The disease or disorder is "improved" if the frequency with which it is experienced, or both, is reduced. "It is being done."

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

[0054] As used herein, "cancer" refers to the abnormal growth or division of cells. The growth and / or lifespan exceeds that of surrounding normal cells and tissues, and Cancer can be benign, pre-malignant, or malignant. Cancer can occur in the oral cavity (e.g., , 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 tumors (e.g., cystic, squamous cell, 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 gland, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, etc.) various types of leukemia, including leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, and chronic myeloid leukemia Occurs in cells and tissues.

[0055] (Method for modulating gamma delta T cells) According to a first aspect of the present invention, an ex vivo method for modulating variable delta chain 1 (Vδ1) T cells is provided. and administering an anti-V51 antibody or a fragment thereof as defined herein to a cell population comprising V51 T cells. "Administering" an antibody or fragment thereof is also provided. , it will be understood to include "contacting" Vδ1 T cells.

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

[0057] Such modulation of V51 T cells includes, for example, V51 T cell activation or V51 T cell inhibition. In one embodiment, the V51 T cells may be produced by an anti-V51 antibody as defined herein or an antibody thereof. In an alternative embodiment, V51 T cells are activated by administering a fragment of This is inhibited by administering an anti-V51 antibody or fragment thereof as defined herein. In this embodiment, the V51 T cells are administered to the patient by administration of an anti-V51 antibody or its is not inhibited by administration of the fragment.

[0058] In one embodiment, the modulation of V51 T cells is achieved by culturing an anti-TCR delta1 variable antibody or fragment thereof. This includes administering to V51 T cells in vitro (i.e., in vitro or ex vivo). T cells may be present in mixed cell populations, e.g., with other lymphoid cell types (e.g., αβ T cells or NK cells). It may be present in a cell population containing nuclei (cells).

[0059] In one embodiment, the cell population comprising V51 T cells is selected from the group consisting of: In a further embodiment, the antibody is isolated from a sample as described herein. The cell population is enriched for T cells prior to administration of the anti-V51 antibody or fragment thereof. In a further embodiment, the cell population is selected from the group consisting of γδ T cells, γδ T cells, and γδ T cells, prior to administration of the anti-Vδ1 antibody or fragment thereof. It is enriched for cells.

[0060] The method can also be performed on a cell population comprising a purified fraction of γδ T cells. In such an embodiment, the cell population is cultured prior to administration of the anti-V51 antibody or fragment thereof. In this case, cell types other than γδ T cells present in the sample, such as αβ T cells and / or NK cells, are removed. The cell population may also or instead be treated with an anti-Vδ1 antibody or a fragment thereof. Prior to administration, the subject may be enriched for cell types that may contain V51, e.g., T cells and / or γδ cells. For example, before culturing the sample, the sample can be enriched for T cells or γδ T cells. Enrich for cells or deplete αβ T cells or deplete non-γδ T cells from a sample. In one embodiment, the sample is first depleted of αβ T cells, and then Enrichment or depletion for CD3+ cells can be performed using techniques known in the art, e.g., Antibodies that bind to molecules on the cell surface associated with the phenotype to be enriched / removed are used. This can be achieved using coated magnetic beads.

[0061] The presence of cell types other than lymphocytes in cell culture can inhibit Vδ1 cell expansion. Unwanted cells, e.g., stromal, epithelial, tumor, and / or feeder cells, may be removed prior to culturing. Thus, in one embodiment, the cell population is directly attached to the stromal cells during culture. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, and keratinocytes. , endothelial cells, and non-hematopoietic tumor cells. Preferably, lymphocytes are fibroblasts in culture. In one embodiment, the cell population is not in direct contact with epithelial cells during culture. In one embodiment, the cell population is not contacted with tumor cells and / or feeder cells during culture. Do not come into direct contact with the cells.

[0062] In one embodiment, the method comprises culturing V51 T cells in the absence of substantial stromal cell contact. In a further embodiment, the method comprises: The method includes culturing Vδ1 T cells in the presence of a VEGF-dependent agonist.

[0063] In one embodiment, the method comprises using a medium that is substantially free of serum (e.g., a serum-free medium). or medium containing serum replacement (SR). In one embodiment, the method comprises culturing in serum-free medium. For the supernatant medium, serum substitutes are chemically formulated to avoid the use of human or animal-derived serum. Mention may also be made of serum replacement media, which are based on defined components. In the method, the serum-containing medium (e.g., human AB serum or fetal bovine serum (FBS)) is used. In one embodiment, the medium contains a serum replacement. In embodiments, the medium is free of animal-derived products.

[0064] Samples cultured in serum-free media avoid issues related to serum filtration, precipitation, contamination, and delivery. It will be appreciated that animal-derived products have the advantage of avoiding human It is not preferred for use in the manufacture of clinical-grade therapeutics. The use of serum-free medium to isolate AB serum resulted in fewer cells obtained from the sample compared to the use of medium containing AB serum. Greatly increases the number of cells.

[0065] In one embodiment, the anti-V51 antibody or fragment thereof is in soluble or immobilized form For example, the antibody or fragment thereof can be administered to V51 T cells in a soluble form. Alternatively, the antibody or fragment thereof may be bound to a surface such as a bead or a plate. When combined or covalently bound (i.e., in immobilized form), administered to Vδ1 T cells In one embodiment, the antibody can be applied to wells, such as Fc-coated wells. Alternatively, the antibody or fragment thereof may be attached to the surface of a cell (e.g., In another embodiment, the antibody is immobilized on the surface of an antigen-presenting cell (APC). When the group is in contact with the antibody, it is not immobilized on the surface.

[0066] The cell population contacted by the anti-Vδ1 antibody or fragment thereof can be obtained from a variety of sample types. (Methods of isolation are further described below.) In one embodiment, the sample , a non-hematopoietic tissue sample. References include skin (e.g., human skin) and intestine (e.g., human intestine). Non-hematopoietic tissues include blood, In one embodiment, the tissue is other than bone marrow, lymphoid tissue, lymph node tissue, or thymus tissue. In some embodiments, the non-hematopoietic tissue sample is skin (e.g., human skin). Cell populations (e.g., γδ T cells) are obtained from certain types of biological fluid samples, such as blood or synovial fluid. In some embodiments, the cell population (e.g., γδ T cells) is not It can be obtained from skin (e.g., human skin) that can be obtained by methods known in the art. The cell population is then subjected to a synthetic scanning technique configured to promote cell migration from a non-hematopoietic tissue sample. It can be obtained from non-hematopoietic tissue samples by culturing them on the fold. Alternatively, the method can be used to detect cells in the digestive tract (e.g., colon or intestine), mammary gland, lung, prostate, liver, spleen, etc. , cell populations obtained from the pancreas, uterus, vagina, and other skin membranes, mucosa, or serous membranes (e.g., γ δ T cells).

[0067] In an alternative embodiment, the sample is a hematopoietic sample or a fraction thereof (i.e., a cell population). (The antibody fragment is obtained from a hematopoietic sample or a fraction thereof.) References herein to blood (e.g., peripheral blood or umbilical cord blood), bone marrow, lymphatic tissue, lymph nodes The sample preferably includes peripheral blood, thymus tissue, and fractions or enriched portions thereof. or umbilical cord blood, or blood containing fractions thereof, including buffy coat cells, white blood cells, Hemopheresis products include peripheral blood mononuclear cells (PBMCs) and low-density mononuclear cells (LDMCs) In some embodiments, the sample is human blood or a fraction thereof. The cells are collected by a density gradient. It can be obtained from a sample of blood using techniques known in the art such as fractional centrifugation. For example, whole blood is layered onto an equal volume of FICOLL-HYPAQUE, then centrifuged at 400 × g for 15–30 minutes at room temperature. The material at the interface contains low density mononuclear cells, which can be collected by centrifugation. Then, wash in culture medium and centrifuge at 200 x g and room temperature for 10 min.

[0068] The cell population can be obtained from a cancer tissue sample, for example, a breast or prostate tumor (i.e., (i.e., γδ T cells can also reside in cancer tissue samples.) In some embodiments, The cell population can be obtained from a human cancer tissue sample (e.g., solid tumor tissue). In the present invention, the cell population is a sample other than human cancer tissue (e.g., a sample not containing a significant number of tumor cells). For example, the cell population may be isolated from adjacent or neighboring cancer tissue. It may also be derived from an area of ​​damaged skin (e.g., healthy skin). In one embodiment, the cell population is not obtained from cancer tissue (eg, human cancer tissue).

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

[0070] (expansion of gamma delta T cells) In one embodiment, the modulation comprises activation of V51 T cells, in particular expansion of V51 T cells. Therefore, according to one aspect of the present invention there is provided an ex vivo method for expanding V51 T cells, comprising: and administering an anti-Vδ1 antibody or a fragment thereof as defined in the specification to a cell population comprising Vδ1 T cells. Such expansion of V51 T cells involves the selection of the number of V51 T cells. This can be achieved by selective expansion and / or by promoting survival of V51 T cells. In one embodiment, the expansion of V51 T cells is achieved by injecting an anti-TCR delta1 variable antibody or a fragment thereof into the culture. This includes administering to V51 T cells in vivo (i.e., in vitro or ex vivo). The cells may be added to a mixed cell population, e.g., other lymphoid cell types (e.g., αβ T cells or NK cells). may be present in a cell population comprising:

[0071] Therefore, the present invention provides a method for generating enriched γδ T cell (e.g., Vδ1 T cell) populations. The enriched population may be obtained by ex vivo methods (e.g., by culturing cells collected from a patient / donor). The isolated mixed cell population (obtained from a sample) or a purified fraction thereof is treated with an antibody or a fragment thereof. The antibody can be produced from the mixed cell population by a method comprising contacting the mixed cell population with The antibody (or a fragment thereof) binds to an epitope specific for the Vδ1 chain of the γδ TCR, thereby inhibiting V Selectively expand δ1 T cells.

[0072] Also provided are expanded V51 T cells obtained according to the methods defined herein. According to this aspect of the invention, such an expanded population of V51 T cells is It will be understood that the cells may be obtained and / or expanded in vitro or ex vivo. In one embodiment, the expanded V51 population obtained according to the methods defined herein and a method for isolating and expanding the V51 population in vitro or ex vivo. .

[0073] The antibodies or fragments thereof described herein can be used to expand γδ T cells (e.g., Vδ1 T cells). These methods can be performed in vitro. When this expansion method is carried out in vitro, the antibody (or fragment thereof) obtained as described above is It can be applied to isolated γδ T cells (e.g., Vδ1 T cells). In this study, γδ T cells are expanded from a cell population isolated from a non-hematopoietic tissue sample. In another embodiment, γδ T cells are isolated from a hematopoietic tissue sample, e.g., a blood sample. It is expanded from a population of cells.

[0074] Expansion of γδ T cells (e.g., Vδ1 T cells) can be achieved by treating a sample with an antibody or antibodies described herein. The method may include culturing the cells in the presence of a fragment of the present invention and cytokines, such as: Interleukins, lymphokines, interferons, colony-stimulating factors, and chemokines In one embodiment, the cytokine is interleukin-2 (IL-2). L-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL- 7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-12 (IL-1 2), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-33 ( IL-33), insulin-like growth factor 1 (IGF-1), interleukin-1β (IL-1β), interferon The present invention relates to a method for treating inflammatory bowel disease, and to a method for treating inflammatory bowel disease, and the method is selected from the group consisting of IFN-γ, stromal cell-derived factor-1 (SDF-1). References to cytokines listed in

[14] have similar physiological effects on Vδ1 T cells in culture. The present invention includes any compound that has the same activity as the cytokine in terms of its ability to promote the effect of the cytokine. It is understood that the term "antibody" can be used interchangeably with "antibody" and includes, but is not limited to, a mimetic or any functional equivalent thereof. It will be understood.

[0075] In one embodiment, the cytokine is a common cytokine receptor gamma chain (γ c )Fa In a further embodiment, γ c - Cytokines include IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-21, or a mixture thereof.

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

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

[0078] In one embodiment, the method comprises culturing the cell population in the presence of IL-2, IL-9, and / or IL-15. In a further embodiment, the method comprises cultivating IL-2 and / or IL-15 (i.e., The method includes culturing the cell population in the presence of an IL-1 receptor, i.e., IL-2, IL-15, or a combination thereof. In some embodiments, the methods involve administering IL-9 and / or IL-15 (i.e., IL-9, IL-15, or In one embodiment, the method comprises culturing the cell population in the presence of a soluble or non-soluble form ... in the presence of IL-2, IL-9, and / or IL-15, and additional growth factors (e.g., IL-21). In another embodiment, the method comprises administering to the patient a growth factor other than IL-2 and / or IL-15. In an alternative embodiment, the method comprises culturing the cell population in a medium lacking Culturing the cell population in a medium lacking growth factors other than IL-9 and / or IL-15. In one embodiment, the method comprises culturing cells from a basal medium supplemented with IL-2, IL-9, and / or IL-15. In a further embodiment, the method comprises culturing the cell population in a medium comprising: The method comprises culturing a cell population in a medium consisting of a basal medium supplemented with IL-2 and / or IL-15. nothing.

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

[0080] In one embodiment, the method comprises culturing the cell population in the presence of IL-4. Physiological effects on Vδ1 T cells promoted by (described in WO2016 / 198480) The results showed that NKG2D and NCR expression levels were reduced, cytotoxic function was inhibited, and selective survival was improved. Furthermore, the absence of IL-4 during the later period of culture alters the physiological properties of the cells to anti-tumor or It has previously been shown that use as an antiviral treatment can alter the phenotype appropriately. Thus, in one embodiment, the expansion method comprises growing cells having IL-4-like activity. The method further comprises culturing the sample in the absence of a factor, e.g., IL-4. Therefore, the expansion method involves culturing the sample in the absence of IL-4.

[0081] In one embodiment, the cytokine is a growth factor with interleukin-15-like activity. , i.e., with regard to its ability to promote similar physiological effects on Vδ1 T cells in culture. Any compound that has the same activity as IL-15, including IL-15 and IL-15 mimetics, or IL-15 These include, but are not limited to, any functional equivalent of IL-15, including IL-2 and IL-7. Physiological effects on cultured Vδ1 T cells promoted by IL-15, IL-2, and IL-7 (WO20 16 / 198480) are essentially equivalent, i.e., more cytotoxic, expressions. Furthermore, IL-2, IL-7, and IL-1 during the initial period of culture were The absence of -15 inhibits the proliferation of contaminating cells (TCRαβ+ T cells and It has previously been shown that this contributes to starvation and apoptosis of Vδ2+ T cells (including Vδ2+ and Vδ2+ T cells). Therefore, in one embodiment, the expansion method first involves inducing a growth factor having IL-15-like activity. This involves culturing the sample in the absence of the antigen.

[0082] Thus, in one embodiment, the method comprises culturing a cell population in a first culture medium comprising IL-4. and then culturing the cell population in a second culture medium containing IL-15. nothing.

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

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

[0085] As described herein, the culture medium can further enhance the expansion of V51 T cells. It may also contain other growth factors, including cytokines that can stimulate the growth of cells. Examples of cytokines include (i) IFN-γ and any growth factor with IFN-γ-like activity, (ii) IL-21 and any growth factor having IL-21-like activity, and (iii) IL-1β and any growth factor having IL-1β-like activity. Other components that can be added include, but are not limited to, any growth factors. Examples of long-term factors include costimulatory molecules, such as human anti-SLAM antibodies, any soluble ligand of CD27, and the like. Any combination of these growth factors may be used. It can be included in the culture medium.

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

[0087] In one embodiment, the method comprises administering to a subject a subject a therapeutically effective amount of IL-15 as well as IL-2, IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, IFN-γ, human platelet lysate (HPL), and and culturing the cell population in the presence of a factor selected from the group consisting of stromal cell-derived factor-1 (SDF-1). This includes:

[0088] The expansion of γδ T cells is performed by culturing the sample in the presence of at least one additional T cell mitogen. The method may include culturing the cells containing "T cell mitogen" (which may be referred to as "γδ TCR antigen"). The term "agonist" (which may also be referred to as "agonist") includes, but is not limited to, phytohemagglutinins. Plant lectins such as phenobarbital (PHA) and concanavalin A (ConA), as well as lectins of non-plant origin, It means any agent that can stimulate T cells through TCR signaling, including In an embodiment, the T cell mitogen is an anti-CD3 monoclonal antibody (mAb). Other mitogens include phorbol 12-myristate-13-acetate (TPA) and its related compounds such as mezerein, or bacterial compounds (e.g., Staphylococcus aureus enterotoxin T cell mitogens include soluble T cell mitogens (STPs) and streptococcal protein A (SEA). Multiple T cell mitogens may be used in this expansion method. This may also be done.

[0089] As used herein, "expanded" or "expanded population of γδ T cells" refers to A reference to a group that is larger than or contains more than the unexpanded group. Such a population may be large or small in number. The population may be a mixed population, or may involve expansion of a subset or specific cell types within the population. The term "expanded" will be understood to refer to the process of producing an expanded or expanded population. Therefore, the expanded or expanded population may be affected if the expansion step is not performed or is not performed properly. The population may be larger in number or contain more cells than the population before any expansion step. Any number given herein to indicate magnification (e.g., a factor increase or magnification) may be used. Magnification (fold increase) refers to an increase in the number or size of a group of cells or the number of cells, and is It will be further understood that the amount of

[0090] In one embodiment, the method comprises culturing the cell population for at least 5 days (e.g., at least 6 days). days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 18 days days, at least 21 days, at least 28 days, or longer, e.g., 5 to 40 days , 7 to 35 days, 14 to 28 days, or about 21 days). The method includes culturing the cell population for at least 7 days, for example, at least 11 days, or at least 1 This involves culturing for 4 days.

[0091] In a further embodiment, the method comprises culturing the cell population for a sustained period (e.g., at least at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 18 days, at least 21 days, at least 28 days, or longer , e.g., 5 to 40 days, 7 to 35 days, 14 to 28 days, or about 21 days), expanded γδ T cells The method includes culturing the strain in an amount effective to produce a population of the strain.

[0092] In one embodiment, the cell population is cultured for 5 to 60 days, for example, at least 7 to 45 days, for example, 7 to 21 days. The cells are cultured for 1-40 days, or 7-18 days. When including a culture period, the isolation and expansion process may, in some embodiments, last from 21 to 39 days. It is possible.

[0093] This method involves periodically adding an anti-Vδ1 antibody or a fragment thereof and / or a growth factor during the culture period. For example, the anti-Vδ1 antibody or a fragment thereof and / or growth factor may be administered every 2 to 5 days. More preferably, it can be added every 3 to 4 days. or fragments thereof and / or growth factors after 7 days of culture and every 3-4 days thereafter; It is added.

[0094] The expansion method provides a population of expanded γδ T cells that are greater in number than the reference population. In some embodiments, the expanded population of γδ T cells (e.g., Vδ1 T cells) is subjected to an expansion step. the number of isolated γδ T cells prior to the expansion step is higher than the number of isolated γδ T cells prior to the expansion step (e.g., At least twice as many, at least five times as many, at least 10 times as many as the γδ T cell population double, at least 25 times the number, at least 50 times the number, at least 60 times the number, at least 70 times the number double, at least 80 times the number, at least 90 times the number, at least 100 times the number, at least 2 times the number 00 times, the number is at least 300 times, the number is at least 400 times, the number is at least 500 times, the number is 600 times, In one embodiment, the expanded A population of γδ T cells (e.g., Vδ1 T cells) was cultured for the same period of time in the absence of the antibody or fragment thereof. In one embodiment, the expanded γδ T cells (e.g., Vδ1 T cells) are more numerous than populations cultured for the same time in the presence of TS8.2 or TS-1.

[0095] The expansion method generates expanded V51 T cells with a higher percentage of V51 T cells than the reference population. In some embodiments, the expanded population of V51 T cells is , greater than about 50% Vδ1 T cells, e.g., about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87% , 90%, 91%, 92%, 93%, 94%, or greater than 95% vδ1 T cells. In such cases, the expanded V51 T cell population may be greater than about 85% V51 T cells, e.g., greater than about 90% V51 T cells. containing Vδ1 T cells.

[0096] In some embodiments, the expanded population of γδ T cells (e.g., Vδ1 T cells) is Less than about 10% αβ T cells, e.g., about 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, In a further embodiment, the expanded The population of Vδ1 T cells contains approximately less than 1% of αβ T cells. T cells with αβ receptors Cell populations that are highly reactive and therefore suitable for administration to patients in the context of the present invention include: It can only contain low levels of αβ T cells. This allows for selective expansion of the Vδ1 T cell population, which allows for the elimination of αβ T cells. This reduces the need for extensive purification methods after expansion.

[0097] In some embodiments, the expanded population of γδ T cells (e.g., Vδ1 T cells) is Less than about 10% Vδ2 T cells, e.g., about 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, In a further embodiment, the expanded The population of V51 T cells contains less than about 0.5% V52 T cells.

[0098] In some embodiments, the expanded population of γδ T cells (e.g., Vδ1 T cells) is Less than about 10% natural killer (NK) cells (also referred to as CD56+CD3− cells), e.g., about 5%, In further embodiments, the NK cells comprise less than 4%, 3%, 2.5%, 2%, 1.5%, or 1% NK cells. In this study, the expanded V51 T cell population contained less than about 2% NK cells.

[0099] Increased or decreased expression of cell surface markers, including CD27, CD69, TIGIT, PD-1, and TIM-3 may also or alternatively be used to characterize one or more expanded V51 T cell populations. In some embodiments, the expanded population of V51 T cells expresses high levels of CD27 expressing (CD27 high ) For example, an expanded size of more than about 70%, e.g., more than about 80%, 85%, or 90% A population of V51 T cells expresses CD27 (i.e., CD27+). , the expanded Vδ1 T cell population may be, for example, significantly larger than the isolated Vδ1 T cell population before expansion. In some embodiments, expanded V51 A population of T cells expresses low levels of CD69, TIGIT, PD-1, and / or TIM-3. For example, Less than about 40%, e.g., less than about 30%, of the expanded Vδ1 T cell population expresses CD69, TIGIT, PD-1 In some embodiments, the expanded V51 T cell population expresses TIM-3, TIM-4, TIM-5, TIM-6, TIM-7, TIM-8, TIM-9, TIM-10, TIM-11, TIM-12, TIM-13, TIM-14, TIM-15, TIM-16, TIM-17, TIM-18, TIM-19, TIM-20, TIM-21, TIM-22, The cluster consists of CD69, TIGIT, PD-1, and TIM-3, compared to the isolated Vδ1 T cell population. have a lower average expression of one or more of the markers selected from:

[0100] Many basal culture media are suitable for use in the expansion of γδ T cells, particularly those containing serum or blood. Media in the presence of plasma, such as AIM-V, Iscoves' medium, and RPMI-1640 (Life Technologies); EXVIVO-10, EXVIVO-15, or EXVIVO-20 (Lonza) are available. Other media factors as defined, e.g., serum, serum proteins, and selection agents, e.g., antibiotics For example, in some embodiments, 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Li fe Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and and Ser; 1×MEM non-essential amino acids (Life Technologies), and 10 μl / L β-mercaptoethanol In an alternative embodiment, AIM-V medium is added to RPMI-1640 medium containing CTS Immune The medium may be supplemented with a commercially available serum replacement and amphotericin B. In one embodiment, the medium The compositions may be further supplemented with IL-2, IL-4, IL-9, and / or IL-15 as described herein. Advantageously, cells are grown in a suitable culture medium containing 5% CO2 during isolation and / or expansion. They are incubated at 37°C in a humid atmosphere.

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

[0102] The method for expanding γδ T cells can be performed in less than 5 days (e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3 ... Less than 0.8 days, Less than 3.7 days, Less than 3.6 days, Less than 3.5 days, Less than 3.4 days, Less than 3.3 days, Less than 3.2 days, Less than 3.1 Less than days, Less than 3.0 days, Less than 2.9 days, Less than 2.8 days, Less than 2.7 days, Less than 2.6 days, Less than 2.5 days, 2.4 days Less than, Less than 2.3 days, Less than 2.2 days, Less than 2.1 days, Less than 2.0 days, Less than 46 hours, Less than 42 hours, 38 hours The population doubling time may be less than 3 hours, less than 35 hours, or less than 32 hours.

[0103] (Method for isolating γδ T cells) As described herein, the antibodies (or fragments thereof) are capable of inhibiting γδ T cells in culture, i.e., That is, it can be applied to γδ T cells obtained from a sample. The population is isolated from the sample prior to administering the anti-V51 antibody or fragment thereof. A method for modulating (in particular expanding) T cells, comprising administering to a subject an anti-V51 antibody as defined herein or The fragment is then transferred to a population of γδ T cells isolated from the sample (e.g., a cell population containing Vδ1 T cells). The method includes administering to

[0104] The gamma delta T cells that predominate in non-hematopoietic tissues (i.e., tissue-resident) are primarily delta The anti-V51 antibodies described herein contain a variable 1 chain and are therefore isolated from non-hematopoietic tissues. In one embodiment, the sample is particularly useful for γδ T cells. Alternatively, the methods of the present invention can be used to isolate hematopoietic tissue samples containing primarily Vδ1 chains. It is possible to expand the population of V51 T cells in a sample that does not have V51 T cells, for example, a blood sample. Thus, the method can be used to increase the number of V51 T cells in a sample.

[0105] The present invention relates to the "isolation" or "isolating" of cells, particularly γδ T cells. References herein may be made to cells removed, isolated, or purified from a tissue or pool of cells. refers to a method or process by which a substance is extracted, concentrated, or otherwise removed. References to the substance include the use of terms such as "separated," "removed," "purified," and "enriched." It will be understood that the term "γδ T cells" encompasses the terms "γδ T cells" and similar terms. from tissue samples or from stromal cells (e.g., fibroblasts or epithelial cells) of non-hematopoietic tissues Such isolation may alternatively or additionally involve the isolation or separation of cells from other structures. may include isolating or separating γδ T cells from blood cells (e.g., αβ T cells or other lymphocytes). Isolation is performed for a defined period of time, e.g., when tissue explants or biopsies are placed in isolation culture. Starting from when cells are isolated, for example, by centrifugation or by expanding the isolated cell population, may be removed from the culture by other means for transfer or used for other purposes, or may end when the original tissue explant or biopsy is removed from culture. The isolation step may take at least about 3 days to about 45 days. In a further embodiment, the isolation process is from at least about 10 days to at least 28 days. The isolation process is at least 14 days to at least 21 days. Sunday, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th , 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, The incubation period may be 31 days, 32 days, about 35 days, about 40 days, or about 45 days. Although proliferation may be insignificant, this does not necessarily mean that proliferation of the cells is absent. Indeed, it is readily apparent to those skilled in the art that isolated cells are not It may also begin to divide, generating a plurality of such cells within the isolation vessel containing the sample. It is recognized that it is possible.

[0106] Thus, "isolated γδ T cells," "isolated γδ T cell population," or "isolated References herein to a "population of isolated γδ T cells" refer to the presence or absence of a sample, e.g., a non-hematopoietic tissue of origin. Is it isolated, separated, removed, or purified from a tissue sample? or enriched so that the cells are substantially different from the cells contained in the intact (non-hematopoietic tissue) sample. It will be understood that "isolated V51 T cells" refers to γδ cells that have not been in contact with a T cell. , "Isolated Vδ1 T cell population", "Isolated Vδ1 T cell population", "Isolated Vδ1 "Isolated Vδ1 T cell population," "Isolated Vδ1 T cell population," or "Isolated Vδ1 T cell population" References herein to the presence of a cell in a tissue sample include those in which the cell is in substantial contact with cells contained in an intact (non-hematopoietic tissue) sample. The sample, e.g., the non-hematopoietic tissue sample of origin, may be isolated, separated, or removed so as not to It will be understood that this refers to depleted, purified or enriched V51 T cells.

[0107] The cell population may be lymphocytes, particularly from a human or non-human animal sample, such as a non-hematopoietic tissue sample. These can be obtained by any suitable method that allows for the isolation of V51 T cells. Such a method is described in Clark et al. (2006) J. Invest. Dermatol. 126(5): 1059-70. This includes a three-dimensional skin explant protocol for isolating lymphocytes from human skin. The explants are attached to a synthetic scaffold, and the explants are then removed from the scaffold. The synthetic scaffold can promote lymphocyte migration onto the scaffold. Synthetic scaffolds refer to non-natural three-dimensional structures suitable for supporting growth. - (e.g., natural or synthetic polymers, e.g., polyvinylpyrrolidone, polymethylmethacrylate, acrylate, methylcellulose, polystyrene, polypropylene, polyurethane), Calcium phosphate, calcium aluminate, calcium hydroxide siapatite), or metals (tantalum, titanium, platinum, and metals of the same element group as platinum, Constructed from materials such as titanium, hafnium, tungsten, and alloy combinations thereof Biological factors (e.g., collagen (e.g., collagen I or collagen II) I), fibronectin, laminin, integrin, angiogenic factors, anti-inflammatory factors, glycosaminoglycans aminoglycans, vitrogens, antibodies and their fragments, cytokines (e.g., IL-2 or IL-15, and combinations thereof) onto the scaffold according to methods known in the art. The scaffold material is coated on the surface or encapsulated in the scaffold material to promote cell adhesion and migration. This and other methods can be used to enhance the survival or proliferation of some Cell populations can be isolated from other non-hematopoietic tissue types, such as intestine, prostate, and breast. Another example of a suitable isolation method is to isolate a cell population and / or sample from γδ T cells, particularly Vδ1 T cells. The cells are cultured in the presence of cytokines and / or chemokines sufficient to induce cell isolation or separation. The method utilizes a "crawling" technique, which may involve culturing cells from a sample (e.g., a non-hematopoietic tissue sample). Isolating γδ T cells may involve culturing the sample in the presence of IL-2 and IL-15.

[0108] Non-hematopoietic tissue-resident lymphocytes can be isolated, for example, by vigorous pipetting, from interstitial cells, e.g. For example, lymphocytes can be harvested and isolated from skin fibroblasts. To retain fibroblast aggregates that may break apart during The lymphocytes can be further washed by passing them through an iron mesh. They can also be isolated using fluorescence or magnetic related cell sorting.

[0109] Alternatively, the isolation of γδ T cells from a sample (e.g., a hematopoietic tissue sample) can be performed by subjecting the sample to the method described in WO2012 / 1 T cell mitogens (e.g., γδ TCR agonists) and cytotoxic T cell agonists described in US Pat. No. 5,6958. cytokines (especially the common cytokine receptor gamma chain (γ c The existence of cytokines of the ) family Another alternative may involve culturing the cells in the presence of a sample (e.g., a hematopoietic tissue sample). Isolation of γδ T cells can be achieved by subjecting a sample to T cell mitogens as described in WO2016 / 198480. and culturing in the presence of cytokines.

[0110] Isolation of γδ T cells involves culturing the sample in the presence of at least one cytokine. For example, the method may include culturing the sample in the presence of at least one agent, e.g., a chemokine. The chemokine may be selected depending on the γδ T cells to be isolated. It will be further understood that chemokines may be present in the sample used to isolate γδ T cells. The values ​​are varied and selected depending on the

[0111] Isolation of γδ T cells involves culturing the sample in the presence of at least one cytokine. The cytokines may be different from those used in the initial culture. Good too.

[0112] The isolation method may include culturing the sample. Reference may be made to isolated, separated, removed, purified, or concentrated from a sample. of the sample containing the cells, as required and / or preferred by the cells and / or the sample. Such culture conditions include the addition of growth factors and / or essential nutrients to the medium. The cells or cell populations to be isolated from the sample may be adapted according to the cell or cell population to be isolated from the sample. It is understood that the method may be adapted according to the cell or cell population to be isolated and expanded. It will be understood.

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

[0114] In certain embodiments, the γδ T cells are recovered from the culture of the sample after culturing the sample. Recovery of γδ T cells can be achieved by physical recovery of γδ T cells from culture, collection of γδ T cells from other lymphocytes (e.g. isolation of γδ T cells from other T cells present in the sample (αβ T cells and / or NK cells) Isolation and / or differentiation of γδ T cells from cells, e.g., stromal cells, e.g., fibroblasts. In one embodiment, γδ T cells can be isolated by mechanical means (e.g., pipetting). In a further embodiment, the γδ T cells are recovered by magnetic separation and / or In yet a further embodiment, γδ T cells are recovered by labeling. The cells are collected by a fluorescence in situ hybridization (FACS) technique, for example. γδ T cells are recovered by specific labeling of γδ T cells. Recovery may involve physical removal of the sample from the culture, transfer to a separate culture vessel, or collection into a separate or It will be understood that this may involve transfer to different culture conditions.

[0115] Such recovery of γδ T cells is sufficient to obtain a population of isolated γδ T cells from the sample. It will be understood that the treatment is carried out after a suitable period of time. In one embodiment, the γδ T cells , at least 1 week, at least 10 days, at least 11 days, at least 12 days after culturing the sample , at least 13 days, or at least 14 days later. Preferably, the γδ T cells are harvested after 40 days. Within, for example, within 38 days, within 36 days, within 34 days, within 32 days, within 30 days, within 28 days, within 26 days In one embodiment, the γδ T cells are harvested within 24 days or within 24 days from culture of the sample. In a further embodiment, the γδ T cells are harvested after at least 14 days from the culture of the sample. The samples will be collected 14 to 21 days after the procedure.

[0116] In one embodiment, the sample is prepared in a medium that is substantially free of serum (e.g., serum-free medium or The cells are cultured in a medium containing serum replacement (SR). The material is cultured in a serum-free medium. Such serum-free medium is suitable for use in cells cultured in a serum-free medium, such as a medium containing a serum substitute that is suitable for human or animal use. Serum replacement media are also available that are based on chemically defined components to avoid the use of derived serum. In one embodiment, the medium is free of animal-derived products. In the method, the sample is incubated in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). It is cultivated in.

[0117] The culture medium may further comprise other components that can aid in the growth and expansion of γδ T cells. Examples of other components that can be added include plasma or serum, purified proteins such as albumin, etc. , lipid sources such as low-density lipoprotein (LDL), vitamins, amino acids, steroids, and and any other supplementary substances that support or promote cell growth and / or survival, Not limited to these.

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

[0119] (Antibody or its fragment) Provided herein are antibodies specific for the delta variable 1 chain (Vδ1) of the γδ T cell receptor (TCR). It is an antibody or fragment thereof capable of binding to the antibody.

[0120] In one embodiment, the antibody or fragment thereof is an scFv, Fab, Fab', F(ab')2, Fv, variable domain, or The antibody may be a polypeptide (e.g., a VH or VL), a diabody, a minibody, or a monoclonal antibody. In a further embodiment, the antibody or fragment thereof is an scFv.

[0121] The antibodies described herein can be of any class, e.g., IgG, IgA, IgM, IgE, IgD, or can be of these isotypes and contain kappa or lambda light chains. In one embodiment, the antibody is an IgG antibody, e.g., an IgG1, IgG2, or IgG3 antibody. In a further embodiment, the antibody is at least one of: modified to confer properties such as reduced effector function or Mutated to extend the shear life, alter ADCC, or improve hinge stability. Such a format may have an Fc-linked antibody, such as an IgG format. Modifications are well known in the art.

[0122] In one embodiment, the antibody or fragment thereof is human. The fragment may be derived from a human immunoglobulin (Ig) sequence. The framework and / or constant regions may be derived from human Ig sequences, particularly human IgG sequences. The R, framework, and / or constant regions may be selected from the following for human Ig sequences, particularly human IgG sequences: The advantage of using a human antibody is that it may be less immunogenic or less potent in humans. is that it is non-immunogenic.

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

[0124] Alternatively, the antibody or fragment thereof may be derived from a non-human species, e.g., a mouse. The human antibody may be modified to increase its similarity to antibody variants naturally produced in humans. Thus, the antibody or fragment thereof may be partially or fully humanized. Thus, in one embodiment, the antibody or fragment thereof is humanized.

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

[0126] In one embodiment, the epitope may be an activating epitope of γδ T cells. "Activating" epitopes are those that inhibit, for example, TCR function, e.g., degranulation, TCR downregulation, cytotoxicity, , proliferation, recruitment, increased survival or resistance to exhaustion, intracellular signaling, cytokines These include stimulating transcription or growth factor secretion, phenotypic changes, or changes in gene expression. For example, binding of an activating epitope can stimulate a γδ T cell population, preferably Vδ1+ These antibodies can be used to stimulate the expansion (i.e., proliferation) of T cell populations. It can regulate γδ T cell activation and thereby regulate the immune response. In one embodiment, binding of the activating epitope downregulates γδ TCR. In an alternative embodiment, binding of an activating epitope activates degranulation of γδ T cells. In further or alternative embodiments, binding of the activating epitope is γδ Activates T-cell killing.

[0127] Alternatively, the antibody (or fragment thereof) may block by interfering with the binding or interaction of another antibody or molecule. In one embodiment, the present invention provides a method for blocking V51 and inhibiting TCR binding (e.g., The present invention provides an isolated antibody or fragment thereof that blocks (by steric hindrance) Vδ1. The antibody may thereby interfere with TCR activation and / or signal transduction. It can be an inhibitory epitope for T cells. An "inhibitory" epitope is, for example, one that blocks TCR function. This can include disrupting the TCR activation.

[0128] The epitope is preferably located on at least one extracellular, soluble, proteasome of the V51 chain of the γδ TCR. It consists of an aqueous, outer, or cytoplasmic portion.

[0129] In particular, the epitope is found in the hypervariable region of the Vδ1 chain of the γδ TCR, specifically in the CDR3 of the Vδ1 chain. In a preferred embodiment, the epitope does not comprise the Vδ1 chain of the γδ TCR. Such binding occurs within the non-variable regions of the TCR, particularly the highly variable CDR3. It will be understood that, without limitation, the MHC-like peptide allows for specific recognition of the Vδ1 chain. The various γδ TCR complexes that recognize peptides or antigens are capable of this function solely through the presence of the Vδ1 chain. Therefore, any Vδ1 chain-containing γδ TCR can be recognized independently of the specificity of γδ TCR. It is understood that the antibody or fragment thereof defined herein can be used to recognize the antibody or fragment thereof. In one embodiment, the epitope is the amino acid region 1-24 of SEQ ID NO: 1 and / or 35 to 90, for example, one or more amino acids in the portion of the Vδ1 chain that is not part of the CDR1 and / or CDR3 sequence In one embodiment, the epitope comprises amino acid residues in the amino acid region 91-105 of SEQ ID NO: 1. It does not include amino acid residues within (CDR3).

[0130] In a manner similar to the well-characterized αβ T cells, γδ T cells target a different set of somatic cells. utilizes cellularly rearranged variable (V), diversity (D), joining (J), and constant (C) genes, but δ T cells contain fewer V, D, and J segments than αβ T cells. In the present invention, the epitope bound by the antibody (or fragment thereof) is the J region of the Vδ1 chain (e.g., Four J regions encoded in the human Delta 1 chain germline: SEQ ID NO: 131 (J1 * 0) or 132 (J2 * 0) or 133 (J3 * 0) or 134 (J4 * 0) does not contain an epitope found in one of In this case, the epitope bound by the antibody (or its fragment) is located in the C-region of the Vδ1 chain (e.g. For example, SEQ ID NO: 135 (C1 * 0)) epitopes found in In one embodiment, the epitope bound by the antibody (or fragment thereof) is It does not contain the epitope found in the N-terminal leader sequence of the Vδ1 chain (e.g., SEQ ID NO: 129). Therefore, the antibody or fragment binds only in the V region of the Vδ1 chain (e.g., SEQ ID NO: 130). Thus, in one embodiment, the epitope is located in the V region of a γδ TCR (e.g., It consists of an epitope within amino acid residues 1 to 90 of SEQ ID NO:1.

[0131] Reference to the epitope is made to SEQ ID NO:1: [ka] the sequence described in Luoma et al. (2013) Immunity 39: 1032-1042, which is shown as and RCSB Protein Data Bank entries: for Vδ1 sequences derived from 4MNH and 3OMZ It is done.

[0132] SEQ ID NO: 1 contains the V region (also called variable domain), D region, J region, and TCR constant region. The V region comprises amino acid residues 1 to 90, and the D region comprises amino acid residues 91 to 92. 104, the J region comprises amino acid residues 105 to 115, and the constant region comprises amino acid residues 116 to 209. In the V region, CDR1 is defined as amino acid residues 25 to 34 of SEQ ID NO: 1, and CDR2 is CDR1 is defined as amino acid residues 50 to 54 of SEQ ID NO: 1, and CDR2 is defined as amino acid residues 93 to 104 of SEQ ID NO: 1. (Xu et al., PNAS USA 108(6):2414-2419(2011)).

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

[0134] In a further embodiment, the antibody or fragment thereof comprises an antibody having a sequence identical to amino acid residues 1-90 of SEQ ID NO: 128. The polymorphic V region containing the epitope described herein is further recognized. When defining a group, amino acids 1 to 90 of SEQ ID NO: 1 and polymorphic germline variant sequences (amino acids 1 to 90) are used. 90, SEQ ID NO: 128) can be considered interchangeable. The antibodies of the present invention may be used in conjunction with this germline For example, the antibodies defined herein can recognize both variants of the sequence. The fragment contains one or more amino acid residues in the amino acid region 1 to 24 and / or 35 to 90 of SEQ ID NO: 1. When described as recognizing an epitope containing the same region of SEQ ID NO: 128; Specifically, it also refers to the amino acid region 1 to 24 and / or 35 to 90 of SEQ ID NO:128.

[0135] In one embodiment, the antibody or fragment thereof comprises one or more amino acid residues within amino acid region 1-90 of SEQ ID NO: 1. and the amino acids located in the same positions in regions 1 to 90 of SEQ ID NO: 128. Specifically, in one embodiment, the antibody or fragment thereof defined herein is a human germline antibody. recognizes a germline epitope, wherein the germline is an alanine (A) at position 71 of SEQ ID NO: 1 or It encodes either valine (V).

[0136] In one embodiment, the epitope is one or more, e.g., two, three, or more, of the described regions. The amino acid sequence may contain one, four, five, six, seven, eight, nine, ten, or more amino acid residues.

[0137] In a further embodiment, the epitope is one or more of the amino acid regions 3 to 20 of SEQ ID NO: 1 ( In an alternative embodiment, the amino acid sequence of the nucleotide ... The pitope may be one or more amino acid regions (e.g., amino acid regions 37-77) of SEQ ID NO: 1 (e.g., amino acid regions 50-54). In still further embodiments, the amino acid sequence comprises at least 5 amino acid residues, e.g., at least 10 amino acid residues. The epitope may be one or more (e.g., 5-20 or 3-17) amino acid regions of SEQ ID NO: 1. For example, 5 or more, for example, 10 or more) amino acid residues and the amino acid region 37 to 77 (for example, 62-77 or The amino acid sequence includes one or more (for example, five or more, for example, ten or more) amino acid residues within the amino acid sequence of ...

[0138] The antibody (or fragment thereof) need not bind to every amino acid within the defined range. It will be further understood that such epitopes are sometimes referred to as linear epitopes. For example, an epitope containing amino acid residues within the amino acid region 5 to 20 of SEQ ID NO: 1 can be prepared. The binding antibody may be selected from amino acid residues within the range, e.g., optionally, amino acids within the range (i.e., at each end of the range (i.e., amino acids 5, 9, 16, and 20), including and 20).

[0139] In one embodiment, the epitope is selected from amino acid residues 3, 5, 9, 10, 12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 Includes at least one of 17, 20, 37, 42, 50, 53, 59, 62, 64, 68, 69, 72, or 77 In a further embodiment, the epitope is amino acid residues 3, 5, 9, 10, 12 of SEQ ID NO:1. , 16, 17, 20, 37, 42, 50, 53, 59, 62, 64, 68, 69, 72, or 77, or 2 The amino acid sequence may contain one, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acids.

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

[0141] In a further embodiment, the epitope is selected from the amino acid regions: 5-20 and 62- 77; 50-64; 37-53 and 59-72; 59-77; or one or more amino acid residues within 3-17 and 62-69 In a further embodiment, the epitope comprises the amino acid region of SEQ ID NO: 1: and 62-77; 50-64; 37-53 and 59-72; 59-77; or one or more amino acids in the ranges 3-17 and 62-69 It consists of acid residues.

[0142] In a further embodiment, the epitope is selected from amino acid residues 3, 5, 9, 10, or comprising amino acid residues 12, 16, 17, 62, 64, 68, and 69 of SEQ ID NO: 1: 3, 5, 9, 10 , 12, 16, 17, 62, 64, 68, and 69. In a further embodiment, The loop comprises amino acid residues 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO: 1, or Amino acid residues 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO: 1 are preferred. In another embodiment, the epitope is selected from amino acid residues 37, 42, 50, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 9, 64, 68, 69, 72, 73, and 77, or amino acid residues 37, 42, 50 of SEQ ID NO: 1; In a further embodiment, epi is preferred. The topography comprises amino acid residues 50, 53, 59, 62, and 64 of SEQ ID NO: 1, or In a further embodiment, the amino acid residues are: 50, 53, 59, 62, and 64. The epitope comprises amino acid residues 59, 60, 68, and 72 of SEQ ID NO: 1, or The amino acid residues: 59, 60, 68, and 72 are preferred.

[0143] In one embodiment, the epitope is selected from the amino acid regions 5 to 20 and / or 62 to 77 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one or more amino acid residues within SEQ ID NO: 1. The amino acid sequence of the present invention is one or more amino acid residues within the amino acid regions 5-20 and 62-77 of the present invention. In embodiments, the epitope is one or more amino acid regions 5-20 or 62-77 of SEQ ID NO:1. The antibody or fragment thereof having such an epitope contains the amino acid residue 1245_P01_E07 or such antibodies or fragments thereof may comprise part or all of the sequence of 1245_P01 For example, one or more CDR sequences of 1245_P01_E07 or the VH and VL sequences of 1245_P01_E07 An antibody or fragment thereof having one or both of the sequences can bind to such an epitope. do.

[0144] In one embodiment, the epitope is one or more amino acids within the amino acid region 50 to 64 of SEQ ID NO:1. In a further embodiment, the epitope comprises amino acid region 5 of SEQ ID NO: 1. The epitope consists of one or more amino acid residues 0 to 64. An antibody having such an epitope or a fragment thereof The fragment may comprise part or all of the sequence of 1252_P01_C08 or may be a fragment of such an antibody or fragments thereof. A fragment of 1252_P01_C08 can be derived from 1252_P01_C08. For example, a fragment of 1252_P01_C08 can be derived from one or more CDR sequences of 1252_P01_C08 or from 1252_P01_C08. An antibody or a fragment thereof having one or both of the VH and VL sequences of _P01_C08 is suitable for use in such an antibody. capable of binding to a pitope.

[0145] In one embodiment, the epitope is selected from the amino acid region 37-53 and / or 59-77 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one or more amino acid residues within SEQ ID NO: 1. The amino acid sequence of the present invention is a sequence of one or more amino acid residues within the amino acid regions 37-53 and 59-77 of the present invention. In one embodiment, the epitope is one or more of the amino acid regions 37 to 53 or 59 to 77 of SEQ ID NO: 1. The antibody or fragment thereof having such an epitope contains the amino acid residues 1245_P02_G 04, or such antibodies or fragments thereof may comprise part or all of the sequence of 1245_P For example, one or more CDR sequences of 1245_P02_G04 or the VH and VH sequences of 1245_P02_G04 may be derived from 1245_P02_G04. An antibody or fragment thereof having one or both of the VL sequences binds to such an epitope. obtain.

[0146] In one embodiment, the epitope is one or more amino acids within the amino acid region 59-72 of SEQ ID NO:1. In a further embodiment, the epitope comprises amino acid region 5 of SEQ ID NO: 1. The antibody or fragment thereof having such an epitope is composed of one or more amino acid residues in the range of 9 to 72. The fragment may comprise part or all of the sequence of 1251_P02_C05 or may be a fragment of such an antibody or fragments thereof. A fragment of 1251_P02_C05 can be derived from 1251_P02_C05. For example, a fragment of 1251_P02_C05 can be derived from one or more CDR sequences of 1251_P02_C05 or from 1251 An antibody or a fragment thereof having one or both of the VH and VL sequences of _P02_C05 is suitable for use in such an antibody. capable of binding to a pitope.

[0147] In one embodiment, the epitope is selected from the group consisting of amino acid residues within amino acid region 11 to 21 of SEQ ID NO:1. In one embodiment, the epitope is within amino acid region 21-28 of SEQ ID NO: 1. In one embodiment, the epitope does not include the amino acid residue of SEQ ID NO: 1. does not include amino acid residues within regions 59 and 60. In one embodiment, the epitope is It does not contain amino acid residues within the amino acid region 67 to 82 of No. 1.

[0148] In one embodiment, the epitope is identified by a commercially available anti-V51 antibody, e.g., TS-1 or TS8.2. As described in WO2017197347, the epitope bound by soluble TS-1 and TS8.2 binding to the Vδ1 TCR is mediated by the Vδ1 J1 and Vδ1 chains, but not the Vδ1 J3 chain. The binding of TS-1 and TS8.2 was detected when the delta J1 and delta J2 sequences were included, and the binding of TS-1 and TS8.2 was detected when the delta J1 and delta J2 sequences were included. It was shown that essential residues are required.

[0149] References herein to "within" include the ends of the defined range. "Within the acid region 5-20" refers to all acids from residue 5 (inclusive) to residue 20 (inclusive). It refers to amino acid residues.

[0150] Various techniques are available for determining which epitope is bound by an antibody. Exemplary techniques include, for example, routine cross-blocking algorithms. Assay, alanine scanning mutation analysis, peptide blot analysis, peptide cleavage analysis These include crystallographic studies, and NMR analysis. Methods such as antibody extraction and chemical modification can be used. Another method that can be used to identify amino acids within a peptide is mass spectrometry. Hydrogen / deuterium exchange detected by chromatometry (described in Example 9). In general, hydrogen / deuterium exchange involves deuterium labeling of the protein of interest, followed by deuterium labeling of the antibody. This involves binding to a hydrogen-labeled protein. The protein / antibody complex is then transferred to water. The exchangeable protons in the amino acids protected by the antibody complex are undergo back exchange of deuterium to hydrogen at a slower rate than exchangeable protons in amino acids As a result, amino acids that form part of the protein / antibody interface will retain deuterium. and therefore exhibit a relatively large mass compared to amino acids not included in the interface. After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis. This provides deuterium-labeled residues corresponding to specific amino acids with which the antibody interacts. To do.

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

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

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

[0154] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 2 to 25. It comprises a CDR3 comprising a sequence with 85%, 90%, 95%, 97%, 98%, or 99% sequence identity. In one embodiment, the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 26 to 37 and sequences: A1 to A12 (in Table 2). have at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of In one embodiment, the antibody or fragment thereof comprises a CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 38 to 61. have at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of It contains a CDR1 containing a sequence

[0155] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 2 to 25. Contains a CDR3 consisting of a sequence having 85%, 90%, 95%, 97%, 98%, or 99% sequence identity In one embodiment, the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 26 to 37 and sequences A1 to A12 (in Table 2). having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of In one embodiment, the antibody or fragment thereof comprises a CDR2 consisting of a sequence selected from SEQ ID NOs: 38 to 39. 61 having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of The CDR1 comprises a sequence having the following structure:

[0156] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 2 to 13. A VH region containing a CDR3 sequence that shares 80% sequence identity with the VH region and / or any of SEQ ID NOs: 14 to 25 and a VL region comprising a CDR3 comprising a sequence having at least 80% sequence identity with one of the VL regions. In some embodiments, the antibody or fragment thereof has at least 80% identity with any one of SEQ ID NOs: 2-13. A VH region comprising a CDR3 consisting of a sequence having sequence identity, and / or any of SEQ ID NOs: 14 to 25 The VL region includes a CDR3 consisting of a sequence having at least 80% sequence identity with one of the VL regions.

[0157] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 2 to 13. A VH region comprising a CDR3 containing a sequence having 90% sequence identity, and / or any of SEQ ID NOs: 14 to 25 The VL region includes a CDR3 having a sequence that has at least 90% sequence identity with any one of the VL regions. In one embodiment, the antibody or fragment thereof has at least 90% homology with any one of SEQ ID NOs: 2 to 13. % sequence identity, and / or a VH region comprising a CDR3 consisting of a sequence having any one of SEQ ID NOs: 14 to 25 It contains a VL region containing a CDR3 consisting of a sequence having at least 90% sequence identity with any one of .

[0158] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 2 to 13. A VH region comprising a CDR3 containing a sequence having 95% sequence identity, and / or any of SEQ ID NOs: 14 to 25 The VL region includes a CDR3 having a sequence that has at least 95% sequence identity with any one of the VL regions. In one embodiment, the antibody or fragment thereof has at least 95% repeat sequences with any one of SEQ ID NOs: 2 to 13. % sequence identity, and / or a VH region comprising a CDR3 consisting of a sequence having any one of SEQ ID NOs: 14 to 25 It contains a VL region containing a CDR3 consisting of a sequence having at least 95% sequence identity with any one of .

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

[0160] In one embodiment, any one of SEQ ID NOs: 2 to 7, in particular 2 to 6, for example 2, 3, or 4 a VH region comprising a CDR3 comprising a sequence having at least 80% sequence identity with SEQ ID NO: 14 to SEQ ID NO: 15; 19, particularly 14 to 18, e.g., at least 80% sequence identity to any one of 14, 15, or 16 In one embodiment, the antibody or fragment thereof comprises a VL region comprising a CDR3 having a sequence having the following functional groups: , SEQ ID NO: 2 to 7, particularly SEQ ID NO: 2 to 6, for example, SEQ ID NO: 2, 3, or 4, and at least 80% sequence identity. a VH region comprising a CDR3 consisting of a sequence having sequence identity with a VH region represented by SEQ ID NOs: 14 to 19, particularly SEQ ID NOs: 14 to 18, For example, from a sequence having at least 80% sequence identity with any one of 14, 15, or 16 An antibody or fragment thereof comprising a VL region comprising a CDR3 comprising:

[0161] In one embodiment, any one of SEQ ID NOs: 2 to 7, in particular 2 to 6, for example 2, 3, or 4 a VH region comprising a CDR3 comprising a sequence having at least 90% sequence identity with SEQ ID NO: Nos. 14 to 19, particularly 14 to 18, for example, any one of 14, 15, or 16, and at least 90% In one embodiment, the antibody or fragment thereof comprises a VL region comprising a CDR3 comprising a sequence having sequence identity with the VL region of the antibody or fragment thereof. In this case, any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4 and at least 90 % sequence identity, and / or SEQ ID NOs: 14 to 19, particularly 14 to 18, for example, 14, 15, or 16, and have at least 90% sequence identity with any one of An antibody or fragment thereof comprising a VL region including a CDR3 consisting of a sequence

[0162] In one embodiment, any one of SEQ ID NOs: 2 to 7, in particular 2 to 6, for example 2, 3, or 4 a VH region comprising a CDR3 comprising a sequence having at least 95% sequence identity with SEQ ID NO: Nos. 14 to 19, particularly 14 to 18, e.g., at least 95% of any one of Nos. 14, 15, or 16. In one embodiment, the antibody or fragment thereof comprises a VL region comprising a CDR3 comprising a sequence having sequence identity with the VL region of the antibody or fragment thereof. In this case, any one of SEQ ID NOs: 2 to 7, particularly 2 to 6, for example, 2, 3, or 4 and at least 95 % sequence identity, and / or SEQ ID NOs: 14 to 19, particularly 14 to 18, e.g., 14, 15, or 16, and having at least 95% sequence identity with any one of these. An antibody or fragment thereof comprising a VL region including a CDR3 consisting of a sequence

[0163] In one embodiment, at least one of SEQ ID NOs: 8 to 13, in particular 8, 9, 10, or 11. a VH region comprising CDR3 containing a sequence having 80% sequence identity with any of SEQ ID NOs: 20 to 25, and / or SEQ ID NOs: 20 to 25; In particular, a sequence having at least 80% sequence identity with any one of 20, 21, 22, or 23 In one embodiment, the antibody or fragment thereof comprises a VL region comprising a CDR3 comprising: , particularly a sequence having at least 80% sequence identity with any one of 8, 9, 10, or 11. and / or a VH region comprising a CDR3 consisting of SEQ ID NO: 20 to 25, particularly any of SEQ ID NO: 20, 21, 22, or 23. an antibody comprising a VL region containing a CDR3 consisting of a sequence having at least 80% sequence identity with one of Body or fragment thereof.

[0164] In one embodiment, at least one of SEQ ID NOs: 8 to 13, in particular 8, 9, 10, or 11. a VH region comprising a CDR3 containing a sequence having 90% sequence identity with any of SEQ ID NOs: 20 to 25, and / or SEQ ID NOs: 20 to 25; In particular, a sequence having at least 90% sequence identity with any one of 20, 21, 22, or 23 In one embodiment, the antibody or fragment thereof comprises a VL region comprising a CDR3 comprising: , particularly a sequence having at least 90% sequence identity with any one of 8, 9, 10, or 11. and / or a VH region comprising a CDR3 consisting of SEQ ID NO: 20 to 25, particularly any of SEQ ID NO: 20, 21, 22, or 23. an antibody comprising a VL region containing a CDR3 consisting of a sequence having at least 90% sequence identity with one of Body or fragment thereof.

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

[0166] As used herein, embodiments referring to "at least 80%" or "80% or more" include 80% or more , for example, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity. In one embodiment, the antibody or fragment thereof is At least 85%, e.g., at least 90%, at least 95%, at least 97%, It contains at least 98%, or at least 99% sequence identity.

[0167] Instead of percentage sequence identity, embodiments may include one or more amino acid changes, e.g., one The above additions, substitutions, and / or deletions may also be used to define the amino acid sequence. , the sequence may contain up to five amino acid changes, e.g., up to three amino acid changes, particularly up to two In a further embodiment, the sequence may contain up to five amino acid substitutions. , for example, may contain up to three amino acid substitutions, in particular up to one or two amino acid substitutions. For example, the CDR3 of the antibody or a fragment thereof has 2 or less amino acids compared to any one of SEQ ID NOs: 2 to 25. More preferably, it comprises or consists of a sequence having one or fewer substitutions.

[0168] Preferably, CDR1, CDR2 residues differ from the corresponding residues in SEQ ID NOs: 2 to 61 and SEQ ID NOs: A1 to A12. or any residue in CDR3 is a conservative substitution for the corresponding residue. Any residue in CDR3 that differs from its corresponding residue in numbers 2-25 is This is a conservative substitution.

[0169] In one embodiment, the antibody or fragment thereof (i) CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 13 a VH region comprising: (ii) CDRs comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 VH region containing 2; (iii) A CD containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 49. VH region including R1; (iv) CDRs comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 14 to 25 VL region containing 3; (v) Sequence: CDR2 containing a sequence having at least 80% sequence identity with any one of A1 to A12 and / or a VL region comprising (vi) CDRs comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 50 to 61 VL region containing 1 Contains:

[0170] In one embodiment, the antibody or fragment thereof (i) CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 13 a VH region comprising: (ii) CDRs comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 2; and (iii) A CD containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 49. VH region containing R1 :

[0171] In one embodiment, the antibody or fragment thereof (i) CDR3 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 14 to 25 the VL region containing; (ii) Sequence: CDR2 containing a sequence having at least 80% sequence identity with any one of A1 to A12 a VL region comprising: (iii) A CD containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 50 to 61. VL region including R1 :

[0172] In one embodiment, the antibody or fragment thereof is SEQ ID NO: 2, 3, 4, 5, or 6, e.g., 2, 3, 4, or 5, particularly 2, 3, or 4, having at least 80% sequence identity with any one of In one embodiment, the antibody or its The fragment may be SEQ ID NO: 26, 27, 28, 29, or 30, for example 26, 27, 28, or 29, in particular 26, 27 or a VH comprising a CDR2 comprising a sequence having at least 80% sequence identity to any one of the 28 In one embodiment, the antibody or fragment thereof comprises (or consists of) the region SEQ ID NO: 38, 3 9, 40, 41, or 42, for example, 38, 39, 40, or 41, in particular, any one of 38, 39, or 40 A VH region comprising a CDR1 having a sequence having at least 80% sequence identity with (or consisting of) (This is the case.)

[0173] In one embodiment, the antibody or fragment thereof has any one of SEQ ID NOs: 8, 9, 10, or 11. and a VH region comprising a CDR3 having a sequence having at least 80% sequence identity with (or consisting of) In one embodiment, the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 32, 33, 34, and 35. a VH region comprising a CDR2 having a sequence with at least 80% sequence identity to (or from) In one embodiment, the antibody or fragment thereof has a sequence similar to that of SEQ ID NO: 44, 45, 46, or 47. a VH region comprising a CDR1 having a sequence having at least 80% sequence identity with one of the (consisting of

[0174] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO:2, a CDR4 comprising the sequence of SEQ ID NO:26, In one embodiment, the CDR2 comprises the sequence of SEQ ID NO: 38, and the CDR1 comprises the sequence of SEQ ID NO: 39. CDR2 consists of the sequence of SEQ ID NO: 26, and CDR1 consists of the sequence of SEQ ID NO: 38. It consists of columns.

[0175] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 3, a CDR4 comprising the sequence of SEQ ID NO: 27, In one embodiment, the CDR3 comprises a CDR2 comprising the sequence of SEQ ID NO: 39, and a CDR1 comprising the sequence of SEQ ID NO: 39. CDR2 consists of the sequence of SEQ ID NO: 27, and CDR1 consists of the sequence of SEQ ID NO: 39. It consists of columns.

[0176] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 4, a CDR4 comprising the sequence of SEQ ID NO: 28, In one embodiment, the CDR3 comprises a CDR2 comprising the sequence of SEQ ID NO: 40, and a CDR1 comprising the sequence of SEQ ID NO: 40. CDR2 consists of the sequence of SEQ ID NO: 28, and CDR1 consists of the sequence of SEQ ID NO: 40. It consists of columns.

[0177] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 5, a CDR4 comprising the sequence of SEQ ID NO: 29, In one embodiment, the CDR3 comprises a CDR2 comprising the sequence of SEQ ID NO: 41, and a CDR1 comprising the sequence of SEQ ID NO: 42. CDR2 consists of the sequence of SEQ ID NO: 29, and CDR1 consists of the sequence of SEQ ID NO: 41. It consists of columns.

[0178] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 6, a CDR4 comprising the sequence of SEQ ID NO: 30, In one embodiment, the CDR2 comprises the sequence of SEQ ID NO: 42, and the CDR1 comprises the sequence of SEQ ID NO: 43. CDR2 consists of the sequence of SEQ ID NO: 30, and CDR1 consists of the sequence of SEQ ID NO: 42. It consists of columns.

[0179] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 8, a CDR4 comprising the sequence of SEQ ID NO: 32, In one embodiment, the CDR2 comprises the sequence of SEQ ID NO: 44, and the CDR1 comprises the sequence of SEQ ID NO: 44. CDR2 consists of the sequence of SEQ ID NO: 32, and CDR1 consists of the sequence of SEQ ID NO: 44. It consists of columns.

[0180] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 9, a CDR4 comprising the sequence of SEQ ID NO: 33, In one embodiment, the CDR2 comprises the sequence of SEQ ID NO: 45, and the CDR1 comprises the sequence of SEQ ID NO: 46. CDR2 consists of the sequence of SEQ ID NO: 33, and CDR1 consists of the sequence of SEQ ID NO: 45. It consists of columns.

[0181] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 10, a CDR2 sequence of SEQ ID NO: 34 and the CDR1 sequence of SEQ ID NO: 46. In one embodiment, the CDR3 comprises the sequence of SEQ ID NO: 10. CDR2 consists of the sequence of SEQ ID NO: 34 and CDR1 consists of the sequence of SEQ ID NO: 46.

[0182] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 11, a CDR2 sequence of SEQ ID NO: 35 and the CDR1 sequence of SEQ ID NO: 47. In one embodiment, the CDR3 comprises the sequence of SEQ ID NO: 11. wherein CDR2 consists of the sequence of SEQ ID NO: 35 and CDR1 consists of the sequence of SEQ ID NO: 47.

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

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

[0185] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 15, a sequence of the sequence: A2 In one embodiment, the CDR2 comprises a CDR1 comprising the sequence of SEQ ID NO: 51. In one embodiment, the CDR3 comprises a CDR2 comprising the sequence of SEQ ID NO: 52. 15, CDR2 consists of the sequence: A2, and CDR1 consists of the sequence of SEQ ID NO: 51. Or rather.

[0186] In one embodiment, the VL region comprises a CDR3 comprising the sequence of SEQ ID NO: 16, the sequence: A3 In one embodiment, the CDR2 comprises a CDR1 comprising the sequence of SEQ ID NO: 52. In one embodiment, the CDR3 comprises a CDR2 comprising the sequence of SEQ ID NO: 53. 16, CDR2 consists of the sequence: A3, and CDR1 consists of the sequence: SEQ ID NO: 52. Or rather.

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

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

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

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

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

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

[0193] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO:2, a CDR4 comprising the sequence of SEQ ID NO:26, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 38, a CDR1 comprising the sequence of SEQ ID NO: 14, R3, CDR2 comprising the sequence of sequence A1, and CDR1 comprising the sequence of SEQ ID NO: 50. wherein HCDR3 consists of the sequence of SEQ ID NO: 2, HCDR2 consists of the sequence of SEQ ID NO: 26, R1 consists of the sequence of SEQ ID NO: 38, LCDR3 consists of the sequence of SEQ ID NO: 14, and LCDR2 consists of the sequence : A1, and LCDR1 consists of the sequence of SEQ ID NO: 50.

[0194] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 3, a CDR4 comprising the sequence of SEQ ID NO: 27, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 39, a CDR1 comprising the sequence of SEQ ID NO: 15, R3, CDR2 comprising the sequence of sequence A2, and CDR1 comprising the sequence of SEQ ID NO: 51. wherein HCDR3 consists of the sequence of SEQ ID NO: 3, HCDR2 consists of the sequence of SEQ ID NO: 27, R1 consists of the sequence of SEQ ID NO: 39, LCDR3 consists of the sequence of SEQ ID NO: 15, and LCDR2 consists of the sequence : A2, and LCDR1 consists of the sequence of SEQ ID NO: 51.

[0195] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 4, a CDR4 comprising the sequence of SEQ ID NO: 28, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 40, a CDR1 comprising the sequence of SEQ ID NO: 16, R3, CDR2 comprising the sequence of sequence A3, and CDR1 comprising the sequence of SEQ ID NO: 52. wherein HCDR3 consists of the sequence of SEQ ID NO: 4, HCDR2 consists of the sequence of SEQ ID NO: 28, R1 consists of the sequence of SEQ ID NO: 40, LCDR3 consists of the sequence of SEQ ID NO: 16, and LCDR2 consists of the sequence : A3, and LCDR1 consists of the sequence of SEQ ID NO: 52.

[0196] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 5, a CDR4 comprising the sequence of SEQ ID NO: 29, and a VL region comprising a CDR2 comprising the sequence of SEQ ID NO: 41, a CDR1 comprising the sequence of SEQ ID NO: 17. R3, CDR2 comprising the sequence of sequence A4, and CDR1 comprising the sequence of SEQ ID NO: 53. wherein HCDR3 consists of the sequence of SEQ ID NO: 5, HCDR2 consists of the sequence of SEQ ID NO: 29, R1 consists of the sequence of SEQ ID NO: 41, LCDR3 consists of the sequence of SEQ ID NO: 17, and LCDR2 consists of the sequence : A4, and LCDR1 consists of the sequence of SEQ ID NO: 53.

[0197] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 6, a CDR4 comprising the sequence of SEQ ID NO: 30, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 42, a CDR1 comprising the sequence of SEQ ID NO: 18, R3, CDR2 comprising the sequence of sequence A5, and CDR1 comprising the sequence of SEQ ID NO: 54. wherein HCDR3 consists of the sequence of SEQ ID NO: 6, HCDR2 consists of the sequence of SEQ ID NO: 30, R1 consists of the sequence of SEQ ID NO: 42, LCDR3 consists of the sequence of SEQ ID NO: 18, and LCDR2 consists of the sequence : A5, and LCDR1 consists of the sequence of SEQ ID NO: 54.

[0198] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 7, a CDR4 comprising the sequence of SEQ ID NO: 31, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 43, a CDR1 comprising the sequence of SEQ ID NO: 19, R3, CDR2 comprising the sequence of sequence A6, and CDR1 comprising the sequence of SEQ ID NO: 55. wherein HCDR3 consists of the sequence of SEQ ID NO: 7, HCDR2 consists of the sequence of SEQ ID NO: 31, R1 consists of the sequence of SEQ ID NO: 43, LCDR3 consists of the sequence of SEQ ID NO: 19, and LCDR2 consists of the sequence : A6, and LCDR1 consists of the sequence of SEQ ID NO: 55.

[0199] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 8, a CDR4 comprising the sequence of SEQ ID NO: 32, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 44, a CDR1 comprising the sequence of SEQ ID NO: 20, R3, CDR2 comprising the sequence of sequence A7, and CDR1 comprising the sequence of SEQ ID NO: 56. wherein HCDR3 consists of the sequence of SEQ ID NO: 8, HCDR2 consists of the sequence of SEQ ID NO: 32, R1 consists of the sequence of SEQ ID NO: 44, LCDR3 consists of the sequence of SEQ ID NO: 20, and LCDR2 consists of the sequence : A7, and LCDR1 consists of the sequence of SEQ ID NO: 56.

[0200] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 9, a CDR4 comprising the sequence of SEQ ID NO: 33, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 45, a CDR1 comprising the sequence of SEQ ID NO: 21, R3, CDR2 comprising the sequence of sequence A8, and CDR1 comprising the sequence of SEQ ID NO: 57. HCDR3 consists of the sequence of SEQ ID NO: 9, HCDR2 consists of the sequence of SEQ ID NO: 33, R1 consists of the sequence of SEQ ID NO: 45, LCDR3 consists of the sequence of SEQ ID NO: 21, and LCDR2 consists of the sequence : A8, and LCDR1 consists of the sequence of SEQ ID NO: 57.

[0201] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 10, a CDR4 comprising the sequence of SEQ ID NO: 34, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 46, a CDR1 comprising the sequence of SEQ ID NO: 22, R3, CDR2 comprising the sequence of sequence A9, and CDR1 comprising the sequence of SEQ ID NO: 58. wherein HCDR3 consists of the sequence of SEQ ID NO: 10, HCDR2 consists of the sequence of SEQ ID NO: 34, DR1 consists of the sequence of SEQ ID NO: 46, LCDR3 consists of the sequence of SEQ ID NO: 22, and LCDR2 consists of the sequence Column: A9 and LCDR1 consists of the sequence of SEQ ID NO: 58.

[0202] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 11, a CDR4 comprising the sequence of SEQ ID NO: 35, and a VL region comprising a CDR2 comprising the sequence of SEQ ID NO: 47, a CDR1 comprising the sequence of SEQ ID NO: 23. R3, a CDR2 comprising the sequence of sequence A10, and a CDR1 comprising the sequence of SEQ ID NO: 59. wherein HCDR3 consists of the sequence of SEQ ID NO: 11 and HCDR2 consists of the sequence of SEQ ID NO: 35, HCDR1 consists of the sequence of SEQ ID NO: 47, LCDR3 consists of the sequence of SEQ ID NO: 23, and LCDR2 consists of the sequence of Sequence: A10 and LCDR1 consists of the sequence of SEQ ID NO: 59.

[0203] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 12, a CDR4 comprising the sequence of SEQ ID NO: 36, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 48, a CDR1 comprising the sequence of SEQ ID NO: 24, R3, a CDR2 comprising the sequence of sequence A11, and a CDR1 comprising the sequence of SEQ ID NO: 60. wherein HCDR3 consists of the sequence of SEQ ID NO: 12 and HCDR2 consists of the sequence of SEQ ID NO: 36, HCDR1 consists of the sequence of SEQ ID NO: 48, LCDR3 consists of the sequence of SEQ ID NO: 24, and LCDR2 consists of the sequence of Sequence: A11 and LCDR1 consists of the sequence of SEQ ID NO: 60.

[0204] In one embodiment, the VH region comprises a CDR3 comprising the sequence of SEQ ID NO: 13, a CDR4 comprising the sequence of SEQ ID NO: 37, the VL region comprises a CDR2 comprising the sequence of SEQ ID NO: 49, a CDR1 comprising the sequence of SEQ ID NO: 25, R3, a CDR2 comprising the sequence of sequence A12, and a CDR1 comprising the sequence of SEQ ID NO: 61. wherein HCDR3 consists of the sequence of SEQ ID NO: 13 and HCDR2 consists of the sequence of SEQ ID NO: 37, HCDR1 consists of the sequence of SEQ ID NO: 49, LCDR3 consists of the sequence of SEQ ID NO: 25, and LCDR2 consists of the sequence of A12 and LCDR1 consists of the sequence of SEQ ID NO: 61.

[0205] In one embodiment, the antibody or fragment thereof comprises one or more CDR sequences set forth in Table 2. In a further embodiment, the antibody or fragment thereof is selected from the group consisting of clone 125, described in Table 2. In an alternative embodiment, the antibody or The fragment may comprise one or more (e.g., all) CDRs of clone 1245_P01_E07 listed in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises a sequence listed in Table 2. In an alternative embodiment, the CDR sequences of the clone 1245_P02_G04 are included. The antibody or fragment thereof may be one or more of clones 1245_P02_B07 listed in Table 2 (e.g., all of In an alternative embodiment, the antibody or fragment thereof comprises the CDR sequences of any of the antibodies set forth in Table 2. The present invention also includes one or more (e.g., all) CDR sequences of clone 1251_P02_C05. In the above, the antibody or fragment thereof is one or more of clones 1139_P01_E04 listed in Table 2 (e.g., In an alternative embodiment, the antibody or fragment thereof comprises the CDR sequences of any of the CDRs listed in Table 2. Contains one or more (e.g., all) of the CDR sequences of clone 1245_P02_F07 described. In one embodiment, the antibody or fragment thereof is selected from the group consisting of clone 1245_P01_G06 as described in Table 2. In an alternative embodiment, the antibody or fragment thereof comprises one or more (e.g., all) of the CDR sequences. , comprising one or more (e.g., all) CDR sequences of clone 1245_P01_G09 listed in Table 2. In an alternative embodiment, the antibody or fragment thereof is clone 1138_P0, as described in Table 2. In an alternative embodiment, the antibody or its The fragment may contain one or more (e.g., all) of the CDR sequences of clone 1251_P02_G10 listed in Table 2. Includes:

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

[0207] The antibodies described herein are defined by their complete light and / or heavy chain variable sequences. In one embodiment, the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 62 to 85. In one embodiment, the amino acid sequence has at least 80% sequence identity with one of The antibody or fragment thereof has at least 80% sequence identity with any one of SEQ ID NOs: 62 to 85. It consists of an amino acid sequence that

[0208] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 62-73. In one embodiment, the antibody comprises a VH region comprising an amino acid sequence having at least 80% sequence identity with the VH region. The antibody or a fragment thereof has at least 80% sequence identity with any one of SEQ ID NOs: 62 to 73. In a further embodiment, the VH region comprises a VH region consisting of the amino acid sequence set forth in SEQ ID NO: 62, 63, 64, 65, or 66, for example, 62, 63, 64, or 65, in particular, any of 62, 63, or 64 or one of the amino acid sequences having at least 80% sequence identity. In this case, the VH region is SEQ ID NO: 62, 63, 64, 65, or 66, for example, 62, 63, 64, or 65, particularly an amino acid sequence having at least 80% sequence identity with any one of 62, 63, or 64; In a further embodiment, the VH region consists of SEQ ID NO: 68, 69, 70, 71, 72, or 73. , e.g., 68, 69, 70, or 71. In a further embodiment, the VH region comprises the amino acid sequence of SEQ ID NOs: 68, 69, 70, 71, 72. or 73, e.g., 68, 69, 70, or 71. It consists of an amino acid sequence having the following structure:

[0209] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 74 to 85. In one embodiment, the antibody comprises a VL region comprising an amino acid sequence having at least 80% sequence identity. The antibody or a fragment thereof has at least 80% sequence identity with any one of SEQ ID NOs: 74 to 85. In a further embodiment, the VL region comprises a VL region consisting of the amino acid sequence set forth in SEQ ID NO: 74, 75, 76, 77, or 78, for example, 74, 75, 76, or 77, in particular, any of 74, 75, or 76 or one of the amino acid sequences having at least 80% sequence identity. In this case, the VL region is SEQ ID NO: 74, 75, 76, 77, or 78, for example, 74, 75, 76, or 77, particularly an amino acid sequence having at least 80% sequence identity with any one of 74, 75, or 76; In a further embodiment, the VL region consists of SEQ ID NO: 80, 81, 82, 83, 84, or 85. , e.g., an amino acid sequence having at least 80% sequence identity with any one of 80, 81, 82, or 83. In a further embodiment, the VL region comprises the amino acid sequence of SEQ ID NO: 80, 81, 82, 83, 84. or 85, e.g., 80, 81, 82, or 83, having at least 80% sequence identity with any one of It consists of an amino acid sequence having the following structure:

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

[0211] In one embodiment, the antibody or fragment thereof has 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 SEQ ID NO: 62 (124 In an alternative embodiment, the antibody or its VH region comprises the amino acid sequence of The fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04). In embodiments, the antibody or fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises the sequence of SEQ ID NO: 69 (1245_P02_F0 7). In an alternative embodiment, the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of , and a VH region comprising the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06). The antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09). nothing.

[0212] In one embodiment, the antibody or fragment thereof has 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 SEQ ID NO: 62 (1 In an alternative embodiment, the antibody or The fragment comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 64 (1245_P02_G04). In one embodiment, the antibody or fragment thereof has 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 SEQ ID NO: 69 (124 In an alternative embodiment, the antibody or The fragment comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 70 (1245_P01_G06). In one embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 71 (1245_P01_G09). The VH region comprises:

[0213] In one embodiment, the antibody or fragment thereof has 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 SEQ ID NO: 74 (124 In an alternative embodiment, the antibody or its VL region comprises the amino acid sequence of The fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In embodiments, the antibody or fragment thereof comprises a VL comprising the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In an alternative embodiment, the antibody or fragment thereof comprises the sequence of SEQ ID NO: 81 (1245_P02_F0 7). In an alternative embodiment, the antibody or fragment thereof comprises a VL region comprising the amino acid sequence of , and a VL region comprising the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). The antibody or fragment thereof comprises a VL region comprising the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09). nothing.

[0214] In one embodiment, the antibody or fragment thereof has 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 SEQ ID NO: 74 (1 In an alternative embodiment, the antibody or The fragment comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 76 (1245_P02_G04). In one embodiment, the antibody or fragment thereof has 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 SEQ ID NO: 81 (124 In an alternative embodiment, the antibody or The fragment comprises a VL region consisting of the amino acid sequence of SEQ ID NO: 82 (1245_P01_G06). In one embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09). The VL region comprises:

[0215] In one embodiment, the antibody or fragment thereof has 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 one embodiment, the antibody or fragment thereof has 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 one embodiment, the antibody or fragment thereof comprises 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 embodiments, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 68 (1139_P01_E04). It comprises a VH region and a VL region comprising the amino acid sequence of SEQ ID NO: 80 (1139_P01_E04). In embodiments, the antibody or fragment thereof comprises a VH 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 the above, the antibody or fragment thereof has 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). wherein the antibody or fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 71 (1245_P01_G06). and a VL region comprising the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09).

[0216] In one embodiment, the antibody or fragment thereof has 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 another embodiment, the antibody or fragment thereof has the amino acid sequence of SEQ ID NO: 62 (1245_P01_E07). It comprises a VH region consisting of the amino acid sequence of SEQ ID NO: 74 (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 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 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 has the amino acid sequence of SEQ ID NO: 69 (1245_P02_F07). a VH region consisting of the amino acid sequence of SEQ ID NO: 81 (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 the antibody of SEQ ID NO: 70 (1245_P01_G06). A VH region consisting of the amino acid sequence of SEQ ID NO: 82 (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 the sequence of SEQ ID NO: 71 (1245_P01_G09). A VH region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09) and a VL region consisting of the amino acid sequence of SEQ ID NO: 83 (1245_P01_G09) Includes the area.

[0217] For fragments containing both VH and VL regions, these may be linked together either covalently (e.g., by disulfide bonding). either covalently (through sulfide bonds or linkers) or non-covalently. The antibody fragments described herein may be scFvs, i.e., fragments connected by a linker. In one embodiment, the VH and VL regions may comprise a fragment comprising: The polypeptide linkers are connected by (e.g., synthetic) polypeptide linkers (G ly4Ser) n It may contain a linker (where n=1 to 8, for example, 2, 3, 4, 5, or 7). The peptide linker is [(Gly4Ser) n (Gly3AlaSer) m ] p Linker (where n=1 to 8, e.g. , 2, 3, 4, 5, or 7, m=1 to 8, for example, 0, 1, 2, or 3, and p=1 to 8, for example, In further embodiments, the linker may comprise SEQ ID NO: In a further embodiment, the linker consists of SEQ ID NO:98.

[0218] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 86 to 97. In a further embodiment, the antibody or its The fragment comprises the amino acid sequence of any one of SEQ ID NOs: 86 to 97. wherein the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 87 (1252_P01_C08). In another embodiment, the antibody or fragment thereof has the amino acid sequence of SEQ ID NO: 86 (1245_P01_E07). In an alternative embodiment, the antibody or fragment thereof comprises the 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 (113 In an alternative embodiment, the antibody or fragment thereof comprises the amino acid sequence of In an alternative embodiment, the antibody or its The fragment comprises the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). The antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0219] In one embodiment, the antibody or fragment thereof has at least one sequence identical to any one of SEQ ID NOs: 86-97. In a further embodiment, the antibody or The fragment consists of any one of the amino acid sequences of SEQ ID NOs: 86 to 97. In embodiments, 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 has 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 In an alternative embodiment, the antibody or fragment thereof consists of the amino acid sequence of In an alternative embodiment, the antibody or The fragment consists of the amino acid sequence of SEQ ID NO: 93 (1245_P02_F07). wherein the antibody or fragment thereof consists of the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In one embodiment, the antibody or fragment thereof has the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09). Or rather.

[0220] The scFv constructs were engineered with N- and C-terminal modifications to aid in translation, purification, and detection. It will be understood by those skilled in the art that the present invention can be designed and manufactured to include such features. At the N-terminus of the scFv sequence, additional methionine and / or alanine amino acid residues may be introduced as standard. The VH sequence may be included before the C-terminus (i.e., beginning with QVQ or EVQ). (i) a constant domain portion at the C-terminus of the canonical VL domain sequence according to the IMGT definition; (ii) the addition of tags such as His-tags and Flag-tags to aid in purification and detection; In one embodiment, additional sequences may be included, such as additional synthetic sequences comprising the sequence The number 124 is added to the C-terminus of any one of SEQ ID NOs: 86, 88-90, and 92-97. In the above, SEQ ID NO: 125 is added to the C-terminus of any one of SEQ ID NOs: 86, 88 to 90, and 92 to 97. In one embodiment, SEQ ID NO: 126 is the C-terminal amino acid of any one of SEQ ID NOs: 87 or 91. In one embodiment, SEQ ID NO: 127 is added to any one of SEQ ID NOs: 87 or 91. The N- or C-terminal sequences of the scFv are optional and may be modified by other scFv design, translation, When purification or detection strategies are employed, these may be removed, modified, or replaced. It is well understood that this is possible.

[0221] As described herein, the antibody can be in any format. In a preferred embodiment, the antibody is of the IgG1 format. In the above, the antibody or fragment thereof has at least 80% identity with any one of SEQ ID NOs: 111 to 122. In a further embodiment, the antibody or fragment thereof comprises an amino acid sequence having sequence identity. In a further embodiment, the amino acid sequence of any one of SEQ ID NOs: 111 to 122 is In this case, the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 111 to 116, for example, SEQ ID NOs: 111 to 113 and 116. In yet a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 117. to 122, for example, the amino acid sequences of SEQ ID NOs: 117 to 120. The antibody or fragment thereof may be selected from the group consisting of SEQ ID NOs: 111, 112, 116 to 120, for example, SEQ ID NOs: 111, 112, or The amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 117 to 120 is included.

[0222] In one embodiment, the antibody or fragment thereof has at least one of SEQ ID NOs: 111 to 122. In a further embodiment, the antibody comprises an amino acid sequence having 80% sequence identity with the antibody. Or a fragment thereof consists of any one of the amino acid sequences of SEQ ID NOs: 111 to 122. In some embodiments, the antibody or fragment thereof is selected from SEQ ID NOs: 111-116, e.g., SEQ ID NOs: 111-116. In yet a further embodiment, the antibody or fragment thereof comprises the amino acid sequence of 13 and 116. The amino acid sequences of SEQ ID NOs: 117 to 122, for example, SEQ ID NOs: 117 to 120, are also In some embodiments, the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 111, 112, 116-120, e.g., SEQ ID NOs: It consists of the amino acid sequence of SEQ ID NO: 111, 112 or 116, or SEQ ID NO: 117 to 120.

[0223] In one embodiment, the antibody is identical to or essentially an antibody or fragment thereof as defined herein. or compete with said antibody or fragment thereof. By using routine methods known in the art, it is possible to determine whether the antibody binds to the same epitope as the reference anti-Vδ1 antibody. It can be readily determined whether the antibody binds to a reference anti-Vδ1 antibody or competes for binding with a reference anti-Vδ1 antibody. For example, it is possible to determine whether a test antibody binds to the same epitope as a reference anti-Vδ1 antibody. To determine this, a reference antibody is allowed to bind to the V51 protein or peptide under saturating conditions. The ability of the test antibody to bind to the Vδ1 chain is then assessed. If the test antibody is able to bind to V51 after ligation, then the test antibody has a different epitope than the reference anti-V51 antibody. On the other hand, it can be concluded that the test antibody binds to the reference anti-Vδ1 antibody with saturation binding. If the test antibody is subsequently unable to bind to the Vδ1 chain, it is bound by the reference anti-Vδ1 antibody. The antibody may bind to the same epitope as the antigen-binding protein.

[0224] The present invention relates to antibodies or fragments thereof as defined herein, or to the exemplary antibodies described herein. Also included are anti-V51 antibodies that compete for V51 binding with antibodies having any of the CDR sequences of the antibodies. For example, , which proteins, antibodies, and other antagonists compete with the antibody for binding to the Vδ1 chain Competitive assays using antibodies to determine which genes share the same epitope and / or These assays are readily apparent to those skilled in the art; antagonists or ligands for the limited number of binding sites on proteins, e.g., Vδ1 The competition between the antibodies is assessed. The antibodies (or their fragments) are immobilized or insolubilized before or after the competition. The sample bound to the Vδ1 chain can be recovered, for example, by decanting (if the antibody was previously insolubilized) or by centrifugation. Separate the antibody from the unbound sample by centrifugation (if the antibody precipitates after the competitive reaction) or by centrifugation (if the antibody precipitates after the competitive reaction). Competitive binding also means that a function is determined by the binding or lack of binding of an antibody to a protein. whether the antibody molecule inhibits or enhances, for example, the enzyme activity of the label. This can be determined by ELISA, as known in the art and described herein. and other functional assays can be used.

[0225] The two antibodies competitively inhibit (block) each other's binding to the target antigen In the case of antibodies that bind to the same or overlapping epitopes, i.e., 1-fold, 5-fold, 10-fold, 20-fold, or A 100-fold excess of one antibody inhibits the binding of the other by at least At least 50%, but preferably 75%, 90%, or even 99% inhibition. The body can eliminate essentially all amino acid abruptions in the target antigen that reduce or eliminate binding of one antibody. If the mutation reduces or eliminates binding of the other, then they have the same epitope.

[0226] Additional routine experiments (e.g., peptide mutations and binding analysis) are then performed to The observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody. whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, and other methods. -cytometry, or any other quantitative or qualitative method available in the art. This can be done using a binding assay.

[0227] In some embodiments, the antibody or fragment thereof comprises a nucleotide sequence linked to Asn 297 (Kabat numbering system). The modified sugars provide modified effector functions. In addition, Asn 297 is either non-fucosylated or exhibits reduced fucosylation (i.e., defucosylated). Fucosylated or non-fucosylated antibodies). Fucosylation involves the addition of the sugar fucose to a molecule, For example, this includes the attachment of fucose to N-glycans, O-glycans, and glycolipids. Thus, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. Antibodies can be modified to prevent or inhibit antibody fucosylation. Chemical modifications may be made to the antibody or fragment thereof by targeted engineering or by targeting or Either by chance or by clonal selection, different glycosylation capabilities may be present. These and other effectors include expressing them in host cells containing the effector. -Modifications are, for example, as described in Xinhua Wang et al. (2018) Protein & Cell 9: 63-73 and Per This is further discussed in a recent review by Eira et al. (2018) mAbs 10(5): 693-711. These are incorporated by reference.

[0228] (Antibody sequence modification) Antibodies and fragments thereof can be modified using known methods. Sequence modifications to the antibody molecule can be readily incorporated by one skilled in the art. The examples are non-limiting.

[0229] During antibody discovery and sequence recovery from phage libraries, desired antibody variable domains are identified. It can be reformatted by cloning to give a full-length IgG. To speed up the process, variable domains are often transferred using restriction enzymes. These unique restriction sites introduce additional / alternative amino acids away from the standard sequence. (Such reference sequences can be found, for example, in the International ImMunoGeneTics [IMGT] information system.) (See http: / / www.imgt.org.) These are kappa or lambda light It can be introduced as a strand sequence modification.

[0230] (Kappa light chain modification) The variable kappa light chain variable sequence was reformatted using restriction sites (e.g., Nhe1-Not1). More specifically, the kappa light chain N-terminus can be cloned into a full-length IgG. At the ends, additional Ala-Ser sequences were introduced to aid cloning. This additional AS sequence is then further expanded to generate the canonical N-terminal sequence. Thus, in one embodiment, a method for removing the antibody described herein is The kappa light chain does not contain an AS sequence at its N-terminus, i.e., SEQ ID NOs: 74, 76-78, and In a further embodiment, SEQ ID NOs: 74 and 76-78 do not contain the first AS sequence. does not contain the first AS sequence. This embodiment is included herein containing this sequence. It will be understood that this also applies to other sequences (e.g., SEQ ID NOS: 86, 88-90, and 92-97). There will be.

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

[0232] (lambda light chain modification) Similar to the kappa example above, the lambda light chain variable domain also undergoes a restriction site upon reformatting. By introducing a site (e.g., Nhe1-Not1) into the IgG fragment, it can be cloned to produce a full-length IgG. More specifically, an additional Ala-Ser sequence can be introduced at the N-terminus of the lambda light chain to enhance the cleavage of the cDNA. Preferably, this additional AS sequence is then cloned into a standard In further development, the N-terminal sequence is removed to generate a suitable N-terminal sequence. In embodiments, the lambda light chain containing antibodies described herein contains the AS sequence at its N-terminus In other words, SEQ ID NOs: 75 and 79 do not contain the first AS sequence. Similar sequences are also included herein that contain this sequence (e.g., SEQ ID NOs: 87, 91, 115, and 116). In one embodiment, SEQ ID NO: 75 is , not containing the first six residues, i.e., [ka] The sequence has been removed.

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

[0234] (heavy chain modification) Typically, human variable heavy chain sequences contain either a basic glutamine (Q) or an acidic glutamic acid (E). However, both such sequences then begin with a pyrophosphate group of acidic amino acid residues. It is known that Q converts to glutamic acid (pE). The conversion of Q to pE changes the charge of the antibody. However, the conversion of E to pE does not change the charge of the antibody. To avoid charge changes, one option is to first change the starting heavy chain sequence from Q to E. Thus, in one embodiment, the heavy chain of the antibody described herein is modified. The chain contains a Q to E modification at the N-terminus. In particular, the chains of SEQ ID NOs: 62, 64, and / or 67-71 The first residue can be modified from Q to E. This embodiment is also applicable to the present invention containing this sequence. Other sequences included in the specification (e.g., SEQ ID NOS: 86, 88, 91-97 and 111, 112, 115, 117-118) It will be understood that this also applies to 20).

[0235] Furthermore, the C-terminus of the IgG1 constant domain ends with PGK. The gene (K) is then often cleaved during expression (e.g., in CHO cells). This in turn results in a charge change of the antibody due to the variable loss of the C-terminal lysine residue. Therefore, one option is to first remove the lysine and produce a uniform and consistent product that ends with PG. Thus, in one embodiment, the present invention provides a method for producing a heavy chain C-terminal sequence that is The heavy chains of the antibodies described herein have the terminal K removed from their C-terminus. The antibody may comprise any one of SEQ ID NOs: 111-122 in which the terminal lysine residue has been removed.

[0236] (any allotype modification) Specific human allotypes can be utilized during antibody discovery. In some cases, it can be converted to a different human allotype. The three Km alleles are designated (using allotype numbering) as Km1, Km1,2, and Km3. There are three human allotypes that are known to be involved: Km1, which has valine 153 (IMGT V45.1) and leucine 191 (I Km1,2 is related to alanine 153 (IMGT A45.1) and leucine 191 (IMG Km3 is related to alanine 153 (IMGT A45.1) and valine 191 (IMGT V 101) and are related to each other. Optionally, therefore, the sequence can be modified from one allotype to another, e.g., L191V (IMGT L101V) The change converts the Km1,2 allotype to the Km3 allotype. For further references, see Jefferis and Lefranc, which is incorporated herein by reference. See (2009) MAbs 1(4):332-8.

[0237] Thus, in one embodiment, the antibodies described herein are directed against other antibodies of the same gene. In a further embodiment, the antibody contains amino acid substitutions derived from the allotype. To convert the c-domain from km1,2 to km3 allotype, L191V (IMGT L 101V) substitutions.

[0238] (antibody binding) The antibody or fragment thereof has a molecular weight of 1.5 x 10 as measured by surface plasmon resonance. -7 M (i.e., 1 In a preferred embodiment, the Vδ1 chain of the γδ TCR can be bound with a binding affinity (KD) of less than 50 nM. The KD is 1.5 x 10 -7 In a further embodiment, the KD is less than 100 nM (i.e., 150 nM). , 1.3 × 10 -7 M (i.e., 130 nM) or less, e.g., 1.0 x 10 -7 M (i.e., 100 nM) or less In still further embodiments, the KD is 5.0 x 10 -8 M (i.e., less than 50 nM), e.g., 4. 0×10 -8 M (i.e., less than 40 nM), 3.0 × 10 -8 M (i.e., 30 nM), or 2.0 × 10 -8 M (Sunawa For example, according to one embodiment, the concentration of α-glucan in the α-glucan is less than 20 nM as measured by surface plasmon resonance. 1.5 x 10 -7 Human γδ TCRs bind to the Vδ1 chain with a binding affinity (KD) of less than 1 M (i.e., 150 nM). Anti-Vδ1 antibodies are provided.

[0239] In one embodiment, the antibody or fragment thereof has a 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 M(su i.e., it binds to the Vδ1 chain of the γδ TCR with a binding affinity (KD) of less than 20 nM.

[0240] In one embodiment, the binding affinity of an antibody or fragment thereof is determined by directly binding the antibody or fragment thereof. directly or indirectly (e.g., by capture with anti-human IgG Fc), a sensor (e.g., an amine The anti-reflection coating is applied to the surface of a high-capacity chip or equivalent, where The target bound by the antibody or a fragment thereof (i.e., the Vδ1 chain of the γδ TCR) is detected by Preferably, the MASS-2 instrument (also called the Sierra SPR-32) is used. (can also be used) was used in PBS + 0.02% Tween 20 running buffer at 30 μl / min at 25°C. do.

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

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

[0243] In such functional assays, the output is expressed as an "EC50" or "50 percent effective concentration" The half-maximal concentration, also called the "IC50," can be calculated. The term refers to inhibitory concentration. Both EC50 and IC50 can be determined by methods known in the art, e.g., For the avoidance of doubt, in this application EC50 values ​​for the antibody are provided using an IgG1 formatted antibody. Equivalent values ​​can be easily converted based on the molecular weight of the antibody format, such as Cut: (μg / ml) / (MW in kDa) = μM

[0244] The EC50 for down-regulation of the γδ TCR upon antibody (or fragment) binding is less than 0.50 μg / ml, e.g. For example, 0.40 μg / ml, 0.30 μg / ml, 0.20 μg / ml, 0.15 μg / ml, 0.10 μg / ml, or less than 0.05 μg / ml. In a preferred embodiment, downregulation of γδ TCR upon antibody (or fragment) binding. The EC50 for the antibody (or fragment) is less than 0.10 μg / ml. The EC50 for modulation of the agonist activity is less than 0.06 μg / ml, e.g., 0.05 μg / ml, 0.04 μg / ml, or 0.03 μg / ml. In particular, the EC50 value may be less than 1 μg / ml when the antibody is measured in an IgG1 format. For example, EC50 values ​​for γδ TCR down-regulation can be determined by flow cytometry (e.g., The assay can be measured using the assay described in Example 6.

[0245] The EC50 for γδ T cell degranulation upon antibody (or fragment) binding is less than 0.050 μg / ml, e.g. For example, 0.040 μg / ml, 0.030 μg / ml, 0.020 μg / ml, 0.015 μg / ml, 0.010 μg / ml, or 0.008 μg / ml In particular, the EC50 for γδ T cell degranulation upon antibody (or fragment) binding may be less than 1000 mg / ml. In a preferred embodiment, the concentration may be less than 0.005 μg / ml, for example less than 0.002 μg / ml. The EC50 for γδ T cell degranulation upon antibody (or fragment) binding is less than 0.007 μg / ml. In particular, the EC50 values ​​are those when the antibody is measured in an IgG1 format. EC50 values ​​for δ T cell degranulation were determined by flow cytometry (e.g., in the assay of Example 7). CD107a expression (i.e., a marker of cell degranulation) was measured using a chromatin assay (described previously). In one embodiment, CD107a expression can be measured by measuring the expression of anti-CD10 7a antibody, for example, anti-human CD107a BV421 (clone H4A3) (BD Biosciences). can be.

[0246] The EC50 for γδ T cell killing upon antibody (or fragment) binding is less than 0.50 μg / ml, e.g., Less than 0.40 μg / ml, 0.30 μg / ml, 0.20 μg / ml, 0.15 μg / ml, 0.10 μg / ml, or 0.07 μg / ml In a preferred embodiment, for γδ T cell killing upon antibody (or fragment) binding The EC50 of the antibody (or fragment) is less than 0.10 μg / ml. The EC50 for all of the compounds is less than 0.060 μg / ml, for example less than 0.055 μg / ml, in particular less than 0.020 μg / ml or 0.010 μg / ml. In particular, the EC50 value may be less than 1 μg / ml when the antibody is measured in an IgG1 format. For example, the EC50 value for γδ T cell killing is calculated based on the interaction between the antibody, γδ T cells, and target cells. After incubation, flow cytometry (e.g., as described in the assay in Example 8) was performed. Detect the proportion of dead cells (i.e., use a cell viability dye) using a In one embodiment, target cell death can be measured by viability. Viability is measured using the cell viability dye eFluor™ 520 (ThermoFisher).

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

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

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

[0250] Monoclonal antibodies against a given antigen can be, for example, a) from the peripheral blood of an animal pre-immunized with a given antigen to form hybridomas immortalizing the resulting lymphocytes with immortal cells, preferably myeloma cells; b) Cultivating the resulting immortalized cells (hybridomas) to produce antibodies with the desired specificity To collect cells that : can be obtained by

[0251] Alternatively, the use of hybridoma cells is not required. Antibodies capable of binding to the original can be prepared by routine practice, e.g., by methods known in the art. Phage display, yeast display, ribosome display, or mammalian display The antibody can be isolated from a suitable antibody library using display technology. Monoclonal antibodies can be, for example, a) vectors, in particular phages, more particularly filamentous bacteriophages, lymphocytes, particularly peripheral blood lymphocytes (preferably those previously immunized with a predetermined antigen) cloning the obtained DNA or cDNA sequence. b) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the antibody; Process c) selecting the antibody by subjecting it to antigen-affinity selection d) recovering antibodies with the desired specificity : can be obtained by a process including

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

[0253] The pharmaceutical composition comprises V51 T cells, in particular expanded V51 T cells, as described herein, in a In combination with the above pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers, such as neutral buffered saline, phosphate buffered saline, water, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, maize nitritol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelates an adjuvant (e.g., aluminum hydroxide); and Cryoprotective solutions that may be used in the pharmaceutical compositions of the present invention include, for example, For example, DMSO. The composition can be formulated, for example, for intravenous administration.

[0254] In one embodiment, the pharmaceutical composition is free of endotoxin or mycoplasma contamination. Qualitatively free from detectable levels of contaminants, e.g., endotoxin or mycoplasma does not exist.

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

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

[0257] In a further embodiment of the invention, the cell population, composition or pharmaceutical composition comprises at least one The two active agents may be administered sequentially, simultaneously, or separately.

[0258] (Therapeutic method using cell populations) According to a further aspect of the present invention there is provided a method as defined herein for use as a medicament. According to a further aspect of the present invention, a cell population obtained by the method is provided. There is provided an expanded cell population as defined herein for use as a pharmaceutical agent or References herein to a cell population "for use" in a therapy include administering the cells to a subject. Such use is limited to administration to a population of patients. Such use may include direct administration of antibodies or fragments thereof to a patient. In this case, the antibody is used as a therapeutic agent.

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

[0260] In one embodiment, the cell population for use as a medicament comprises more than 50% V51 T cells, e.g. For example, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% Vδ1 T cells. In certain embodiments, the cell population for use as a medicament consists of V51 T cells.

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

[0262] According to a further aspect of the present invention there is provided a method for treating a pulmonary arthritis, as defined herein, for use as a medicament. In one embodiment, a pharmaceutical composition comprising the cell population is provided. The product is for use in the treatment of cancer, an infectious disease, or an inflammatory disease. In certain embodiments, a pharmaceutical composition comprising the cell population is provided for use in the treatment of cancer. It is something.

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

[0264] According to a further aspect of the present invention, a method for treating cancer, infectious diseases, or a method of treating an inflammatory disease, comprising administering a therapeutically effective amount of a cell population as defined herein. Alternatively, a method for the treatment of a patient is provided, comprising administering to the patient a pharmaceutical composition comprising the cell population. An effective dose is administered.

[0265] According to a further aspect of the invention, the present invention provides a method for treating, for example, cancer, infectious diseases, or inflammatory diseases. There is provided the use of the cell population as defined herein for the manufacture of a medicament in

[0266] (Adoptive T cell therapy) The gamma delta T cells obtained by the expansion method of the present invention can be used as pharmaceutical agents, e.g., as adoptive T It can be used for cell therapy, which involves the transplantation of γδ T cells into a patient. Therapy may be autologous, i.e., γδ T cells are administered to the same host as they were obtained. The cells may be transplanted back into the patient, or the therapy may be allogeneic, i.e. That is, γδ T cells from one person may be transplanted into a different patient. Alternatively, γδ T cells may be substantially free of αβ T cells. The cells may be, for example, after expansion, dissected using any suitable means known in the art (e.g., magnetic They may be removed from the γδ T cell population using negative selection (e.g., using beads). The therapeutic method involves providing a sample of tissue (e.g., a non-hematopoietic tissue sample) obtained from a donor individual. culturing γδ T cells obtained from the samples described herein to produce expanded populations; and administering the population of γδ T cells to a recipient individual.

[0267] The patient or subject to be treated is preferably a human cancer patient (e.g., a patient with a solid tumor). Human cancer patients undergoing treatment) or virally infected patients (e.g., CMV-infected or HIV-infected patients) Optionally, the patient has a solid tumor and / or has received treatment for a solid tumor. Tissue-resident Vδ1 T cells are normally present in non-hematopoietic tissues, so these cells also its systemic blood-resident counterparts that may home to and be retained within the tumor mass Higher than conventional methods, adoptive transfer of these cells has potential applications in solid tumors and potentially other non-hematopoietic tissue-associated diseases. It may be more effective in targeting immunopathology.

[0268] Because γδ T cells are not MHC-restricted, they do not recognize the host into which they are transplanted as a foreign body. This means that γδ T cells are less likely to cause graft-versus-host disease. γδ T cells can be used “off the shelf” and used as, for example, allogeneic adoptive T cells This means that the cells can be transplanted into any recipient for therapy.

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

[0270] In some embodiments, the method of treating an individual with a tumor comprises administering to the individual a tumor-containing tissue obtained from a donor individual. providing a sample of a tumor comprising the γδ T cells of interest; culturing the γδ T cells obtained from said sample; and administering the population of γδ T cells to a tumor-bearing individual. In the method for treating an individual having a tumor in a non-hematopoietic tissue, providing a sample of hematopoietic tissue; culturing gamma delta T cells obtained from said sample; and and; administering the population of γδ T cells to an individual with a tumor.

[0271] Optionally, a therapeutically effective amount of γδ T cells obtained by any of the above methods (e.g., administered to a subject in a therapeutically effective amount, e.g., for the treatment of cancer, e.g., for the treatment of solid tumors. Optionally, γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) can be used. The therapeutically effective dose of 10 x 10 cells per dose 12 Fewer than 9 x 10 cells (e.g., 9 x 10 per dose) 12 Not yet available Fully 8 x 10 cells per dose 12 Less than 7 x 10 cells 12 Less than 6 x 10 cells per dose 12 Less than 5 x 10 cells per dose 12 Less than 4 x 10 cells per dose 12 Less than 1 cell, dose 3 x 10 12 Less than 2 x 10 cells per dose 12 Less than 1 x 10 cells per dose 12 Not yet available Fully 9 x 10 cells per dose 11 Less than 8 x 10 cells per dose 11 Less than 10 cells per dose 7×10 11 Less than 6 x 10 cells per dose 11 Less than 5 x 10 cells per dose 11 Less than one cells, 4 x 10 per dose 11 Less than 3 x 10 cells per dose 11 Less than 2 cells per dose x10 11 Less than 1 x 10 cells per dose 11Less than 9 x 10 cells per dose 10 Fewer than one cell , 7.5 x 10 per dose 10 Less than 5 x 10 cells per dose 10 Less than 2.5 cells per dose x10 10 Less than 1 x 10 cells per dose 10 Less than 7.5 x 10 cells per dose 9 Less than one piece cells, 5 x 10 per dose 9 Less than 2.5 x 10 cells per dose 9 <1 x 1 cells per dose 0 9 Less than 7.5 x 10 cells per dose 8 Less than 5 x 10 cells per dose 8 For less than 1 cell 2.5 x 10 per quantity 8 Less than 1 x 10 cells per dose 8 Less than 7.5 x 10 cells per dose 7 pieces Less than 5 x 10 cells per dose 7 Less than 2.5 x 10 cells per dose 7 Less than 10 cells per dose 1 x 10 7 Less than 7.5 x 10 cells per dose 6 Less than 5 x 10 cells per dose 6 Less than one cells, 2.5 x 10 per dose 6 Less than 1 x 10 cells per dose 6 Less than 7 cells per dose. 5×10 5 Less than 5 x 10 cells per dose 5 Less than 2.5 x 10 cells per dose 5 Less than one cell , or 1 x 10 per dose 5 (less than 100 cells).

[0272] In some embodiments, γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 The therapeutically effective dose of T cells is 10 × 10 12 Fewer than 9 × 1 cells (e.g., during the course of treatment) 0 12 Less than 8 x 10 cells 12 Less than 7 x 10 cells 12 Less than 6 x 10 cells 12 Less than one cell, 5×10 12 Less than 4 x 10 cells 12 Less than 3 x 10 cells 12 Less than 2 x 10 cells 12 Less than one piece cells, 1×10 12 Less than 9 x 10 cells 11 Less than 8 x 10 cells 11 Less than 7 x 10 cells 11 Less than cells, 6 × 10 11 Less than 5 x 10 cells 11 Less than 4 x 10 cells 11 Less than 3 x 10 cells 11 pieces Less than 2 x 10 cells 11 Less than 1 x 10 cells 11 Less than 9 x 10 cells 10 Less than 7.5x cells 10 10 Less than 5 x 10 cells 10 Less than 2.5 x 10 cells 10 Less than 1 x 10 cells 10 Less than one piece cell, 7.5×10 9 Less than 5 x 10 cells 9 Less than 2.5 x 10 cells 9 Less than 1 x 10 cells 9 Less than cells, 7.5 × 10 8 Less than 5 x 10 cells 8 Less than 2.5 x 10 cells 8 Less than 1 x 10 cells 8 pieces Less than 7.5 x 10 cells 7 Less than 5 x 10 cells 7 Less than 2.5 x 10 cells 7Less than 1 cell, 1 x 1 0 7 Less than 7.5 x 10 cells 6 Less than 5 x 10 cells 6 Less than 2.5 x 10 cells 6 Less than one cell, 1×10 6 Less than 7.5 x 10 cells 5 Less than 5 x 10 cells 5 Less than 2.5 x 10 cells 5 Less than one piece cells, or 1 x 10 5 (less than 100 cells).

[0273] In some embodiments, the γδ T cells described herein (e.g., skin-derived γδ The dose of Vδ1 T cells and / or Vδ1 T cells is approximately 1 × 10 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , or 5 x 10 8 Contains cells / kg. In some embodiments, γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) ) doses up to approximately 1 × 10 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 x10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , or 5 x 10 8 In some embodiments, Thus, the dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) is about 1.1×10 6 ~1.8×10 7 In some embodiments, γδ T cells (e.g., skin-derived γδ T cells) are administered intracellularly. The dose of Vδ1 T cells and / or Vδ1 T cells is approximately 1 × 10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 In some embodiments, the γ The dose of γδ T cells (e.g., skin-derived γδ T cells and / or Vδ1 T cells) is at least about 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 Individual cells In some embodiments, γδ T cells (e.g., skin-derived γδ T cells and / or V δ1 T cells) doses up to approximately 1 × 10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 x 10 9 Contains cells.

[0274] In one embodiment, the subject receives 10 mg of 10 ... 4 ~10 6 γδ T cells (e.g., In one embodiment, the subject is administered: An initial administration of a population of γδ T cells (e.g., 10 per kg body weight of the subject) 4 ~10 6 γδ T cells, e.g. For example, 10 per kg of subject's body weight 4 ~10 5 initial administration of γδ T cells), and more than one administration of γδ T cells or more (e.g., 2, 3, 4, or 5) subsequent doses (e.g., 10 per kg body weight of the subject). 4 ~10 6 γ δ T cells, e.g., 10 per kg body weight of the subject 4 ~10 5 and one or more subsequent administrations of γδ T cells In one embodiment, one or more subsequent administrations are administered less than 15 days after the previous administration, e.g. , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days or less since the previous administration, e.g., In one embodiment, the subject is administered a small number of γδ T cells in less than 1, 2, or 3 days. During the course of at least three doses, a total of approximately 10 6 γδ T cells, e.g. For example, the target is 1×10 5 Initial dose of γδ T cells, 3 × 10 5 a second administration of γδ T cells, and and 6×10 5 and a third administration of γδ T cells, e.g., each administration being 4 or more times longer than the previous administration. , 3, or less than 2 days.

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

[0276] Administration of the compositions can be carried out in any convenient manner. The composition may be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, or intravenously. Administering the compound to a patient by intravenous injection, or intraperitoneally, e.g., by intradermal or subcutaneous injection. The gamma delta T cell composition can be injected directly into a tumor, lymph node, or site of infection. can.

[0277] (genetic modification) The γδ T cells obtained by the methods of the present invention may have enhanced therapeutic properties, e.g., chimeric antibodies They can also be genetically modified for carcinoma receptor T cell (CAR-T) therapy, which has novel specificities For example, modifications to reprogram T cells with the specificity of a monoclonal antibody. This involves the generation of altered T cell receptors (TCRs). The altered TCRs are specific for the malignant cells. Therefore, it is possible to create T cells that are useful for cancer immunotherapy. For example, T cells can be generated by using T cells derived from target tissues. Cancer cells that express tumor antigens, e.g., tumor-associated antigens, that are not expressed by normal somatic cells. Therefore, CAR-modified T cells can be used for, for example, adoptive T cell therapy for cancer patients. can be used for.

[0278] Other uses of antibodies or fragments thereof According to a further aspect of the present invention, antigen recognition, activation, and Anti-V51 antibodies or fragments thereof described herein for testing signal transduction or function As described herein, the antibody may be used to investigate γδ T cell function. It has been shown to be active in assays that can be used to Such antibodies may also be useful in inducing proliferation of γδ T cells and therefore γδ It can be used in methods to expand T cells (e.g., V51 T cells).

[0279] Antibodies that bind to the Vδ1 chain can be used to detect γδ T cells (i.e., as a label). Preferably, the antibody used as a label is one that targets V51 T cells and is affected upon antibody binding. For example, the antibody may be attached to a detectable label or receptor and not stimulate cell proliferation so that the antibody is not subject to These can be labeled with a target molecule or used as a capture ligand to selectively detect Vδ1 T cells in a sample. Labeled antibodies can be isolated and / or purified by a number of methods known in the art, including For example, it is used in immunohistochemistry and ELISA.

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

[0281] Polynucleotides and Expression Vectors Also provided are polynucleotides encoding the anti-Vδ1 antibodies or fragments of the invention. In one embodiment, the anti-Vδ1 antibody or fragment has a sequence identical to that of SEQ ID NOs: 99 to 110 by at least 70%. , e.g., at least 80%, e.g., at least 90%, e.g., at least 95%, e.g. a polynucleotide comprising or consisting of a sequence having at least 99% sequence identity In one embodiment, the anti-Vδ1 antibody or fragment is encoded by SEQ ID NO: 99 In another embodiment, the anti-Vδ antibody is encoded by an expression vector comprising the VH region of Vδ 110. The antibody or fragment thereof is encoded by an expression vector comprising the VL region of any of SEQ ID NOs: 99 to 110. In a further embodiment, the polynucleotide comprises SEQ ID NOs: 99-110 or the sequences In a further embodiment, a cDNA comprising the polynucleotide is provided. can be.

[0282] In one embodiment, the polynucleotide has at least 70% identical sequence to SEQ ID NOs: 99-110, e.g. For example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least In one embodiment, the sequence comprises or consists of a sequence having at least 99% sequence identity with the In another embodiment, the expression vector comprises a VH region of any of SEQ ID NOs: 99 to 110. In a further embodiment, the polynucleotide comprises a VL region of SEQ ID NO: 99 to 110. In a further aspect, the code comprises or consists of SEQ ID NOs: 99 to 110. A cDNA comprising the polynucleotide is provided.

[0283] In one embodiment, the polynucleotide has at least one sequence identical to SEQ ID NOs: 99-101 or 105-108. at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, For example, it comprises or consists of a sequence having at least 99% sequence identity. In another embodiment, the expression vector comprises a VH region of SEQ ID NO: 99 to 101 or 105 to 108. In one embodiment, the expression vector comprises a VL region of SEQ ID NO: 99 to 101 or 105 to 108. In certain embodiments, the polynucleotide comprises SEQ ID NOs: 99-101 or 105-108. or consisting of SEQ ID NOs: 99 to 101 or 105 to 108. A cDNA containing the nucleotide is provided.

[0284] In one embodiment, the polynucleotide has at least 70% identical sequence to SEQ ID NOs: 99-101, e.g., For example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least In one embodiment, the sequence comprises or consists of a sequence having at least 99% sequence identity. The expression vector comprises the VH regions of SEQ ID NOs: 99 to 101. In another embodiment, the expression vector In a further embodiment, the polynucleotide comprises a VL region of SEQ ID NOs: 99 to 101. In a further aspect, the A cDNA containing polynucleotide is provided.

[0285] In one embodiment, the polynucleotide encodes an immunoglobulin chain variable domain. any one of the portions of SEQ ID NOS: 99 to 110 encoding CDR1, CDR2, and / or CDR3 of the amino acid sequence of at least 70%, for example at least 80%, for example at least 90%, for example at least It comprises or consists of a sequence having 95%, for example at least 99%, sequence identity. In one embodiment, the polynucleotide encodes an immunoglobulin chain variable domain. Any of the portions of SEQ ID NOs: 99 to 101 or 105 to 108 encoding CDR1, CDR2, and / or CDR3 of or one and at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., or a sequence having at least 95%, for example at least 99%, sequence identity with the sequence In one embodiment, the polynucleotide comprises an encoded immunoglobulin chain. Any of the portions of SEQ ID NOS: 99 to 101 encoding CDR1, CDR2, and / or CDR3 of the variant domain One and at least 70%, for example, at least 80%, for example, at least 90%, for example, or a sequence having at least 95%, for example at least 99%, sequence identity thereto. Or rather.

[0286] In one embodiment, the polynucleotide encodes an immunoglobulin chain variable domain. any one of the portions of SEQ ID NOS: 99 to 110 encoding FR1, FR2, FR3, and / or FR4 of the At least 70%, for example, at least 80%, for example, at least 90%, for example, at least or consisting of a sequence having at least 95%, e.g., at least 99%, sequence identity. In one embodiment, the polynucleotide encodes an immunoglobulin chain variable domain. Any of the portions of SEQ ID NOs: 99 to 101 or 105 to 108 encoding FR1, FR2, FR3, and / or FR4 of the One of them and at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., For example, a sequence having at least 95%, for example at least 99%, sequence identity with or comprising the sequence In one embodiment, the polynucleotide comprises the encoded immunoglobulin Any of the portions of SEQ ID NOS: 99 to 101 encoding FR1, FR2, FR3, and / or FR4 of the chain variable domain One of them and at least 70%, e.g., at least 80%, e.g., at least 90%, e.g., For example, a sequence having at least 95%, for example at least 99%, sequence identity with or comprising the sequence It consists of an array.

[0287] The polynucleotides and expression vectors of the present invention may be used in conjunction with the encoded amino acid sequence. Thus, in one embodiment, the polynucleotide may be described as follows: It comprises or consists of a sequence encoding any one of the amino acid sequences of Nos. 62 to 85. In one embodiment, the expression vector comprises an amino acid sequence selected from the group consisting of: In another embodiment, the expression vector comprises a sequence encoding any one of SEQ ID NOs: 74 to 85. It includes a sequence encoding any one of the amino acid sequences.

[0288] To express an antibody or fragment thereof, a partial or full-length light chain and The polynucleotide encoding the heavy chain is a gene operably linked to transcriptional and translational control sequences. Therefore, in one aspect of the present invention, In one embodiment, an expression vector is provided comprising the polynucleotide sequence defined in The expression vector is selected from the group consisting of SEQ ID NOs: 99 to 110, for example, SEQ ID NOs: 99, 100, 101, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 In another embodiment, the expression vector comprises a VH region of SEQ ID NO: 99-108. 10, for example, the VL region of SEQ ID NO: 99, 100, 101, 105, 106, 107, or 108.

[0289] The nucleotide sequences described herein may be synthesized using amino acid sequences to aid in translation, purification, and detection. It contains additional sequences encoding amino acid residues, but depending on the expression system used, different sequences may be used. For example, the first 9 (5'-end) sequences of SEQ ID NOs: 99 to 110 can be used. and the last (3'-end) 36 nucleotides of SEQ ID NOs: 99-100, 102-103, and 105-110. The last (3'-end) 39 nucleotides of the sequence or SEQ ID NOs: 101 and 104 are arbitrary sequences. If alternative design, translation, purification, or detection strategies are employed, these optional sequences may be removed or It can be modified or substituted.

[0290] It is silent with respect to the amino acid sequence of the polypeptide, but is Mutations to the DNA or cDNA encoding the polypeptide provide preferred codons for For example, Escherichia coli and S. cerevisiae, as well as mammalian Preferred codons for translation of nucleic acids in organisms, particularly humans, are known.

[0291] Mutations in a polypeptide can include, for example, substitutions, additions, or modifications to the nucleic acid encoding the polypeptide. This can be achieved by addition or deletion to the nucleic acid encoding the polypeptide. The substitutions, additions, or deletions can be made by, for example, error-prone PCR, shuffling, oligonucleotide synthesis, or the like. Tide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, Set mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific Targeted mutagenesis, gene reassembly, artificial gene synthesis, gene site saturation mutagenesis (G Many methods are available, including synthetic ligation reassembly (SSM), synthetic ligation reassembly (SLR), or a combination of these methods. Modifications, additions, or deletions to nucleic acids can be introduced by methods such as recombination, Recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis Induction, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strains Mutagenesis, chemical mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction selection Mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimerization, or It can also be introduced by methods including combinations of these.

[0292] In particular, artificial gene synthesis can be used. The gene can be produced synthetically, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo without the need for a To obtain a oligonucleotide, the building blocks are added to a growing oligonucleotide chain according to the sequence of the product. Once chain assembly is complete, the product is transferred to a solid support. The product is then released into solution, deprotected, and recovered. Thus, the desired oligonucleotide can be isolated and obtained in high purity.

[0293] Examples of expression vectors include plasmids, retroviruses, cosmids, and yeast artificial dyes. Polynucleotides include chromatids (YACs), and Epstein-Barr virus (EBV)-derived episomes. The nucleotide is a transcriptional and translational regulatory sequence within the vector that controls the transcription and translation of the polynucleotide. It is ligated into a vector so that it performs its intended function of regulating expression. and / or regulatory sequences, such as promoters, enhancers, transcription terminators, coding The initiation codon (i.e., ATG) 5' of the sequence, the splicing signals of the introns, and The expression vector and expression control sequence may be used in the expression SEQ ID NOs: 99-110 are selected to be compatible with the host cell. A single chain variable fragment of the present invention comprising a VH domain and a VL domain connected by a VH domain (encoding sequence number 98). The polynucleotide or expression vector of the present invention comprises a nucleotide sequence encoding the fragment. It will be understood that the VH region, VL region, or both (optionally including a linker) may be included. Therefore, the polynucleotides encoding the VH and VL regions are inserted into separate vectors. Alternatively, sequences encoding both regions can be inserted into the same expression vector. Polynucleotides can be prepared by standard methods (e.g., polynucleotides and vectors). Ligation of complementary restriction sites, or, if no restriction sites are present, blunt-end ligation It is inserted into an expression vector by the following procedure:

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

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

[0296] Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammals are known in the art. These methods are well known in the art and include dextran-mediated transfection, phosphorylation, and Acid calcium precipitation, polybrene-mediated transfection, protoplast fusion, elastase Microporation, encapsulation of polynucleotides in liposomes, biolistic injections and direct microinjection of DNA into the nucleus. , can be introduced into mammals by viral vectors.

[0297] Mammalian cell lines available as hosts for expression are well known in the art; This includes the American Type Culture Collection These include, among others, the many immortalized cell lines available from the American College of Cancer Cells (ATCC). Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney ( BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells Mammalian host cells include human, mouse, rat, and several other cell lines. , dog, monkey, pig, goat, bovine, horse, and hamster cells. Cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used include insect cell lines, e.g., Sf9 cells, amphibian cells, bacterial cells, plant cells, and the like. Antigen-binding fragments of antibodies, such as scFv and Fv fragments, are known in the art. The vector can be isolated and expressed in E. coli using methods known in the art.

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

[0299] The antibodies (or fragments) of the present invention can be prepared by any of the methods described in, for example, Green and Sambrook, Molecular Cloning: Experimental Methods. Molecular Cloning: A Laboratory Manual (2012), 4th ed., Cold Spring It can be obtained and manipulated using techniques disclosed in Harbour Laboratory Press. do.

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

[0301] Monoclonal antibodies against a given antigen can be, for example, a) from the peripheral blood of an animal pre-immunized with a given antigen to form hybridomas immortalizing the resulting lymphocytes with immortal cells, preferably myeloma cells; b) Cultivating the resulting immortalized cells (hybridomas) to produce antibodies with the desired specificity To collect cells that : can be obtained by

[0302] Alternatively, the use of hybridoma cells is not required. Antibodies capable of binding to the original can be prepared by routine practice, e.g., by methods known in the art. Phage display, yeast display, ribosome display, or mammalian display The antibody can be isolated from a suitable antibody library using display technology. Monoclonal antibodies can be, for example, a) vectors, in particular phages, more particularly filamentous bacteriophages, lymphocytes, particularly peripheral blood lymphocytes (preferably those previously immunized with a predetermined antigen) cloning the obtained DNA or cDNA sequence. b) transforming a prokaryotic cell with the vector described above under conditions that allow the production of the antibody; Process c) selecting the antibody by subjecting it to antigen-affinity selection d) recovering antibodies with the desired specificity : can be obtained by a process including

[0303] It is understood that all embodiments described herein are applicable to all aspects of the present invention. It will be possible.

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

[0305] (Example) Example 1. Materials and Methods (Human antibody discovery) Human phage display can be used to generate the human anti-human variable V51+ domains described herein. The main antibody was produced. The library was prepared according to the method described by Schofield et al. (Genome biology 2007, 8(11) : R254) and contains ~40 billion single-chain fragments of human clones. This library was used to generate the antibodies described herein. Original,methods, selection, deselection, screening, and screening using characterization strategies did.

[0306] (antigen preparation) Soluble γδ TCR heterodimers comprising TCR α and TCR β constant regions used in the examples below The design was made according to Xu et al. (2011) PNAS 108: 2414-2419. Vγ or Vδ domain The TCRα or TCRβ constant region lacking the transmembrane domain is followed by a leucine zipper sequence. The nucleotide sequence was fused in-frame to the Fc or Fc sequence and a histidine tag / linker.

[0307] Expression constructs were transfected into mammalian EXPI HEK293 suspension cells (single transfectant for heterodimers). transiently transfected (either as transfection or cotransfection) The secreted recombinant protein was purified from the culture supernatant by affinity chromatography. To ensure good recovery of the monomeric antigen, the sample was purified by preparative size exclusion chromatography. The purified antigen was further purified using chromatography (SEC). The aggregates were analyzed by DS-PAGE and the aggregation state by analytical SEC.

[0308] (Functional validation of antigen) The specificity of antigens containing the delta variable 1 (Vδ1) chain was determined using the DELFIA immunoassay (Perkin Elmer). and REA173, a flow-based antibody produced by Miltenyi Biotec that competes with γδ T cells. This was confirmed by the assay.

[0309] (Dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)) For confirmation of antigen specificity, the DELFIA immunoassay was performed on plates (3 μg / ml in 50 μL PBS). Antigens were directly coated onto a 1000 nM plate (Nunc #437111) overnight at 4°C, and the 1000 nM plate was then incubated at 4°C for 1 hour. For the detection of DELFIA Eu-N1, anti-human IgG (Perkin Elmer # 1244-330) as the secondary antibody, in 50 μL of 3% MPBS (PBS + 3% (w / V) skim milk powder). The color was developed using 50 μL of DELFIA Enhancement Solution (Perkin Elmer #4001-001) at a 1 / 500 dilution. 0).

[0310] The affinity ranking of the target antibody is determined by aligning the antibody with the protein coated on the plate. Captured with PEG and soluble biotinylated L1 (DV1-GV4) antigen was added at 5 nM in 50 μL (3M PBS). For detection, 50 μL of streptavidin-Eu was used. (1:500 in assay buffer, Perkin Elmer) and the signal was enhanced with DELFIA enhancement solution. D1.3 hIgG1 (described in England et al. (1999) J. Immunol. 162: 2129-2136) (previously known as 'anti-cancer drug') was used as a negative control.

[0311] The output of the phage display selection was transformed into the scFv expression vector pSANG10 (Martin et al., 2006). The soluble scFv was expressed and directly immobilized. Hits were screened for binding to the identified targets by DELFIA. was defined as a DELFIA signal above the first level.

[0312] (Antibody preparation) The selected scFvs were subcloned into an IgG1 framework using commercially available plasmids. For antibody expression, the plasmid was transfected into expi293F suspension cells. For convenience, unless otherwise noted, the antibodies characterized in these examples are expressed as scFvs. It refers to an IgG1 formatted antibody selected from phage display. The antibody may be of any of the antibody formats discussed above.

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

[0314] (γδ T cell preparation) The enriched population of γδ T cells is referred to as WO2016 / 198480 (i.e., blood-derived γδ T cells) or W γδ T cells were prepared according to the method described in O2020 / 095059 (i.e., skin-derived γδ T cells). Briefly, for blood-derived γδ T cells, PBMCs were obtained from blood and magnetically isolated. The αβ-depleted PBMCs were then subjected to depletion of αβ T cells. Anti-Vδ1 antibody), IL-4, IFN-γ, IL-21, and IL-1β were added to CTS OpTmiser medium (ThermoFis On day 7 of the culture, OKT-3 (or the respective anti-Vδ1 antibodies), IL- On day 11 of culture, the medium was supplemented with OKT-3 (or each of the antibodies). On day 14 of culture, half of the medium was replaced with fresh complete OpT The medium was replaced with miser and supplemented with OKT-3 (or the respective anti-Vδ1 antibody), IL-15, and IFN-γ. From day 17 onwards, cultures were supplemented with OKT-3 (or the respective anti-Vδ1 antibody) and IL-15 every 3–4 days. Half of the medium was replaced with fresh medium every 7 days.

[0315] For skin-derived γδ T cells, skin samples were prepared by removing subcutaneous fat and incubated for 3 min. A number of biopsy punches are prepared using the biopsy punch. The punches are then placed on a carbon matrix grid. Each well was filled with AIM-V medium (Gibco, Life Technologies), serum replacement from CTS Immune (Life Technologies), IL-2, and IL-15 During the first 7 days of culture, amphotericin B (Life Technologies) was added to the culture medium. Complete isolation medium containing the AMP-containing AMP-1000 (Primer's technology) was used ("+AMP"). Gently aspirate the top medium without disturbing the cells at the bottom of the tube and add 2x complete isolation medium (AM The medium was changed every 7 days by replacing it with fresh medium (without P). After more than 3 weeks in culture, Afterwards, the resulting transmigrated cells were transferred to a new tissue culture vessel and fresh medium (e.g., AIM-V medium or TexM Cells were collected after passage in AX medium (Miltenyi) plus recombinant IL-2, IL-4, IL-15, and IL-21. The αβ T cells also present in the culture were then depleted using an αβ T cell depletion kit and associated protocols. Protocols such as those provided by Miltenyi are used. For reference, see WO2020 / 095059.

[0316] (γδ T cell binding assay) Antibody binding to γδ T cells was assessed by incubating a fixed concentration of purified antibody with 250,000 γδ T cells. This incubation was performed to determine whether Fc receptor-mediated signaling was involved. Detection was performed under blocking conditions to prevent nonspecific binding of antibodies. Negative controls were performed by adding a fluorescent dye-conjugated secondary antibody against , cells were incubated with a) isotype antibody alone (recombinant human IgG), b) fluorochrome-conjugated anti-human I c) prepared using only IgG antibody, and c) a combination of a) and b). Control wells were also prepared and analyzed. As a positive control, purified mouse monoclonal IgG2 anti-human CD Three antibodies and a purified mouse monoclonal IgG1 anti-human TCR Vδ1 antibody were used at two different concentrations. The assay was performed in the FITC channel, followed by staining with a fluorescent dye-conjugated goat anti-mouse secondary antibody. The mean fluorescence intensity of the lower concentration positive control is at least 10 times that of the highest negative control. In some cases it was accepted.

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

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

[0319] γδ TCR Down-Regulation and Degranulation Assay THP-1 (TIB-202™, A) cells were loaded with or without test antibodies. TCC) target cells were labeled with CellTracker™ Orange CMTMR (ThermoFisher, C2927) and expressed CD107 a In the presence of antibody (anti-human CD107a BV421 (clone H4A3) BD Biosciences 562623) at a ratio of 2:1 After 2 hours of incubation, γδ T cells were incubated with Surface expression of γδ TCR (to measure TCR downregulation) and expression of CD107a (to measure degranulation) on cells were measured. The activity of the IgG4-dependent ...

[0320] Killing assay The effect of the test antibody on the killing activity of gamma delta T cells and gamma delta T cells was examined. After 4 hours of in vitro co-culture, live target THP cells were assessed by immunocytometry. To distinguish between THP-1 cells and dead target THP-1 cells, γδ T cells and CellTracker were used at a ratio of 20:1. (Trademark) Orange CMTMR (ThermoFisher, C2927) labeled THP-1 cells (antibody loaded) Loaded or unloaded) was treated with Viability Dye eFluor™ 520 (ThermoFisher At the time of sample acquisition, target cells were stained with CellTracker™ Orange. Cells were gated on CMTMR-positive cells and examined for cell death based on viability dye uptake. MTMR and eFluor™ 520 double positive cells were recognized as dead target cells. γδ T cell killing Activity was expressed as % of dead target cells.

[0321] (epitope mapping) All protein samples used for epitope mapping (antigen L1 (DV1-GV4) and antibody 12 45_P01_E07, 1245_P02_G04, 1252_P01_C08, 1251_P02_C05, and 1141_P01_E01) Protein integrity and aggregation levels were analyzed using high-mass MALDI.

[0322] L1(DV1-GV4) / 1245_P01_E07, L1(DV1-GV4) / 1245_P02_G04, L1(DV1-GV4) / 1252_P01_C08, High resolution L1(DV1-GV4) / 1251_P02_C05 and L1(DV1-GV4) / 1141_P01_E01 complex epitopes To determine the kinetics of ATP binding, the protein complex is incubated with a deuterated cross-linker. Trypsin, chymotrypsin, Asp-N, elastase, and thermolysin were used. After enrichment of cross-linked peptides, the samples were analyzed by high-resolution mass spectrometry. The data was analyzed by chromatometry (nLC-LTQ-Orbitrap MS), and the generated data was analyzed using XQuest and Stavr Analysis was performed using ox software.

[0323] SYTOX-Flow Killing Assay The SYTOX assay is a method for quantifying T cell-mediated lysis of target cells using flow cytometry. It allows the amount of dead / dying cells to penetrate only cells with defective plasma membranes. , a dead cell stain (SYTOX® AAD) that cannot cross the intact cell membrane of healthy cells. NALM-6 target cells were detected by CTV colorimetry (vanced™, Life Technologies, S10274). The enzyme was labeled with Cell Trace Violet™, Life Technologies, C34557, thereby preventing the formation of unlabeled Dead / dying target cells were distinguishable from effector T cells by death dye and Identified by double staining with cell tracking dyes.

[0324] Effector and CTV labeling at the indicated effector-to-target ratios (E:T, 1:1, or 10:1) After 16 hours of in vitro co-culture of target cells, the cells were stained with SYTOX® AADvanced™. The killing results were compared between the addition of effector cells and the control cells. The ratio of live target cells in the test sample to live target cells in the control wells (maximum count) Shown as % target cell reduction, calculated by considering the number of target cells (sample count) Currently:

number

[0325] Example 2. Antigen design Gamma delta (γδ) T cells are polyclonal with respect to CDR3 polyclonality. To avoid the situation where antibodies are generated that are selected against their CDR3 sequences (which are TCR clones), (As CDR3 fragments vary across formats, antigen design requires maintaining a consistent CDR3 across various formats.) This design is germline encoded and therefore all clones and generate antibodies that recognize sequences within the variable domains that are identical in γδ T cells. The goal was to provide antibodies that recognize a broader subset.

[0326] Another important aspect of the antigen preparation process is to design an antigen that is suitable for expression as a protein. The gamma delta TCR has intrachain and interchain disulfide bonds. It is a complex protein containing a heterodimer containing leucine zipper (LZ) format and Fc The format is used to generate soluble phage markers to be used in phage display selection. Both the LZ and Fc formats were well expressed and expressed in TCR. (In particular, heterodimeric TCRs, such as Vδ1Vγ4, were well presented.

[0327] The CDR3 sequences obtained from public database entries of γδ TCRs were identified as proteins (RCSB proteins). It was found to be well expressed as a protein (protein data bank entry: 3OMZ). This was selected for antigen preparation.

[0328] The antigen containing the delta variable 1 chain was expressed as a heterodimer in the LZ format (i.e., heterodimer). and a heterodimer in Fc format (combined with gamma variable chains - "L1", "L2", "L3"). either as monodimers (“F1”, “F2”, “F3”) or as homodimers (i.e., different delta The antigen was expressed either in combination with the delta variable 1 chain ("Fc1 / 1"). All contained the 30MZ CDR3. Another series of γδ TCR antibodies using a similar format The antibodies were designed to contain different delta variable chains (e.g., delta variable 2 and delta variable 3) to generate non-specific were used to deselect antibodies with target or off-target binding ("L4", "F 9, Fc4 / 4, Fc8 / 8). Antibody binding in the CDR3 region is also deselected. To ensure this, these antigens were designed to contain the 30MZ CDR3.

[0329] The designed antigen was confirmed to be suitable for generating anti-TRDV1 (TCR delta variable 1) antibodies. To detect the antigen function, we performed a verification test. was observed (Figure 1).

[0330] Example 3. Phage display Either heterodimeric LZ TCR format was used in rounds 1 and 2, and both rounds Phage display selection was performed using the heterodimeric LZ TCR for deselection and the human scFv Alternatively, round 1 was performed using a homodimeric Fc-fused TCR and a human IgG1 F round 2 was performed with heterodimeric LZ TCR and round 3 was performed with heterodimeric LZ TCR. deselected (see Table 1). Table 1. Summary of phage display selection [Table 3] bt = biotin

[0331] Selection was performed in solution phase using 100 nM biotinylated protein. Deselection was performed using 1 μM This was performed using non-biotinylated proteins.

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

[0333] Example 4. Antibody Selection The hits obtained in Example 3 were sequenced (using standard methods known in the art). 130 unique clones were identified, which consisted of the VH CDR3 and VL CDR3 unified sequences. Of these 130 unique clones, 125 were unique. VH CDR3s were shown, and 109 showed unique VL CDR3s.

[0334] Unique clones were rearranged and specificity was analyzed by ELISA (DELFIA). , L2, L3, F1, F2, F3) but not TRDV2 (L4) A panel of Fv binders was identified from the selection.

[0335] Affinity ranking of selected binders helps guide clone selection forward. Many binders showed affinities in the nanomolar range, between 25 and 100 nM. A few binders showed strong reactions with 5 nM of antigen, with single-digit binding activity. Some binders showed nanomolar affinity potential, with reactions with 100 nM of antigen. showed no cleavage and showed affinity in the micromolar range.

[0336] To advance clone selection to IgG conversion, the goal is to identify as many germline lineages as possible. The goal was to include as many different CDR3s as possible. Sequence trends such as the cysteine-binding site, CD11c / CD18-binding site, and unpaired cysteine ​​were avoided. Additionally, various affinities were included.

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

[0338] Example 5: Antibody SPR analysis The prepared IgG antibodies were subjected to a γδ cell binding assay, and the five best binders were selected. The equilibrium dissociation constant (K D ) to determine SPR analysis was performed. The sensorgram of the interaction between the test antibody and the analyte was obtained as a steady-state flow. The binding of TS8.2 was detected in the TS8.2 antibody, along with the binding of the antibody to the TS8.2 antibody (where available). The results are summarized in Table 3. Table 3. IgG capture results [Table 5] * The binding of 1252_P02_C05 did not reach saturation, therefore the data were extrapolated.

[0339] Example 6: TCR engagement assay We have identified several assays that should be used for the functional characterization of selected antibodies. The first assay was designed to measure downregulation of γδ TCR upon antibody binding. γδ TCR engagement was assessed. Selected antibodies were used as positive controls. Commercially available anti-CD3 and anti-Vδ1 antibodies, or (1139_P01_E04, 1245_P02_F07, 1245_P01 The antibodies were tested against 1252_P01_C08 as a positive control (1245_P01_G06, and 1245_P01_G09). Pan-γδ is a pan-γδ antibody that recognizes all γδ T cells, regardless of variable chain, and therefore , which was used as a negative control since it likely has a different mode of action.

[0340] This assay was performed on three different donor samples (samples with purity of 94%, 80%, and 57%). The results are shown in Figure 4. EC50 The values ​​are summarized in Table 4 below.

[0341] Example 7: T cell degranulation assay The second assay assessed the degranulation of γδ T cells. γδ T cells are perforating It is believed that it may mediate target cell killing by ranzyme-mediated activation of apoptosis. Lytic granules in the cytoplasm of γδ T cells are released toward target cells upon T cell activation. Therefore, we performed labeling of target cells with antibodies against CD107a and flow cytometry. Measurement of expression by γδ T cells can be used to identify degranulating γδ T cells.

[0342] Regarding Example 6, the selected antibodies were compared with a commercially available anti-CD3 antibody and an anti-Vδ1 antibody as positive controls. as antibodies or positive controls (1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09). All 1252_P01_C08 antibodies were tested against IgG2a, IgG1, and D1.3 antibodies as negative controls. The assay was performed on three different donor samples (samples with purities of 94%, 80%, and 57%). The results are shown in Figure 5. The 50 values ​​are summarized in Table 4 below.

[0343] Example 8: Killing Assay The third assay measures the ability of selected antibody-activated γδ T cells to kill target cells. strength was evaluated.

[0344] Regarding Example 6, the selected antibodies were compared with a commercially available anti-CD3 antibody and an anti-Vδ1 antibody as positive controls. Positive for the body or (1139_P01_E04, 1245_P02_F07, 1245_P01_G06, and 1245_P01_G09) Tested against 1252_P01_C08 as a control and anti-pan-γδ as a negative control. IgG2a, I The gG1 and D1.3 antibodies were also used as isotype controls. The results are shown in Figure 6. is shown in.

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

[0346] Example 9: Epitope Mapping To determine the epitopes of antigen / antibody complexes with high resolution, protein complexes were purified using deuterium oxide. The cross-linked peptide was incubated with a hydroxylated cross-linker and subjected to multiple enzymatic cleavage. After enrichment, the samples were analyzed by high-resolution mass spectrometry (nLC-LTQ-Orbitrap MS). The generated data was then analyzed using XQuest (version 2.0) and Stavrox (version 3.6) software. The analysis was performed using a

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

[0348] The protein complex L1(DV1-GV4) / 1252_P01_C08 was purified with trypsin along with deuterated d0d12. After proteolysis with chymotrypsin, Asp-N, elastase, and thermolysin, nLC -orbitrap MS / MS analysis identified five cross-linked peptides between L1 (DV1-GV4) and antibody 1252_P01_C08 was detected. The results are shown in Figure 8.

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

[0350] The protein complex L1(DV1-GV4) / 1251_P02_C05 was purified with trypsin along with deuterated d0d12. After proteolysis with chymotrypsin, Asp-N, elastase, and thermolysin, nLC -orbitrap MS / MS analysis identified five cross-linked peptides between L1 (DV1-GV4) and antibody 1251_P02_C05 was detected. The results are shown in Figure 10.

[0351] Epitope binding by another antibody, clone ID 1141_P01_E01, was also tested. The protein complex L1(DV1-GV4) / 1141_P01_E01 was treated with trypsin, chymotrypsin, Proteolysis with Asp-N, elastase, and thermolysin was followed by nLC-orbitrap MS / MS Analysis detected 20 cross-linked peptides between L1 (DV1-GV4) and antibody 1141_P01_E01. The results are shown in FIG.

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

[0353] Example 10: Expansion of V51 T cells The expansion of isolated γδ T cells was investigated in the presence of selected and comparator antibodies. The comparator antibodies were OKT3 anti-CD3 antibody as a positive control and antibody as a negative control. The antibody was selected from the following: none, or an IgG1 antibody as an isotype control. S-1 and TS8.2 were also tested for comparison.

[0354] Test 1: 70,000 cells / well were cultured using the complete Optimizer and the "γδ T cells" of blood-derived γδ T cells of Example 1. Initial studies were performed by seeding the cells with cytokines as described in "Preparation" The selected antibody and comparator antibody were incubated at various concentrations ranging from 4.2 ng / ml to 420 ng / ml. This experiment was performed using a tissue culture plate that allows for antibody binding / immobilization to plastic. The experiment was carried out using the rate.

[0355] Cells were harvested on days 7, 14, and 18, and total cell count was determined using a cell counter (NC250, ChemoMetec). Cell counts were determined, and the results are shown in Figure 12. The cell viability of Vδ1 T cells was also All antibodies were shown to maintain cell viability throughout the experiment, as measured by cell harvest. On day 18, the percentage of Vδ1 T cells, cell count, and transformation were measured. The fold increase was also analyzed, and the results are shown in Figure 13.

[0356] As can be seen in Figure 12, the total number of cells produced in culture with antibody increased throughout the culture. The antibody titer increased steadily with increasing dose, and was comparable to or greater than that of a commercially available anti-Vδ1 antibody. The maximum concentrations of 1245_P02_G04 ("G04"), 1245_P01_E07 ("E07"), and 1245_P01_B07 ("B07") The percentage of Vδ1-positive cells in the presence of OKT3, TS-1, or 1252_P01_C08 ("C08") antibodies was significantly higher than that in the presence of 1252_P01_C08 ("C08") antibodies. was greater than in cultures in the presence of TS8.2 control antibody (see Figure 13A).

[0357] Test 2: Subsequent experiments were performed using cytokine-containing culture media as described in Example 1, "γδ T cell preparation." This was performed on isolated cells in a culture vessel. Compared to experiment 1, the surface was more suitable for antibody binding / fixation. A different culture vessel that does not promote chromatinization was used. The selected antibodies and comparator antibodies were Various concentrations ranging from 42 pg / ml to 42 ng / ml were tested. In experiment 2, the results were obtained in triplicate. obtained from the experiments carried out.

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

[0359] The cell composition, including non-Vδ1 cells, was also measured in Experiment 2. Cells were collected on day 17 and flow cytometry was performed. Surface expression of V51, V52, and αβ TCRs was analyzed by cytometry. The proportion of each cell type in the culture is shown graphically in Figure 16, where percentage values ​​are are provided in Table 6. Table 6. Cellular composition at day 17 - percentage of viable cells in each subset [Table 8]

[0360] As can be seen from these results, the percentage of Vδ1-positive cells was significantly higher than that of OKT3, TS-1, or TS8.2 controls. is greater in cultures in which B07, C08, E07, and G04 are present compared to The tested antibodies inhibited V more efficiently than commercially available antibodies, even when present at low concentrations in culture. Produces and expands δ1-positive cells.

[0361] The presence of CD27-expressing (i.e., CD27+) natural killer (NK) cells and Vδ1 T cells To confirm this, cells from day 17 of experiment 2 were analyzed for additional cell markers, including CD3-CD56+. The results are summarized in Table 7. Table 7. Cellular composition on day 17 - percentage of NK and CD27+ cells [Table 9] SEM: Standard error of the mean

[0362] Example 11: Functionality of V51 T cells Vδ1 T cells expanded in the presence of selected antibodies express polyclonal replicative groups in the CDR3 region. The cells were then subjected to a SYTOX-flow killing assay to confirm their functionality. The results showed that in Experiment 1, cells were used at an effector to target (E:T) ratio of 10:1. Cells obtained on day 14 (Figure 17A) and in experiment 2, cells were used at E:T ratios of 1:1 and 10:1. , shown for cells obtained on day 17 (post-freeze-thaw) (FIG. 17B).

[0363] As can be seen in Figure 17, Vδ1-positive cells expanded in the presence of all antibodies were significantly higher than the target cells. Effectively lysing the cells, demonstrating that the cells remain functional even after freezing and thawing. Ta.

[0364] Example 12: Functionality of cells after storage The functionality of the cells after a preservation process involving freezing and then thawing was also investigated. A portion of the culture was removed from the culture on day 17 of experiment 2 and frozen. The cells were then thawed and incubated with IL-15-containing medium. Figure 18 shows the results of the 500 cells incubated with B07, C08, E07, G04, or OKT-3 antibodies before freezing. For the contacted cultures, total cell counts were performed 7 days after the cells were cultured after freezing and thawing. All cultures showed the ability to grow after storage. Culture was continued for up to 42 days. Total cell counts were monitored over the period (results are shown in Figure 19). was maintained or increased in cultures pre-exposed to the selected antibodies.

[0365] Example 13: Anti-Vδ1 antibodies conferred immune cell regulation and proliferation in TILs A study to investigate the regulation and proliferation of human tumor-infiltrating lymphocytes (TILs) conferred by anti-Vδ1 antibodies For these studies, human renal cell carcinoma (RCC) tumor biopsies were shipped fresh. The tissue was processed immediately upon receipt. Specifically, the tissue was minced into ~2 mm pieces. 2 The maximum weight is 1g. The tissue was resuspended in 4.7 mL of RPMI and 0.2x the concentration used to prevent shedding of relevant cell surface molecules. Use Miltenyi's Tumor Dissociation Enzyme at the concentrations recommended by the manufacturer, except for Enzyme R, which is used. The enzymes from the kit were placed in Miltenyi C-tubes. The tissue was placed on a gentleMACS™ Octo Dissociator, which was programmed for the dissociation of soft tissue tumors. The digest was then filtered through a 70 mm filter to obtain a single cell. A cell suspension was prepared. RPMI containing 10% FBS was added to the digest to quench the enzyme activity. The cells were washed twice with RPMI / 10% FBS and resuspended for counting. Cells were seeded at 2.5 × 10e6 per well in TC wells (24-well G-REX, Wilson Wolf). The cells were then incubated with or without cytokines and with or without antibodies for 18 days. The antibodies included in the study are summarized in Figure 20. These included OKT3 (50 ng / ml or and 1252_P01_C08 (up to 500 ng / ml), also known herein as "C08" When included, bolus doses of these antibodies were added on days 0, 7, 11, and 14. During the incubation, the medium was replaced with fresh medium on days 11 and 14. Lymphocyte expression Flow cytometry analysis was performed on days 0 and 18 to determine fold changes in cell type and number. Cells were first screened for live CD45+ cells and then screened for CD45+ cells as shown. In the groups containing recombinant cytokines, these were added as follows: Days 7, 11, and 14: IL-4, IFN-γ, IL-21, and IL-1β. Additional IL-15 was included on days 7, 11, and 14. 21 and IFN-γ were included on days 7 and 14, respectively. Figure 20(A) shows the results of the IFN-γ treatments when indicated. , after 18 days of culture in the presence of C08 or OKT3 with and without cytokine supplementation (CK). These results show that the fold increase in TIL Vδ1 cells was significantly greater than that observed with either antibody or cytokine alone. The effect of either C08 or the comparator OKT3 antibody in the presence of cytokines compared to Figure 20(B) shows a significant fold increase in TIL Vδ1 cells upon application of either These results demonstrate an increase in total Vδ1 cell numbers compared with antibody or cytokine alone. In addition, TIL V after incubation with C08 or comparator OKT3 antibody in the presence of cytokines. Figure 20(C) shows a significant increase in the number of δ1+ cells. An example of the gating strategy used in this study is shown. From the live CD45+ cell population, cells Then, lymphocytes were gated based on their forward and side scatter characteristics (not shown), and then γδ T cells were separated from αβ T cells by staining for T cell receptors. The proportion of Vδ1 cells within the total γδ T cell population was subsequently determined. Data from the example at day 18 are shown in Table For the two conditions shown (+ / -1252_P01_C08): 64.3% of cells were CD45+ Of these CD45% cells, 53.1% were γδ+, and of these γδ cells, 89.7 were Vδ Figure 20(D) shows the cell surface phenotype profile of TIL Vδ1 cells upon harvest. Higher levels of CD69 were observed after incubation with C08 antibody. This shows the analysis of the TIL γδ-negative CD8-positive lymphocyte fraction within the CD45-positive gate. Thus, the combined results demonstrate the efficacy of the anti-V51 antibodies of the invention described herein on TIL populations. This emphasizes the regulatory effect that is imparted by

Claims

1. 1. An ex vivo method for modulating V51 T cells, comprising administering to a subject the amino acid region: (i) sequences 3 to 20 of SEQ ID NO: 1; and / or (ii) 37 to 77 of SEQ ID NO: 1 An epitope of the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) containing one or more amino acid residues within A human anti-TCR delta variable 1 (anti-Vδ1) antibody or a fragment thereof that binds to the target polypeptide is administered to a subject, including Vδ1 T cells. The ex vivo method comprising administering to the cell population.

2. The epitope is located in the amino acid regions: 5-20 and 62-77; 50-64; 37-53 and 5 of SEQ ID NO:

1.

2. The method of claim 1, comprising one or more amino acid residues within 9 to 72; 59 to 77; or 3 to 17 and 62 to 69. Law.

3. The method according to claim 1 or claim 2, wherein the epitope is an activating epitope for γδ T cells. Law.

4. 4. The antibody of claim 1, wherein the antibody binds only to an epitope in the V region of the Vδ1 chain of the γδ TCR. The method described.

5. 5. The antibody of claim 1, wherein the antibody does not bind to an epitope found in the CDR3 of the Vδ1 chain of the γδ TCR. The method described in section.

6. 1. An ex vivo method of modulating V51 T cells, comprising: a CDR3 comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 25; At least 80% sequence identity with any one of SEQ ID NOs: 26-37 and sequences A1-A12 (in Table 2) and / or a CDR2 comprising a sequence having the formula: CDR1 comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61, administering to a cell population comprising V51 T cells an anti-V51 antibody or a fragment thereof comprising one or more of: The ex vivo method comprising:

7. The antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 62-73, for example, SEQ ID NOs: 63, 62, or 64. or one of the VH regions having at least 80% sequence identity. The method described.

8. The antibody or fragment thereof is any of SEQ ID NOs: 74-85, for example, SEQ ID NOs: 75, 74, or 76. or one of the VL regions comprising an amino acid sequence having at least 80% sequence identity. The method described.

9. The antibody or fragment thereof is any of SEQ ID NOs: 86-97, for example, SEQ ID NOs: 87, 86, or 88. The method of any one of claims 6 to 8, comprising one or more amino acid sequences.

10. The antibody or fragment thereof is the same as the antibody or fragment thereof according to any one of claims 6 to 9. or a combination of any one of claims 6 to 9, which binds to the same or essentially the same epitope. The method of any one of claims 6 to 9, wherein the antibody or fragment thereof competes.

11. The antibody or fragment thereof has a surface plasmon resonance (SPR) content of 1.5×10 -7 Less than M Binds to the variable delta 1 (Vδ1) chain of the gamma delta T cell receptor (TCR) with a high affinity (KD) according to claims 1 to 1 10. The method of any one of claims 1 to 0.

12. The antibody or fragment thereof may be 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, according to any one of claims 1 to 11. The method described in claim 1.

13. The method of any one of claims 1 to 12, wherein said modulation comprises expansion of Vδ1 T cells.

14. The method may involve producing an expanded population containing more than about 85% V51 T cells, such as more than about 90% V51 T cells. The method of claim 13, further comprising providing a population of V51 T cells isolated from the host.

15. 15. The method of claim 1, wherein the method comprises culturing the cell population for at least 5 days. or the method described in any one of claims 1 to 5.

16. the method comprising culturing the cell population in the presence of at least one cytokine; 16. The method of any one of claims 1 to 15.

17. The cytokines include interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-6), interleukin-6 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin- Interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-13 (IL-14), interleukin-15 (IL-16), interleukin-16 (IL-18), interleukin-17 (IL-19), interleukin-18 (IL-20), interleukin-19 (IL-21), interleukin-19 (IL-22), interleukin-19 (IL-23), interleukin-19 (IL-24), interleukin-19 (IL-25), interleukin-19 (IL-26), interleukin-19 (IL-27), interleukin-19 (IL-28), interleukin-19 (IL-29 ... interleukin-15 (IL-15), interleukin-21 (IL-21), or a mixture thereof.

17. The method of claim 16, wherein

18. the method comprising culturing the cell population in the presence of IL-2, IL-9, and / or IL-15.

18. The method of any one of claims 1 to 17.

19. 19. The method of claim 1, wherein the method comprises culturing the cell population in the presence of IL-21. The method described in section.

20. 20. The method of claim 1, wherein the method comprises culturing the cell population in the presence of IL-4. The method described in section.

21. The method comprises culturing the cell population in a first culture medium containing IL-4, and then 18. The method of claim 1, further comprising culturing the population in a second culture medium comprising IL-15. The method described in section.

22. 22. The method of claim 21, wherein the first culture medium lacks IL-15, IL-2, and / or IL-7. Law.

23. 22. The method of claim 21, wherein the second culture medium lacks IL-4.

24. the first or second culture medium, or both culture media, containing one or more additional cytokines 24. The method of any one of claims 21 to 23, comprising:

25. the additional cytokine is selected from the group consisting of: IL-21, IFN-γ, and IL-1β; 25. The method of claim 24.

26. 26. The method of claim 15, wherein the cell population is not in direct contact with stromal and / or epithelial cells during culture. The method described in any one of claims 1 to 4.

27. 27. The method of claim 26, wherein the cell population is not in direct contact with fibroblasts during culture.

28. wherein the cell population is not in direct contact with tumor cells and / or feeder cells during culture.

28. The method according to any one of paragraphs 15 to 27.

29. 29. The method of claim 1, wherein the method comprises culturing the cell population in serum-free medium. or the method described in any one of claims 1 to 5.

30. the cell population is enriched for T cells prior to administration of the antibody or fragment thereof.

30. The method according to any one of paragraphs 1 to 29.

31. the cell population is enriched for γδ T cells prior to administration of the antibody or fragment thereof.

31. The method of any one of claims 1 to 30.

32. The cell population is depleted of αβ T cells or NK cells prior to administration of the antibody or fragment thereof. The method of any one of claims 1 to 31,

33. 33. The method of any one of claims 1 to 32, wherein the cell population is obtained from a hematopoietic sample or a fraction thereof. How to do it.

34. The hematopoietic sample is peripheral blood, umbilical cord blood, lymphoid tissue, thymus, bone marrow, lymph node tissue, or any of these.

34. The method of claim 33, wherein the fraction is selected from the following:

35. 33. The method of claim 32, wherein the hematopoietic sample comprises low density mononuclear cells (LDMCs) or peripheral blood mononuclear cells (PBMCs). The method of claim 34.

36. The cell population is derived from a non-hematopoietic tissue sample, such as skin, colon, intestine, breast, lung, prostate, liver, spleen, pancreas, uterus, vagina, or other skin membrane, mucous membrane, or serous membrane sample. Item 33. The method according to any one of items 1 to 32.

37. The cell population is a synthetic By culturing the non-hematopoietic tissue sample on the scaffold, a cell membrane derived from the non-hematopoietic tissue sample can be obtained.

37. The method of claim 36,

38. The method of any one of claims 1 to 37, wherein the cell population is obtained from a cancer tissue sample.

39. 39. The method of claim 1, wherein the cell population is obtained from human or non-human animal tissue. How to post.

40. the cell population is isolated from the sample prior to administering the anti-V51 antibody or fragment thereof.

40. The method of any one of claims 1 to 39.

41. A Vδ1 T cell population obtained by the ex vivo method of any one of claims 1 to 40.

42. A composition comprising the Vδ1 T cell population of claim 41.

43. A pharmaceutical composition comprising the Vδ1 T cell population of claim 41.

44. 44. The pharmaceutical composition of claim 43 for use as a medicament.

45. 44. The medicament of claim 43 for use in the treatment of cancer, an infectious disease, or an inflammatory disease. composition.

46. Methods for treating cancer, infectious diseases, or inflammatory diseases in a subject in need thereof 42. The V51 T cell population of claim 41 . Or, administering a therapeutically effective amount of the pharmaceutical composition according to claim 43. The method comprising administering.

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