Method of inhibiting or activating gamma delta t cells

Abstract

To provide methods of inhibiting or activating γδ T cells.SOLUTION: The present disclosure relates to methods for inhibiting activation of γδ T cells that express a Vγ9+ TCR in a subject by administering a BTN2A1 antagonist to the subject, as well as to methods for inducing or enhancing γδ T cells that express a Vγ9+ TCR in a subject by administering a BTN2A1 antagonist to the subject. The present disclosure further relates to BTN2A1 antagonists and BTN2A1 agonists.SELECTED DRAWING: None

Description

Technical Field

[0001] Data of related applications This application claims priority from Australian Patent Application No. 2019902308, entitled "Methods of Inhibiting or Activating Gamma Delta T Cells", filed on June 28, 2019, Australian Patent Application No. 2019904771, entitled "Methods of Inhibiting or Activating Gamma Delta T Cells", filed on December 17, 2019, and Australian Patent Application No. 2019904773, entitled "Methods of Inhibiting or Activating Gamma Delta T Cells", filed on December 17, 2019. The entire contents of each application are incorporated herein by reference.

[0002] Sequence listing This application is filed with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference.

[0003] The present disclosure relates to reagents and methods for inhibiting or activating γδ T cells.

Background Art

[0004] Recognition of an antigen (Ag) by alpha-beta (αβ) T cells is through a T cell receptor (TCR) that is encoded by the TCR-α locus and the TCR-β locus and binds to the Ag presented by an Ag-presenting molecule. This basic principle applies to αβ T cells that recognize peptide Ags presented by MHC molecules, NKT cells that recognize lipid Ags presented by CD1d, and mucosal-associated invariant T (MAIT) cells that recognize vitamin B metabolites presented by MR1 (J. Rossjohn et al. (2015)). Gamma-delta (γδ) T cells are a unique lineage that expresses TCRs derived from separate variable (v), diversity (D), joining (J), and constant (C) TCR-γ and TCR-δ loci. Most circulating human γδ T cells are Vγ9+ They express TCR, and most of them react with a distinct class of Ag called phosphoantigen (pAg) (P. Constant et al. (1994); Y. Tanaka et al., (1995)).

[0005] pAg is an intermediate in isoprenoid biosynthesis and is present in almost all cellular organisms. Vertebrates produce isoprenoids through the mevalonate pathway, while microorganisms utilize the non-mevalonate pathway, generating chemically distinct pAg intermediates (L. Zhao et al. (2013)). Vγ9 + Vγ9 T cells sense pAg produced through either pathway (including isopentenyl pyrophosphate (IPP) from the mevalonate pathway and 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) from the non-mevalonate pathway), but are approximately 1000-fold more sensitive to bacterial HMBPP than to vertebrate IPP pAg (A. Sandstrom et al. (2014)). Thus, Vγ9 + T cells can respond not only to HMBPP derived from bacterial infection but also to IPP accumulated in abnormal cells such as cancer cells. During bacterial and parasitic infections, pAg causes + Vγ9 T cells to produce cytokines and + proliferate, coming to account for approximately 10% - 50% of peripheral blood mononuclear cells (PBMC) (Y.L. Wu et al. (2014); J. Zheng et al. (2013)). Vγ9 + This important role of Vγ9 T cells in antibacterial immunity was demonstrated by the fact that transferring human PBMC into immunodeficient mice leads to Vγ9 T cell-dependent protection against bacterial infection (L. Wang et al. (2001)). Vγ9 +T cells can also kill various tumor cell lines in a pAg-dependent manner in vitro, and their anti-cancer ability has been investigated in numerous clinical trials, resulting in some promising outcomes (D.I. Godfrey et al. (2018)). Therefore, Vγ9+γδ T cells become one important and non-redundant weapon of the human immune system.

[0006] Despite the importance of pAg sensed by γδ T cells in protective immunity, the molecular mechanism governing pAg recognition remains unclear.

[0007] From the above, it will be apparent to those skilled in the art that it is necessary to better understand the mechanism governing pAg recognition in order to provide new immunotherapies and drugs capable of inducing or inhibiting γδ T cell responses, for example, in cancer patients or patients with chronic infections.

Summary of the Invention

[0008] In reaching the present invention, the inventors have identified butyrophilin, subfamily 2, member A1 (BTN2A1), a surface protein, as a novel ligand for pAg-reactive γδ TCR. The inventors have demonstrated that the expression of BTN2A1 is essential for an effective pAg response by γδ T cells. The inventors have also shown that BTN2A1 associates tightly with BTN3A1 on the surface of antigen-presenting cells (APCs), and this complex is necessary and sufficient to confer pAg presentation ability to mouse and hamster APCs.

[0009] These findings by the inventors provide a basis for reagents that bind to BTN2A1 and enhance γδ T activation, and the use of such reagents, for example, in the treatment of cancer or infections.

[0010] These findings by the inventors also provide a basis for reagents that bind to BTN2A1 and impair γδ T activation, and the use of such reagents, for example, in autoimmune diseases, transplant rejection, or graft-versus-host disease.

[0011] Accordingly, the present disclosure provides a method for inhibiting the activation of γδ T cells expressing Vγ9+ TCR in a subject, the method comprising administering a BTN2A1 antagonist to the subject, the BTN2A1 antagonist i) inhibits the formation of a BTN2A1 / BTN3 complex (e.g., a BTN2A1 / BTN3A1 complex) on the surface of cells; ii) inhibits the binding of BTN2A1 to Vγ9; iii) inhibits the binding of BTN2A1 / BTN3 (e.g., a BTN2A1 / BTN3A1 complex) to Vγ9+ TCR; and / or iv) decreases the activity and / or survival of cells expressing BTN2A1.

[0012] In one embodiment, the method inhibits the activation of one or more Vγ9+ T cell subsets. For example, the method inhibits the activation of one or more of Vγ9Vδ2+, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ T cells. In another example, the method inhibits the activation of Vγ9Vδ2- T cells.. For example, the method inhibits the activation of one or more of Vγ9Vδ2+, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ or Vγ9Vδ2- T cells. For example, the method inhibits the upregulation of CD25 on the surface of one or more Vγ9+ T cell subsets and / or the production of IFN-γ from the subset. In one embodiment, the method inhibits the activation of Vγ9Vδ2+ γδ T cells. In another embodiment, the method inhibits the activation of Vγ9Vδ2- γδ T cells. In a further embodiment, the method inhibits the activation of Vγ9Vδ2+ γδ T cells and / or Vγ9Vδ2- γδ T cells.

[0013] In one embodiment, BTN2A1 / BTN3 is a BTN2A1 / BTN3A1 complex. This complex can be a heteromeric complex or a multimeric complex.

[0014] In one embodiment, BTN2A1 and BTN3 are expressed on the surface of the same cell.

[0015] In a further embodiment, the BTN2A1 / BTN3A1 complex comprises one or more additional molecules (such as BTN3A2 and / or BTN3A3). The one or more additional molecules can enhance the activation of γδ T cells.

[0016] In one embodiment, the above method inhibits one or more of cell lysis function, cytokine production of one or more cytokines, or proliferation of γδ T cells.

[0017] In one embodiment, the BTN2A1 antagonist inhibits phosphoantigen-mediated activation of γδ T cells.

[0018] In one embodiment, or in a further embodiment, the BTN2A1 antagonist inhibits the association of BTN2A1 and BTN3A1. For example, the BTN2A1 antagonist inhibits the direct association of BTN2A1 and BTN3A1.

[0019] In one embodiment, or in a further embodiment, the BTN2A1 antagonist inhibits BTN2A1 from binding to the region encoded by the germline of Vγ9 and / or distal to the TCR δ-chain. In one embodiment, the BTN2A1 antagonist prevents BTN2A1 from binding to the framework region of Vγ9 and / or a region containing at least one of Arg20, Glu70, and His85. The BTN2A1 antagonist can prevent binding to regions on the outer surfaces of the B, D, and E strands of the ABED antiparallel β-sheet of Vγ9. In one embodiment, the BTN2A1 antagonist binds to a region closer to the Cγ domain than to the CDR loops.

[0020] In one embodiment, the BTN2A1 antagonist inhibits the formation of a BTN2A1 / BTN3 complex from binding to regions encoded by the germline of Vδ2 (such as the CDR2 loop of the TCR δ chain and / or the CDR3 loop of the TCR γ chain). For example, the BTN2A1 antagonist prevents BTN2A1 from binding to regions near Arg51 of Vδ2 and Lys108 of the CDR3 loop encoded by Vγ9-JγP.

[0021] In one embodiment, the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch the BTN2A1 molecule from a stimulatory BTN2A1 to a non-stimulatory BTN2A1.

[0022] In one embodiment, or in a further embodiment, the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to inhibit the activation of phosphoantigens. For example, the BTN2A1 antagonist inhibits the binding of phosphoantigens to the cytoplasmic domain of BTN2A1 and / or the BTN3 molecule.

[0023] In one embodiment, the BTN2A1 antagonist is bispecific for BTN2A1 and the BTN3 molecule (such as BTN3A1). In another embodiment, the BTN2A1 antagonist cross-reacts with the BTN3 molecule (such as BTN3A1). In another embodiment, the BTN2A1 antagonist is a soluble Vγ9+ TCR.

[0024] The present disclosure also provides a method for suppressing or inhibiting a Vγ9+ γδ T cell response in a subject, the method comprising administering a BTN2A1 antagonist to the subject, the BTN2A1 antagonist i) inhibits the formation of a BTN2A1 / BTN3 complex (such as a BTN2A1 / BTN3A1 complex) on the surface of cells; ii) inhibits the binding of BTN2A1 to Vγ9+ TCR; iii) inhibiting the binding of the BTN2A1 / BTN3 complex (e.g., the BTN2A1 / BTN3A1 complex) to the Vγ9+ TCR; and / or iv) decreasing the activity and / or survival of cells expressing BTN2A1.

[0025] In one embodiment, the method suppresses or inhibits one or more of the Vγ9Vδ2+, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ T cell responses. In one embodiment, the method suppresses or inhibits one or more of the Vγ9Vδ2+, Vγ9Vδ2-, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ T cell responses. In one embodiment, the method suppresses or inhibits the Vγ9Vδ2+ γδ T cell response. In another embodiment, the method suppresses or inhibits the Vγ9Vδ2- γδ T cell response. In a further embodiment, the method suppresses or inhibits the Vγ9Vδ2+ γδ T cell response and / or the Vγ9Vδ2- γδ T cell response.

[0026] In one embodiment, BTN2A1 / BTN3 is the BTN2A1 / BTN3A1 complex. The complex can be a heteromeric complex or a multimeric complex.

[0027] In a further embodiment, the BTN2A1 / BTN3A1 complex comprises one or more additional molecules (such as BTN3A2 and / or BTN3A3). The one or more additional molecules can enhance the activation of γδ T cells.

[0028] In one embodiment, the above method suppresses or inhibits one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of γδ T cells.

[0029] In one embodiment, the BTN2A1 antagonist inhibits phosphoantigen-mediated activation of γδ T cells.

[0030] In one embodiment, or in a further embodiment, the BTN2A1 antagonist inhibits the association of BTN2A1 and BTN3A1. For example, the BTN2A1 antagonist inhibits the direct association of BTN2A1 and BTN3A1.

[0031] In one embodiment, or in a further embodiment, the BTN2A1 antagonist inhibits BTN2A1 from binding to the region encoded by the germline of Vγ9 and / or distal to the TCR δ-chain. In one embodiment, the BTN2A1 antagonist prevents BTN2A1 from binding to the framework region of Vγ9 and / or a region containing at least one of Arg20, Glu70, and His85. The BTN2A1 antagonist can prevent BTN2A1 from binding to regions on the outer surface of the B, D, and E strands of the ABED antiparallel β-sheet of Vγ9. In one embodiment, the BTN2A1 antagonist binds to a region closer to the Cγ domain than to the CDR loop.

[0032] In one embodiment, the BTN2A1 antagonist inhibits the BTN2A1 / BTN3 complex from binding to the region encoded by the germline of Vδ2 (such as the CDR2 loop and / or CDR3 loop of the TCR γ-chain). For example, the BTN2A1 antagonist prevents BTN2A1 from binding to a region near at least one of Arg51 and Lys108 of the CDR3 loop encoded by Vγ9-JγP.

[0033] In one embodiment, the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch this BTN2A1 molecule from a stimulatory BTN2A1 to a non-stimulatory BTN2A1.

[0034] In one embodiment, or in a further embodiment, the BTN2A1 antagonist modifies one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to inhibit the activation of phosphoantigens. For example, the BTN2A1 antagonist inhibits the binding of phosphoantigens to the cytoplasmic domains of BTN2A1 and / or BTN3 molecules.

[0035] In one embodiment, the BTN2A1 antagonist is bispecific for BTN2A1 and BTN3 molecules (e.g., BTN3A1). In another embodiment, the BTN2A1 antagonist cross-reacts with BTN3 molecules (e.g., BTN3A1). In another embodiment, the BTN2A1 antagonist is a soluble Vγ9+ TCR.

[0036] The present disclosure also provides a method for inhibiting the activation of γδ T cells expressing Vγ9+ TCR in vitro or ex vivo, the method comprising culturing γδ T cells and cells expressing BTN2A1 in the presence of a BTN2A1 antagonist, the BTN2A1 antagonist being i) inhibiting the formation of a BTN2A1 / BTN3A1 heteromeric complex on the surface of cells; ii) inhibiting the binding of BTN2A1 to Vγ9; and / or iii) inhibiting the binding of the BTN2A1 / BTN3A1 heteromeric complex to Vγ9+ TCR; and / or iv) reducing the activity and / or survival of cells expressing BTN2A1.

[0037] In one embodiment, the method further comprises administering the γδ T cells to a subject in need thereof. For example, the γδ T cells comprise an engineered receptor (e.g., a T cell receptor whose gene has been engineered or modified). For example, the γδ T cells do not comprise an engineered receptor (e.g., a T cell receptor whose gene has been engineered or modified). In a further embodiment, the γδ T cells are engineered γδ T cells. This method may be useful in the context of treating a patient in tissue transplantation or allogeneic blood cell transplantation.

[0038] The present disclosure also provides a method for preventing, treating, delaying the progression of, preventing the recurrence of, or alleviating the symptoms of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect, the method comprising administering a BTN2A1 antagonist to a subject in need thereof in an amount sufficient to prevent, treat, delay the progression of, prevent the recurrence of, or alleviate the said symptoms of the autoimmune disease, transplant rejection or graft-versus-host disease, or graft-versus-tumor effect in the subject.

[0039] The present disclosure also provides a method for preventing, treating, delaying the progression of, preventing the recurrence of, or alleviating the symptoms of cancer or an infectious disease, the method comprising administering a BTN2A1 antagonist to a subject in need thereof in an amount sufficient to prevent, treat, delay the progression of, prevent the recurrence of, or alleviate the said symptoms of cancer or an infectious disease in the subject.

[0040] The present disclosure also provides a method for activating γδ T cells expressing Vγ9+ TCR in a subject, the method comprising administering a BTN2A1 agonist to the subject, wherein the BTN2A1 agonist: i) promotes the formation of BTN2A1 / BTN3 (e.g., BTN2A1 / BTN3A1 complex) on the surface of cells; ii) induces the ligation of Vγ9+ TCR to the surface of γδ T cells; and / or iii) increases the activity and / or survival of cells expressing BTN2A1.

[0041] In one embodiment, the method activates one or more Vγ9+ T cell subsets. For example, one or more of Vγ9Vδ2+, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ T cells. For example, one or more of Vγ9Vδ2+, Vγ9Vδ2-, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ T cells. In one embodiment, the method activates Vγ9Vδ2+ γδ T cells. In another embodiment, the method activates Vγ9Vδ2- γδ T cells. In a further embodiment, the method activates Vγ9Vδ2+ γδ T cells and Vγ9Vδ2- γδ T cells.

[0042] In one embodiment, BTN2A1 / BTN3 is a BTN2A1 / BTN3A1 complex. This complex can be a heteromeric complex or a multimeric complex.

[0043] In a further embodiment, the BTN2A1 / BTN3A1 complex includes one or more additional molecules (such as BTN3A2 and / or BTN3A3). The one or more additional molecules can enhance the activation of γδ T cells.

[0044] In one embodiment, the above method activates one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of γδ T cells.

[0045] In one embodiment, or in a further embodiment, the activated γδ T cells express one or more of CD25, CD40-ligand (CD40-L), CD69, and CD107a.

[0046] In one embodiment, the BTN2A1 agonist activates γδ T cells independently of phosphoantigen binding.

[0047] In one embodiment, or in a further embodiment, the BTN2A1 agonist promotes the association of BTN2A1 and BTN3A1. For example, the BTN2A1 agonist promotes the direct association of BTN2A1 and BTN3A1. For example, the BTN2A1 agonist crosslinks BTN2A1 and BTN3A1.

[0048] In one embodiment, the BTN2A1 agonist is bispecific for BTN2A1 and a BTN3 molecule (e.g., BTN3A1). In another embodiment, the BTN2A1 agonist cross-reacts with a BTN3 molecule (e.g., BTN3A1).

[0049] In one embodiment, the BTN2A1 agonist changes one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch BTN2A1 from a non-stimulatory BTN2A1 to a stimulatory BTN2A1.

[0050] The present disclosure also provides a method of inducing or enhancing a Vγ9+ γδ T cell response in a subject, the method comprising administering a BTN2A1 agonist to the subject, the BTN2A1 agonist i) promoting the formation of BTN2A1 / BTN3 (e.g., BTN2A1 / BTN3A1 complex) on the surface of cells; ii) inducing the ligation of Vγ9+ TCR on the surface of γδ T cells; and / or iii) increasing the activity and / or survival of cells expressing BTN2A1.

[0051] In one embodiment, the method induces one or more Vγ9+ T cell subsets. For example, one or more of Vγ9Vδ2+, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ γδ T cells. For example, one or more of Vγ9Vδ2+, Vγ9Vδ2- γδ, Vγ9Vδ1+, Vγ9Vδ3+, Vγ9Vδ4+, or Vγ9Vδ5+ bispecific T cells. In one embodiment, the method induces a Vγ9Vδ2+ bispecific T cell response. In another embodiment, the method induces a Vγ9Vδ2- bispecific T cell response. In a further embodiment, the method induces a Vγ9Vδ2+ bispecific T cell and a Vγ9Vδ2- bispecific T cell response.

[0052] In one embodiment, BTN2A1 / BTN3 is a BTN2A1 / BTN3A1 complex. This complex can be a heteromeric complex or a multimeric complex.

[0053] In a further embodiment, the BTN2A1 / BTN3A1 complex comprises one or more additional molecules (such as BTN3A2 and / or BTN3A3). The one or more additional molecules can enhance the activation of γδ T cells.

[0054] In one embodiment, the above method activates one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of γδ T cells.

[0055] In one embodiment, or in a further embodiment, the activated γδ T cells express one or more activation-related markers such as CD25, CD69, CD40-ligand (CD40-L), and CD107a.

[0056] In one embodiment, the BTN2A1 agonist activates γδ T cells independently of phosphoantigen binding.

[0057] In one embodiment, or in a further embodiment, the BTN2A1 agonist promotes the association of BTN2A1 and BTN3A1. For example, the BTN2A1 agonist promotes the direct association of BTN2A1 and BTN3A1. For example, the BTN2A1 agonist crosslinks BTN2A1 and BTN3A1.

[0058] In one embodiment, the BTN2A1 agonist is bispecific for BTN2A1 and a BTN3 molecule (e.g., BTN3A1). In another embodiment, the BTN2A1 agonist cross-reacts with a BTN3 molecule (e.g., BTN3A1).

[0059] In one embodiment, the BTN2A1 agonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch BTN2A1 from a non-stimulatory BTN2A1 to a stimulatory BTN2A1.

[0060] Also provided by the present disclosure is a method of activating γδ T cells expressing Vγ9+ TCR in vitro or ex vivo, the method comprising culturing the γδ T cells and cells expressing BTN2A1 in the presence of a BTN2A1 agonist, the BTN2A1 agonist being: i) promoting the formation of a BTN2A1 / BTN3A1 heteromeric complex on the surface of the cells; ii) inducing the ligation of Vγ9+ TCR on the surface of the γδ T cells; and / or iii) increasing the activity and / or survival of the cells expressing BTN2A1.

[0061] In one embodiment, the method further comprises administering the activated γδ T cells to a subject in need thereof. In a further embodiment, the method further comprises administering the engineered γδ T cells to a subject in need thereof.

[0062] The present disclosure also provides a method for preventing, treating, delaying the progression of, preventing recurrence of, or alleviating the symptoms of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect, the method comprising administering a BTN2A1 agonist to a subject in need thereof in an amount sufficient to prevent, treat, delay the progression of, prevent recurrence of, or alleviate said symptoms of the autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect in said subject.

[0063] The present disclosure also provides a method for preventing, treating, delaying the progression of, preventing recurrence of, or alleviating the symptoms of cancer or an infectious disease, the method comprising administering a BTN2A1 agonist to a subject in need thereof in an amount sufficient to prevent, treat, delay the progression of, prevent recurrence of, or alleviate said symptoms of the cancer or infectious disease in said subject.

[0064] The present disclosure also provides a BTN2A1 antagonist, which specifically binds to BTN2A1 and i) inhibits the formation of a BTN2A1 / BTN3 complex (BTN2A1 / BTN3A1 complex) on the surface of cells; ii) inhibits the binding of BTN2A1 to Vγ9; iii) inhibits the binding of a BTN2A1 / BTN3A1 complex to Vγ9+ TCR; and / or iv) inhibits the activity and / or survival of cells expressing BTN2A1.

[0065] The present disclosure also provides a compound that specifically binds to BTN2A1 and i) promotes the formation of a BTN2A1 / BTN3 complex (e.g., BTN2A1 / BTN3A1 complex) on the surface of cells; ii) induces the ligation of Vγ9+ TCR to the surface of γδ T cells; and / or iii) increases the activity and / or survival of cells expressing BTN2A1. ​BTN2A1 agonists are also provided.

[0066] In one embodiment, the antagonist or agonist of BTN2A1 is a protein comprising an antigen-binding domain.

[0067] In one embodiment, the protein is (i) single-chain Fv fragment (scFv); (ii) dimeric scFv; (iii) Fv fragment; (iv) single-domain antibody (sdAb) (e.g., nanobody); (v) diabody, triabody, tetrabody, or higher-order multimer; (vi) Fab fragment; (vii) Fab’ fragment; (viii) F(ab’) fragment; (ix) F(ab’)2 fragment; (x) any one of (i)-(ix) linked to the Fc region of an antibody; (xi) any one of (i)-(ix) fused to an antibody or an antigen-binding fragment thereof that binds to immune effector cells; or (xii) an antibody is.

[0068] In one embodiment, the protein is (i) single-chain Fv fragment (scFv); (ii) dimeric scFv; (iii) Fv fragment; (iv) single-domain antibody (sdAb); (v) nanobody; (vi) diabody, triabody, tetrabody, or higher-order multimer; (vii) Fab fragment; (viii) Fab’ fragment; (ix) F(ab’) fragment; (x) F(ab’)2 fragment; (xi) any one of (i) to (x) linked to the Fc region of an antibody; (xii) any one of (i) to (x) fused to an antibody that binds to immune effector cells or an antigen-binding fragment thereof; or (xiii) an antibody is.

[0069] In one example, the protein of the present disclosure is an affinity matured antibody, a chimeric antibody, a CDR-grafted antibody, or a humanized antibody, or an antigen-binding fragment thereof.

[0070] In one example, the BTN2A1 antagonist is a heavy chain variable region (V H ) comprising the sequence shown in SEQ ID NO: 100 and a light chain variable region (V L ) comprising the sequence shown in SEQ ID NO: 101.

[0071] In another example, the BTN2A1 antagonist is a heavy chain variable region (V H ) comprising the sequence shown in SEQ ID NO: 108 and a light chain variable region (V L ) comprising the sequence shown in SEQ ID NO: 109.

[0072] In another example, the BTN2A1 antagonist is a heavy chain variable region (V H ) comprising the sequence shown in SEQ ID NO: 116 and a light chain variable region (V L ) comprising the sequence shown in SEQ ID NO: 117.

[0073] In another example, the BTN2A1 antagonist is a heavy chain variable region (V H ) comprising the sequence shown in SEQ ID NO: 124 and a light chain variable region (V L ) comprising the sequence shown in SEQ ID NO: 125.

[0074] In another example, the BTN2A1 antagonist is a heavy chain variable region (V H ) comprising the sequence shown in SEQ ID NO: 132 and a light chain variable region (VL An antibody comprising

[0075] In one example, the BTN2A1 antagonist is a V H comprising a complementarity determining region (CDR) of H and a V L comprising a CDR of L An antibody comprising

[0076] For example, this antagonist is (i) (a) CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 100; (b) CDR2 comprising the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 100; and (c) CDR3 comprising the sequence set forth in amino acids 97 - 105 of SEQ ID NO: 100 comprising a V H and / or (ii) (a) CDR1 comprising the sequence set forth in amino acids 27 - 32 of SEQ ID NO: 101; (b) CDR2 comprising the sequence set forth in amino acids 50 - 52 of SEQ ID NO: 101; and (c) CDR3 comprising the sequence set forth in amino acids 89 - 97 of SEQ ID NO: 101 comprising a V L An antibody comprising

[0077] In one example, this antagonist is (i) (a) CDR1 comprising the sequence set forth in SEQ ID NO: 102; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 103; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 104 comprising a V H and / or (ii) (a) CDR1 comprising the sequence set forth in SEQ ID NO: 105; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 106; and (c) A CDR3 containing the sequence set forth in SEQ ID NO: 107 containing a V L is an antibody.

[0078] In another example, this antagonist (i) (a) A CDR1 containing the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 108; (b) A CDR2 containing the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 108; and (c) A CDR3 containing the sequence set forth in amino acids 97 - 105 of SEQ ID NO: 108 containing a V H and / or (ii) (a) A CDR1 containing the sequence set forth in amino acids 27 - 33 of SEQ ID NO: 109; (b) A CDR2 containing the sequence set forth in amino acids 51 - 53 of SEQ ID NO: 109; and (c) A CDR3 containing the sequence set forth in amino acids 90 - 98 of SEQ ID NO: 109 containing a V L is an antibody.

[0079] In one example, this antagonist (i) (a) A CDR1 containing the sequence set forth in SEQ ID NO: 110; (b) A CDR2 containing the sequence set forth in SEQ ID NO: 111; and (c) A CDR3 containing the sequence set forth in SEQ ID NO: 112 containing a V H and / or (ii) (a) A CDR1 containing the sequence set forth in SEQ ID NO: 113; (b) A CDR2 containing the sequence set forth in SEQ ID NO: 114; and (c) A CDR3 containing the sequence set forth in SEQ ID NO: 115 containing a V L is an antibody.

[0080] In another example, this antagonist (i) (a) CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 116; (b) CDR2 comprising the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 116; and (c) CDR3 comprising the sequence set forth in amino acids 97 - 104 of SEQ ID NO: 116 comprising V H , and / or (ii) (a) CDR1 comprising the sequence set forth in amino acids 27 - 32 of SEQ ID NO: 117; (b) CDR2 comprising the sequence set forth in amino acids 24 - 26 of SEQ ID NO: 117; and (c) CDR3 comprising the sequence set forth in amino acids 89 - 97 of SEQ ID NO: 117 comprising V L and is an antibody comprising V

[0081] In one example, this antagonist is (i) (a) CDR1 comprising the sequence set forth in SEQ ID NO: 118; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 119; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 120 comprising V H , and / or (ii) (a) CDR1 comprising the sequence set forth in SEQ ID NO: 121; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 122; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 123 comprising V L and is an antibody comprising V

[0082] In another example, this antagonist is (i) (a) CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 124; (b) CDR2 comprising the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 124; and (c) CDR3 comprising the sequence set forth in amino acids 97 - 105 of SEQ ID NO: 124 V comprising H and / or (ii) (a) CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 125; (b) CDR2 comprising the sequence set forth in amino acids 51 - 53 of SEQ ID NO: 125; and (c) CDR3 comprising the sequence set forth in amino acids 90 - 101 of SEQ ID NO: 125 V comprising L is an antibody comprising.

[0083] In one example, this antagonist is (i) (a) CDR1 comprising the sequence set forth in SEQ ID NO: 126; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 127; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 128 V comprising H and / or (ii) (a) CDR1 comprising the sequence set forth in SEQ ID NO: 129; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 130; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 131 V comprising L is an antibody comprising.

[0084] In another example, this antagonist is (i) (a) CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 132; (b) CDR2 comprising the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 132; and (c) CDR3 comprising the sequence set forth in amino acids 97 - 106 of SEQ ID NO: 132 V comprising H and / or (ii) (a) CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 133; (b) CDR2 comprising the sequence set forth in amino acids 51 - 53 of SEQ ID NO: 133; and (c) CDR3 containing the sequence set forth in amino acids 92 - 100 of SEQ ID NO: 133 and containing a V L is an antibody.

[0085] In one example, this antagonist is (i) (a) CDR1 containing the sequence set forth in SEQ ID NO: 134; (b) CDR2 containing the sequence set forth in SEQ ID NO: 135; and (c) CDR3 containing the sequence set forth in SEQ ID NO: 136 and containing a V H and / or (ii) (a) CDR1 containing the sequence set forth in SEQ ID NO: 137; (b) CDR2 containing the sequence set forth in SEQ ID NO: 138; and (c) CDR3 containing the sequence set forth in SEQ ID NO: 139 and containing a V L is an antibody.

[0086] In one example, the protein of the present disclosure is an affinity - matured antibody, a chimeric antibody, a CDR - grafted antibody, or a humanized antibody, or an antigen - binding fragment thereof.

[0087] In one example, the protein, antibody, or antigen - binding fragment thereof is any form of a protein, antibody, or functional fragment encoded by a nucleic acid encoding any of the above - mentioned proteins, antibodies, or functional fragments.

[0088] In one example, the above - mentioned antagonist is an antibody containing a variable region that competitively inhibits the binding of a protein, such as an antibody disclosed herein.

[0089] In another embodiment, the BTN2A1 antagonist is a soluble Vγ9 + TCR. This soluble Vγ9 + TCR can contain any TCR allele.

[0090] In one embodiment, the soluble Vγ9+ TCR is a monomer.

[0091] In one embodiment, the soluble Vγ9+ TCR is a multimer.

[0092] In one embodiment, the soluble Vγ9+ TCR comprises a γ chain comprising a sequence set forth in any one of SEQ ID NOs: 85 to 89, and / or a δ chain comprising a sequence set forth in any one of SEQ ID NOs: 70 to 74. In one embodiment, the γ chain and the δ chain are cleaved, for example, at a thrombin protease cleavage site (e.g., LVPRGS).

[0093] In one embodiment, the soluble Vγ9+ TCR comprises a γ chain comprising a variable region set forth in any one of SEQ ID NOs: 90 to 94, and / or a δ chain comprising a variable region set forth in any one of SEQ ID NOs: 75 to 79.

[0094] In one embodiment, the soluble Vγ9+ TCR comprises a complementarity determining region 3 (CDR3) of a γ chain variable region comprising a sequence set forth in any one of SEQ ID NOs: 95 to 99, and / or a complementarity determining region 3 (CDR3) of a δ chain variable region comprising a sequence set forth in any one of SEQ ID NOs: 80 to 84.

[0095] In one embodiment, the soluble Vγ9+ TCR comprises a γ chain variable region comprising a CDR3 set forth in any one of SEQ ID NOs: 95 to 99, and / or a δ chain variable region comprising a CDR set forth in any one of SEQ ID NOs: 80 to 84.

[0096] In one embodiment, the soluble Vγ9+ TCR comprises a γ chain variable region comprising the CDR3 set forth in SEQ ID NO: 95 and a δ chain variable region comprising the CDR3 set forth in SEQ ID NO: 80.

[0097] In one embodiment, the soluble Vγ9+ TCR comprises a γ chain variable region comprising the CDR3 set forth in SEQ ID NO: 96 and a δ chain variable region comprising the CDR3 set forth in SEQ ID NO: 81.

[0098] In one embodiment, the soluble Vγ9+ TCR comprises a γ-chain variable region comprising the CDR3 set forth in SEQ ID NO: 97 and a δ-chain variable region comprising the CDR3 set forth in SEQ ID NO: 82.

[0099] In one embodiment, the soluble Vγ9+ TCR comprises a γ-chain variable region comprising the CDR3 set forth in SEQ ID NO: 98 and a δ-chain variable region comprising the CDR3 set forth in SEQ ID NO: 83.

[0100] In one embodiment, the soluble Vγ9+ TCR comprises a γ-chain variable region comprising the CDR3 set forth in SEQ ID NO: 99 and a δ-chain variable region comprising the CDR3 set forth in SEQ ID NO: 84.

[0101] Further provided by the present disclosure is a BTN2A1 agonist that specifically binds to BTN2A1 and activates γδ T cells.

[0102] Further provided by the present disclosure is a BTN2A1 agonist that specifically binds to BTN2A1 and induces the expression of cell surface markers associated with γδ T cell activation.

[0103] Further provided by the present disclosure is a BTN2A1 agonist that specifically binds to BTN2A1 and induces the secretion of one or more cytokines by γδ T cells.

[0104] Further provided by the present disclosure is a BTN2A1 agonist that specifically binds to BTN2A1 and induces γδ T cells to kill cancer cells and / or inhibit the growth of cancer cells and / or kill cells infected with, for example, a virus, bacterium, or parasite and / or inhibit the growth of cells infected with, for example, a virus, bacterium, or parasite.

[0105] Further provided by the present disclosure is a BTN2A1 agonist that specifically binds to BTN2A1 and: (i) Activate γδ T cells and / or increase the number of activated γδ T cells within a population of cells; and / or (i) Increase the proportion of γδ T cells expressing a T cell activation marker; and / or (ii) Increase the secretion of cytokines (such as interferon-γ) by γδ T cells; and / or (iii) Induce γδ T cells to kill cancer cells and / or inhibit the proliferation of cancer cells and / or kill infected cells and / or inhibit the proliferation of infected cells; and / or (iv) Increase the amount of a marker of T cell activation expressed on the cell surface of γδ T cells A BTN2A1 agonist is provided.

[0106] Further provided by the present disclosure, as a BTN2A1 agonist, a compound that specifically binds to BTN2A1 to: (i) Increase the proportion of γδ T cells expressing CD25 on the cell surface; and / or (ii) Increase the secretion of interferon-γ by γδ T cells; and / or (iii) Induce γδ T cells to kill cancer cells and / or inhibit the proliferation of cancer cells; and / or (iv) Increase the amount of CD25 expressed on the cell surface of γδ T cells A BTN2A1 agonist is provided.

[0107] In one example, a BTN2A1 agonist increases the number of γδ T cells expressing CD25 on the cell surface when measured in an assay that includes contacting a population of γδ T cells in vitro with the BTN2A1 agonist for a period of at least 6 hours or 8 hours or 10 hours or 12 hours and measuring the proportion of γδ T cells expressing CD25 in that population by flow cytometry. Such an assay is also useful for assessing the level of CD25 and / or other molecules expressed on the surface of γδ T cells.

[0108] In one example, an increase in the percentage of γδ T cells expressing CD25 on the cell surface is a comparison with: (i) the percentage of γδ T cells expressing CD25 on the cell surface in a population of γδ T cells not contacted with a BTN2A1 agonist; and / or (ii) the percentage of γδ T cells expressing CD25 on the cell surface in a population of γδ T cells contacted with an antibody that specifically binds to BTN2A1 and is not a BTN2A1 agonist or a BTN2A1 antagonist

[0109] In one example, the agonist as described above increases the percentage of γδ T cells expressing one or more additional markers (in addition to CD25) of γδ T cell activation and / or increases the amount of one or more additional markers (in addition to CD25) of activated CD25 expressed on the cell surface of γδ T cells.

[0110] In one example, the BTN2A1 agonist increases the percentage of γδ T cells expressing CD25 on the cell surface to at least 10% of the cells within the population of γδ T cells. In one example, the BTN2A1 agonist increases the percentage of γδ T cells expressing CD25 on the cell surface to at least 15% of the cells within the population of γδ T cells. In one example, the BTN2A1 agonist increases the percentage of γδ T cells expressing CD25 on the cell surface to at least 20% of the cells within the population of γδ T cells. In one example, the BTN2A1 agonist increases the percentage of γδ T cells expressing CD25 on the cell surface to at least 30% of the cells within the population of γδ T cells. In one example, the BTN2A1 agonist increases the percentage of γδ T cells expressing CD25 on the cell surface to at least 40% of the cells within the population of γδ T cells.

[0111] ​In another example, a BTN2A1 agonist increases the secretion of interferon-γ by γδ T cells when measured in an assay that includes culturing a population of γδ T cells in an in vitro cell culture with the BTN2A1 agonist for a period of at least 6 hours or 8 hours or 10 hours or 12 hours and measuring the amount of interferon-γ per mL of the fluid of the cell culture.

[0112] In one example, a BTN2A1 agonist increases the secretion of interferon-γ up to 10 pg / mL of the fluid from the γδ T cell culture. In one example, a BTN2A1 agonist increases the secretion of interferon-γ up to 20 pg / mL of the fluid from the γδ T cell culture. In one example, a BTN2A1 agonist increases the secretion of interferon-γ up to 30 pg / mL of the fluid from the γδ T cell culture. In one example, a BTN2A1 agonist increases the secretion of interferon-γ up to 40 pg / mL of the fluid from the γδ T cell culture.

[0113] In one example, this agonist increases the secretion of one or more additional or alternative cytokines (in addition to, or instead of, interferon-γ).

[0114] In a further example, cells (such as melanoma cells or a melanoma cell line) are cultured in the presence of γδ T cells, a BTN2A1 agonist, and a reagent (such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT]) that is reduced by living cells to a detectable reagent (such as formazan), and when measured in an assay that includes detecting the detectable reagent, the γδ T cells induce killing of the cells (such as cancer cells or infected cells) and / or inhibition of cell proliferation. A decrease in the level of the detectable reagent in the presence of the BTN2A1 agonist compared to its absence indicates that the cells have been killed or that cell proliferation has been inhibited.

[0115] In one example, the BTN2A1 agonist is a protein comprising an antigen-binding domain.

[0116] In one embodiment, the protein is (i) a single-chain Fv fragment (scFv); (ii) a dimeric scFv; (iii) an Fv fragment; (iv) a single-domain antibody (sdAb); (v) a diabody, triabody, tetrabody, or higher-order multimer; (vi) a Fab fragment; (vii) a Fab’ fragment; (viii) an F(ab’) fragment; (ix) an F(ab’)2 fragment; (x) any one of (i) to (ix) linked to the Fc region of an antibody; (xi) any one of (i) to (ix) fused to an antibody or an antigen-binding fragment thereof that binds to immune effector cells; or (xii) an antibody is.

[0117] In one example, the BTN2A1 agonist is an antibody comprising a light chain variable region (V L ) comprising the sequence set forth in SEQ ID NO: 140 and a heavy chain variable region (V H ) comprising the sequence set forth in SEQ ID NO: 144.

[0118] In one example, the BTN2A1 agonist is an antibody comprising a V L comprising the sequence set forth in SEQ ID NO: 148 and a V H comprising the sequence set forth in SEQ ID NO: 152.

[0119] In one example, the BTN2A1 agonist is an antibody comprising a V L comprising the sequence set forth in SEQ ID NO: 156 and a V H comprising the sequence set forth in SEQ ID NO: 160.

[0120] In one example, the BTN2A1 agonist is an antibody comprising the CDRs of any of the above antibodies in V L and V H . For example, the CDRs are defined by the Kabat numbering system (Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991).

[0121] For example, the BTN2A1 agonist is (i) (a) CDR1 comprising the sequence set forth in amino acids 26-33 of SEQ ID NO: 140; (b) CDR2 comprising the sequence set forth in amino acids 51-53 of SEQ ID NO: 140; and (c) CDR3 comprising the sequence set forth in amino acids 90-98 of SEQ ID NO: 140 in V L ; and / or (ii) (a) CDR1 comprising the sequence set forth in amino acids 26-33 of SEQ ID NO: 144; (b) CDR2 comprising the sequence set forth in amino acids 51-58 of SEQ ID NO: 144; and (c) CDR3 comprising the sequence set forth in amino acids 97-109 of SEQ ID NO: 144 in V H and is an antibody comprising the same.

[0122] For example, the BTN2A1 agonist is (i) (a) CDR1 comprising the sequence set forth in amino acids 26-33 of SEQ ID NO: 148; (b) CDR2 comprising the sequence set forth in amino acids 51-53 of SEQ ID NO: 148; and (c) CDR3 comprising the sequence set forth in amino acids 90-100 of SEQ ID NO: 148 in V L ; and / or (ii) (a) CDR1 comprising the sequence set forth in amino acids 26-33 of SEQ ID NO: 152; (b) A CDR2 comprising the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 152; and (c) A CDR3 comprising the sequence set forth in amino acids 97 - 116 of SEQ ID NO: 152 comprising a V H which is an antibody.

[0123] For example, a BTN2A1 agonist is (i) (a) A CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 156; (b) A CDR2 comprising the sequence set forth in amino acids 51 - 53 of SEQ ID NO: 156; and (c) A CDR3 comprising the sequence set forth in amino acids 90 - 100 of SEQ ID NO: 156 comprising a V L ; and / or (ii) (a) A CDR1 comprising the sequence set forth in amino acids 26 - 33 of SEQ ID NO: 160; (b) A CDR2 comprising the sequence set forth in amino acids 51 - 58 of SEQ ID NO: 160; and (c) A CDR3 comprising the sequence set forth in amino acids 97 - 109 of SEQ ID NO: 160 comprising a V H which is an antibody.

[0124] In one example, a BTN2A1 agonist is (i) (a) A CDR1 comprising the sequence set forth in SEQ ID NO: 141; (b) A CDR2 comprising the sequence set forth in SEQ ID NO: 142; and (c) A CDR3 comprising the sequence set forth in SEQ ID NO: 143 comprising a V L ; and / or (ii) (a) A CDR1 comprising the sequence set forth in SEQ ID NO: 145; (b) A CDR2 comprising the sequence set forth in SEQ ID NO: 146; and (c) A CDR3 comprising the sequence set forth in SEQ ID NO: 147 comprising a V H which is an antibody.

[0125] In one example, the BTN2A1 agonist is (i)(a) CDR1 comprising the sequence set forth in SEQ ID NO: 149; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 150; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 151 comprising V L ; and / or (ii)(a) CDR1 comprising the sequence set forth in SEQ ID NO: 153; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 154; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 155 comprising V H and is an antibody comprising the same.

[0126] In one example, the BTN2A1 agonist is (i)(a) CDR1 comprising the sequence set forth in SEQ ID NO: 157; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 158; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 159 comprising V L ; and / or (ii)(a) CDR1 comprising the sequence set forth in SEQ ID NO: 161; (b) CDR2 comprising the sequence set forth in SEQ ID NO: 162; and (c) CDR3 comprising the sequence set forth in SEQ ID NO: 163 comprising V H and is an antibody comprising the same.

[0127] In one example, the BTN2A1 agonist of the present disclosure is an affinity matured antibody, a chimeric antibody, a CDR-grafted antibody, or a humanized antibody, or an antigen-binding fragment thereof.

[0128] In one example, the BTN2A1 agonist is an antibody that competitively inhibits the binding of a protein, such as an antibody disclosed herein, and / or comprises a variable region that binds to the same epitope as an antibody disclosed herein.

[0129] The present disclosure also provides a method of activating γδ T cells expressing Vγ9+ TCR in a subject, the method comprising administering the above-described BTN2A1 agonist to the subject.

[0130] The present disclosure also provides a method of inducing or enhancing a Vγ9+ γδ T cell response in a subject, the method comprising administering the above-described BTN2A1 agonist to the subject.

[0131] The present disclosure also provides a method of activating γδ T cells expressing Vγ9+ TCR in vitro or ex vivo, the method comprising culturing the γδ T cells and cells expressing BTN2A1 in the presence of the above-described BTN2A1 agonist. In one embodiment, the method further comprises administering the activated γδ T cells to a subject in need thereof.

[0132] The present disclosure also provides a method of preventing, treating, delaying the progression of, preventing recurrence of, or alleviating symptoms of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect, the method comprising administering the above-described BTN2A1 agonist to a subject in need thereof in an amount sufficient to prevent, treat, delay the progression of, prevent recurrence of, or alleviate said symptoms of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect in the subject.

[0133] The present disclosure also provides a method for preventing, treating, delaying the progression of, preventing the recurrence of, or alleviating the symptoms of cancer or an infectious disease, the method comprising administering to a subject in need thereof an amount of the BTN2A1 agonist sufficient to prevent, treat, delay the progression of, prevent the recurrence of, or alleviate the symptoms of cancer or an infectious disease in the subject.

[0134] Key to the sequence listing

[0135] SEQ ID NO: 1 is the amino acid sequence of human BTN2A1 isoform 1. SEQ ID NO: 2 is the amino acid sequence of human BTN2A1 isoform 2. SEQ ID NO: 3 is the amino acid sequence of human BTN2A1 isoform 3. SEQ ID NO: 4 is the amino acid sequence of human BTN2A1 isoform 4. SEQ ID NO: 5 is the amino acid sequence of human annexin A5. SEQ ID NO: 6 is the amino acid sequence of human annexin A1. SEQ ID NO: 7 is the amino acid sequence of the lactadherin C1C2 domain. SEQ ID NO: 8 is the amino acid sequence of the PSP1 protein. SEQ ID NOS: 9-69 are nucleotide sequences encoding primers (see Table 2). SEQ ID NO: 70 is the amino acid sequence of δ2 (clone 6). SEQ ID NO: 71 is the amino acid sequence of δ2 (clone 3). SEQ ID NO: 72 is the amino acid sequence of δ2 (clone 4). SEQ ID NO: 73 is the amino acid sequence of δ2 (clone 5). SEQ ID NO: 74 is the amino acid sequence of δ2 (clone 7). SEQ ID NO: 75 is the amino acid sequence of the variable region of δ2 (clone 6). SEQ ID NO: 76 is the amino acid sequence of the variable region of δ2 (clone 3). SEQ ID NO: 77 is the amino acid sequence of the variable region of δ2 (clone 4). Sequence number 78 is the amino acid sequence of the variable region of δ2 (clone 5). Sequence number 79 is the amino acid sequence of the variable region of δ2 (clone 7). Sequence number 80 is the amino acid sequence of CDR3δ (clone 3) Sequence number 81 is the amino acid sequence of CDR3δ (clone 4) Sequence number 82 is the amino acid sequence of CDR3δ (clone 5) Sequence number 83 is the amino acid sequence of CDR3δ (clone 6) Sequence number 84 is the amino acid sequence of CDR3δ (clone 7) Sequence number 85 is the amino acid sequence of γ9 (clone 6). Sequence number 86 is the amino acid sequence of γ9 (clone 3). Sequence number 87 is the amino acid sequence of γ9 (clone 4). Sequence number 88 is the amino acid sequence of γ9 (clone 5). Sequence number 89 is the amino acid sequence of γ9 (clone 7). Sequence number 90 is the amino acid sequence of the variable region of γ9 (clone 6). Sequence number 91 is the amino acid sequence of the variable region of γ9 (clone 3). Sequence number 92 is the amino acid sequence of the variable region of γ9 (clone 4). Sequence number 93 is the amino acid sequence of the variable region of γ9 (clone 5). Sequence number 94 is the amino acid sequence of the variable region of γ9 (clone 7). Sequence number 95 is the amino acid sequence of CDR3γ (clone 3) Sequence number 96 is the amino acid sequence of CDR3γ (clone 4) Sequence number 97 is the amino acid sequence of CDR3γ (clone 5) Sequence number 98 is the amino acid sequence of CDR3γ (clone 6) Sequence number 99 is the amino acid sequence of CDR3γ (clone 7). Sequence number 100 is Hu34C V H is the amino acid sequence of Sequence number 101 is Hu34C VL is the amino acid sequence of SEQ ID NO: 102 is Hu34C V H is the amino acid sequence of CDR1 SEQ ID NO: 103 is Hu34C V H is the amino acid sequence of CDR2 SEQ ID NO: 104 is Hu34C V H is the amino acid sequence of CDR3 SEQ ID NO: 105 is Hu34C V L is the amino acid sequence of CDR1 SEQ ID NO: 106 is Hu34C V L is the amino acid sequence of CDR2 SEQ ID NO: 107 is Hu34C V L is the amino acid sequence of CDR3 SEQ ID NO: 108 is clone 227 V H is the amino acid sequence of SEQ ID NO: 109 is clone 227 V L is the amino acid sequence of SEQ ID NO: 110 is clone 227 V H is the amino acid sequence of CDR1 SEQ ID NO: 111 is clone 227 V H is the amino acid sequence of CDR2 SEQ ID NO: 112 is clone 227 V H is the amino acid sequence of CDR3 SEQ ID NO: 113 is clone 227 V L is the amino acid sequence of CDR1 SEQ ID NO: 114 is clone 227 V L is the amino acid sequence of CDR2 SEQ ID NO: 115 is clone 227 V L is the amino acid sequence of CDR3 SEQ ID NO: 116 is clone 236 V H is the amino acid sequence of SEQ ID NO: 117 is clone 236 V L is the amino acid sequence of SEQ ID NO: 118 is clone 236 V H is the amino acid sequence of CDR1 SEQ ID NO: 119 is clone 236 VH The amino acid sequence of CDR2 SEQ ID NO: 120 is clone 236 V H The amino acid sequence of CDR3 SEQ ID NO: 121 is clone 236 V L The amino acid sequence of CDR1 SEQ ID NO: 122 is clone 236 V L The amino acid sequence of CDR2 SEQ ID NO: 123 is clone 236 V L The amino acid sequence of CDR3 SEQ ID NO: 124 is clone 266 V H is the amino acid sequence of SEQ ID NO: 125 is clone 266 V L is the amino acid sequence of SEQ ID NO: 126 is clone 266 V H The amino acid sequence of CDR1 SEQ ID NO: 127 is clone 266 V H The amino acid sequence of CDR2 SEQ ID NO: 128 is clone 266 V H The amino acid sequence of CDR3 SEQ ID NO: 129 is clone 266 V L The amino acid sequence of CDR1 SEQ ID NO: 130 is clone 266 V L The amino acid sequence of CDR2 SEQ ID NO: 131 is clone 266 V L The amino acid sequence of CDR3 SEQ ID NO: 132 is clone 267 V H is the amino acid sequence of SEQ ID NO: 133 is clone 267 V L is the amino acid sequence of SEQ ID NO: 134 is clone 267 V H The amino acid sequence of CDR1 SEQ ID NO: 135 is clone 267 V H The amino acid sequence of CDR2 SEQ ID NO: 136 is clone 267 V H The amino acid sequence of CDR3 Sequence number 137 is the V of clone 267 L It is the amino acid sequence of CDR1 Sequence number 138 is the V of clone 267 L It is the amino acid sequence of CDR2 Sequence number 139 is the V of clone 267 L It is the amino acid sequence of CDR3 Sequence number 140 is the V of antibody 244 L It is the amino acid sequence of Sequence number 141 is the V of antibody 244 L It is the amino acid sequence of CDR1 of Sequence number 142 is the V of antibody 244 L It is the amino acid sequence of CDR2 of Sequence number 143 is the V of antibody 244 L It is the amino acid sequence of CDR3 of Sequence number 144 is the V of antibody 244 H It is the amino acid sequence of Sequence number 145 is the V of antibody 244 H It is the amino acid sequence of CDR1 of Sequence number 146 is the V of antibody 244 H It is the amino acid sequence of CDR2 of Sequence number 147 is the V of antibody 244 H It is the amino acid sequence of CDR3 of Sequence number 148 is the V of antibody 253 L It is the amino acid sequence of Sequence number 149 is the V of antibody 253 L It is the amino acid sequence of CDR1 of Sequence number 150 is the V of antibody 253 L It is the amino acid sequence of CDR2 of Sequence number 151 is the V of antibody 253 L It is the amino acid sequence of CDR3 of Sequence number 152 is the V of antibody 253 H It is the amino acid sequence of Sequence number 153 is the V of antibody 253 H It is the amino acid sequence of CDR1 of Sequence number 154 is the V of antibody 253 H It is the amino acid sequence of CDR2 of Sequence number 155 is the amino acid sequence of the CDR3 of the V H of antibody 253 Sequence number 156 is the amino acid sequence of the V L of antibody 259 Sequence number 157 is the amino acid sequence of the CDR1 of the V L of antibody 259 Sequence number 158 is the amino acid sequence of the CDR2 of the V L of antibody 259 Sequence number 159 is the amino acid sequence of the CDR3 of the V L of antibody 259 Sequence number 160 is the amino acid sequence of the V H of antibody 259 Sequence number 161 is the amino acid sequence of the CDR1 of the V H of antibody 259 Sequence number 162 is the amino acid sequence of the CDR2 of the V H of antibody 259 Sequence number 163 is the amino acid sequence of the CDR3 of the V H of antibody 259

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0167] General

[0168] Throughout this specification, unless a different description is specifically made or the context requires otherwise, a single step, composition of matter, group of steps, or composition of a group of matters is understood to encompass one and a plurality (i.e., one or more) of these steps, composition of matter, group of steps, or composition of a group of matters.

[0169] Those skilled in the art will recognize that there may be variations and modifications to the present disclosure other than those specifically described. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure includes all of the steps, features, compositions, and compounds individually or collectively mentioned or shown herein, and any combination of any two or more of those steps or features.

[0170] The present disclosure should not be limited in scope by the specific examples described herein, which are intended to be illustrative only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0171] It is understood that any example of the present disclosure, unless otherwise specified, applies to any other example of the present disclosure with the necessary modifications. In other words, any specific example of the present disclosure can be combined with any other specific example of the present disclosure (except when they are mutually exclusive).

[0172] Any example of the present disclosure that discloses a particular feature, or group of features, or method, or method step is understood to provide explicit support for negating that particular feature, or group of features, or method, or method step.

[0173] Unless otherwise specified, all scientific and technical terms used herein are understood to have the same meaning as commonly understood by those skilled in the art in the relevant fields (e.g., cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0174] Unless otherwise specified, the recombinant protein, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in various sources, examples of which include J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors) Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all revisions to date).

[0175] The description and definition of variable regions and portions thereof, antibodies and fragments thereof herein can be further clarified by the discussions in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol Biol. 196:901 -917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

[0176] The expression "and / or", e.g., "X and / or Y", is understood to mean "X and Y" or "X or Y", and is understood to provide explicit support for both meanings, or either meaning.

[0177] Throughout this specification, the term "comprising", or variations such as "comprises" (where the subject is in the third-person singular), "comprising", etc., is understood to include one element, integer, or step, or group of elements, integers, or steps recited, and does not exclude any other one element, integer, or step, or group of elements, integers, or steps.

[0178] As used herein, the expression "derived from" is understood to indicate that the specified integer can be obtained from a particular source, but not necessarily directly from that source.

[0179] Selected Definitions

[0180] The terms "butyrophilin (BTN)" and "butyrophilin-like (BTNL)" molecules refer to regulators of the immune response belonging to the immunoglobulin (Ig) superfamily of transmembrane proteins. These are structurally related to the B7 family of costimulatory molecules and have similar immune regulatory functions. BTN is involved in T cell development, activation, and inhibition, as well as in altering the interaction of T cells with antigen-presenting cells and epithelial cells. Some BTNs are genetically associated with autoimmune and inflammatory diseases. The human butyrophilin family contains seven members, which are divided into three subfamilies, namely BTN1, BTN2, and BTN3. The BTN1 subfamily contains only the prototype single-copy BTN1A1 gene, whereas the BTN2 and BTN3 subfamilies each contain three genes, namely BTN2A1, BTN2A2, and BTN2A3, and BTN3A1, BTN3A2, and BTN3A3. BTNL proteins share significant homology with members of the BTN family. The human genome contains four BTNL genes, namely BTNL2, 3, 8, and 9.

[0181] Butyrophilin and BTNL molecules contain two immunoglobulin-like domains, namely an N-terminal Ig-V-like domain (referred to herein as "IgV") and a C-terminal Ig-C-like domain (referred to herein as "IgC").

[0182] For purposes of name only, and without limitation, the amino acid sequence of BTN2A1 is taught in NCBI RefSeq NP_001184162.1, NP_001184163.1, NP_008980.1, or NP_001184163.1, and / or in SEQ ID NOs: 1-4. In one example, BTN2A1 is human BTN2A1.

[0183] The term "γδ T cell" refers to cells that express γ and δ chains as part of the T cell receptor (TCR) complex. The γδ TCR consists of a γ-chain and a δ-chain, each containing a variable Ig domain and a constant Ig domain. These domains are formed by gene recombination of the variable (v), diversity (D) (for TCRδ only), joining (J), and constant (C) genes in the TCRδ and γ loci. The variable domain of each chain typically contains three solvent-exposed loops (known as the CDR1 region, CDR2 region, and CDR3 region) that typically contact the ligand, and the CDR3 region is highly diverse in composition, which results from the diversity of V-D-J combinations and non-template nucleotide changes (additions and deletions) at the V-D recombination site and the D-J recombination site.

[0184] In humans, γδ T cells can be further divided into "Vδ2 cells" and "non-Vδ2 cells", the latter consisting mostly of cells that express Vδ1, and rarely of cells that express Vδ3-chain or Vδ5-chain, although Vδ4, Vδ6, Vδ7, Vδ8 have also been described. γδ T cells can mediate antibody-dependent cell cytotoxicity (ADCC) and phagocytosis and can respond rapidly to pathogen-specific antigens without prior differentiation or proliferation. Since γδ T cells respond directly to protein and non-peptide antigens, they are not restricted by MHC. At least some γδ T cell-specific antigens present evolutionarily conserved molecular patterns found in bacterial pathogens and induced self-antigens, and such molecular patterns are upregulated by cellular stress, infection, and transformation. Such antigens are generally referred to herein as "phosphoantigens" or pAgs. Vγ9+ γδ T cells can also respond to other antigens and ligands through the TCR and (co)receptors.

[0185] In addition, γδ T cells can be further classified into the following series of multifunctional populations, namely IFN-γ-producing γδ T cells, IL-17A-producing γδ T cells, antigen-presenting γδ T cells, follicular B helper γδ T cells, and regulatory γδ T cells. γδ T cells can promote immune responses that exert direct cytotoxicity, cytokine production, and indirect immune responses. For example, the IFN-γ-producing phenotype is characterized by increased CD56 expression and enhanced cytolytic responses. Some γδ T cell subsets may contribute to disease progression by facilitating inflammation and / or immunosuppression. For example, IL-17A-producing γδ T cells are widely involved in inflammatory reactions and play a role in pathogens between infectious diseases and autoimmune diseases.

[0186] The complementarity-determining region 3 (CDR3) regions of both the γ gene and the δ gene form very large protrusions at the top of the receptor. The human TCR consisting of the Vγ9 chain and the Vδ2 chain is characterized by a bend at the position of the C-V junction. In the CDR2 loop of Vδ, the C" chain forms a pair with the C' chain of the inner β-sheet of its domain.

[0187] The term "BTN2A1 agonist" means a molecule that specifically binds to BTN2A1 and induces or enhances the activation of Vγ9+ γδ TCR. For example, this agonist binds to one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule. The agonist BTN2A1 can + Vδ2 + and / or Vγ9 + Vδ2 - induce or enhance the activation of γδ TCR. For example, the agonist BTN2A1 can induce or enhance the activation of Vγ9+ γδ TCR, and non-limiting examples thereof include Vγ9 + Vδ2 + and / or Vγ9 + Vδ1 +Activation of γδ TCR. The activation may be independent of the antigen. For example, without being bound by theory or motivation, the binding of a BTN2A1 agonist to BTN2A1 can modify one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to mimic antigen (e.g., pAg) activation as a switch from non-stimulatory BTN2A1 to stimulatory BTN2A1. The BTN2A1 agonist can induce Vγ9+ γδ TCR activation with kinetics and efficacy similar to antigen binding. In one embodiment, the binding of the BTN2A1 agonist leads to a change in the organization of the BTN2A1 molecule on the cell surface, for example, on the cell surface of tumor cells, monocytes, macrophages, dendritic cells, and / or natural killer (NK) cells. For example, the BTN2A1 agonist can promote the formation of a BTN2A1 / BTN3 complex (e.g., a BTN2A1 / BTN3A1 complex) on the cell surface. This agonist can cross-react with BTN3A1 or be bispecific for BTN2A1 and a BTN3 molecule (e.g., BTN3A1). In another or further embodiment, the binding of the BTN2A1 agonist induces ligation of Vγ9+ TCR on γδ T cells and / or increases the activity and / or survival of cells expressing BTN2A1. The BTN2A1 agonist is stimulatory to γδ T cells and can activate one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of γδ T cells.

[0188] The term "BTN2A1 antagonist" means a molecule that specifically binds to BTN2A1 and inhibits the activation of Vγ9+ γδ TCR. For example, this antagonist binds to one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule. The BTN2A1 antagonist can inhibit the activation of Vγ9 + Vδ2 + and / or Vγ9 + Vδ2 - γδ TCR. For example, the BTN2A1 antagonist can inhibit the activation of Vγ9 + Vδ2 + and / or Vγ9 +Vδ1 + It can inhibit the activation of γδ TCR. Representative BTN2A1 antagonists bind to one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to inhibit antigen (e.g., pAg) activation, inhibit binding to Vγ9+ γδ TCR, and / or prevent interaction with BTN3 molecules (e.g., BTN3A1). BTN2A1 antagonists induce a conformational change to switch the BTN2A1 molecule from stimulatory BTN2A1 to non-stimulatory BTN2A1, for example, to prevent antigen activation and / or interaction with BTN3A1. BTN2A1 antagonists can bind to the site on the BTN2A1 molecule that interacts with Vγ9+ TCR or the site on the BTN2A1 molecule that interacts with BTN3 molecules (e.g., BTN3A1). For example, soluble TCR can be used as a BTN2A1 antagonist. In another example, a BTN2A1 antagonist can cross-react with BTN3A1 or can be bispecific for BTN2A1 and BTN3 molecules (e.g., BTN3A1). BTN2A1 antagonists are inhibitory to γδ T cells and can inhibit one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of γδ T cells.

[0189] As used herein, the terms "inhibit" or "inhibiting" in the context of γδ T cell activation are understood to mean that the BTN2A1 antagonists of the present disclosure reduce or decrease the level of Vγ9+ γδ TCR activation. From the above, it will be apparent that the BTN2A1 antagonists of the present disclosure do not need to completely inhibit activation, but rather need to decrease the activity by a statistically significant amount (e.g., at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%). Methods for demonstrating inhibition of Vγ9+ γδ TCR activation are known in the art and / or described herein.

[0190] As used herein, the term "BTN2A1 / BTN3 complex" means a complex of BTN2A1 and BTN3 molecules (e.g., a complex of BTN2A1 and BTN3A1) on the surface of a cell (e.g., a tumor cell, monocyte, macrophage, dendritic cell, parenchymal cell, and / or natural killer (NK) cell). The BTN2A1 / BTN3 complex can be a heteromeric complex or a multimeric complex. This complex can include one or more BTN3 molecules (e.g., BTN3A1 and BTN3A2, and / or other proteins such as ATP-binding cassette transporter A1 (ABCA1)). This complex can include a BTN2A1 dimer. Similarly, the BTN3 molecule can exist in monomeric or dimeric form. The BTN2A1 and BTN3 molecules can co-localize on the cell surface and can associate directly (e.g., by cross-linking) or indirectly (e.g., via another molecule or protein). The BTN2A1 / BTN3 complex can bind to an antigen directly or indirectly. For example, the cytoplasmic domain of the BTN2A1 and / or BTN3 molecule can bind to an antigen directly or indirectly.

[0191] As used herein, the term "cancer" means an abnormal condition or condition characterized by cells having the ability to self-proliferate, i.e., rapidly increasing cell growth. Hyperproliferative and neoplastic disease states can be classified as pathological, i.e., characteristic of or constituting a disease state, or non-pathological, i.e., a deviation from normal but not related to a disease state. This term is meant to include all types of cancerous growth or carcinogenic processes, metastatic tissues or cells, tissues, or organs that have undergone malignant transformation, regardless of histopathological type or stage of invasion.

[0192] As used herein, the term "binds" with respect to the interaction between the binding region of an agonist or antagonist of BTN2A1 and the BTN2A1 molecule means that the interaction depends on the presence of a particular structure (e.g., an epitope) on the BTN2A1 molecule. For example, an antibody recognizes and binds to a particular protein structure rather than proteins in general. When an antibody binds to epitope "A", if a molecule containing epitope "A" (or "A" without a free label) is present in a reactant containing labeled "A" and the protein, the amount of labeled "A" that binds to the antibody will decrease.

[0193] As used herein, the term "specifically binds" is understood to mean that the binding interaction between the binding region on the surface of a BTN2A1 agonist or antagonist and the BTN2A1 molecule is dependent on the presence of an antigenic determinant or epitope. The binding region selectively binds to or recognizes a particular antigenic determinant or epitope, even when present in a mixture of other molecules or organisms. In one example, the binding region reacts or associates with a particular component, or cell expressing it, more frequently, more rapidly, for a longer period of time, and / or with greater affinity than alternative antigens or cells. By reading this definition, it is also understood that, for example, a binding region that binds to a particular element may or may not specifically bind to a second antigen. Thus, "specifically binds" does not necessarily require exclusive or undetectable binding of another antigen. The term "specifically binds" may be used interchangeably herein with "selectively binds." In general, references to binding herein are understood to mean specific binding, with each term providing explicit support for the other. Methods for determining specific binding will be apparent to one of skill in the art. For example, a binding protein comprising a binding region of the present disclosure is contacted with a component, or a cell expressing it, or a mutant form thereof, or an alternative antigen. Binding to the component or mutant form or alternative antigen is then determined, and the binding region that binds as described above is considered to specifically bind to the component. In one example, "specific binding" to a component or a cell expressing it is considered to be specific binding to the component when the binding region is 10 μM or less (such as 9 μM or less, 8 μM or less, 7 μM or less, 6 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, or 1 μM or less), such as 100 nM or less, such as 50 nM or less (e.g., 20 nM or less), 1 nM or less (e.g., 0.8 nM or less), 1×10 -8 M or less, 5×10 -9 M or less (e.g. 3×10 -9 M or less), etc., 2.5 x 10 -9 Equilibrium constants (K D ) means to join.

[0194] The term "recombinant" is understood to mean the product of artificial genetic recombination. Thus, in the context of an antibody or its antigen-binding fragment, this term does not include the natural antibodies in the body of the subject that are the products of natural recombination that occur while B cells are maturing. However, if such an antibody is isolated, it is considered an isolated protein containing the antibody variable region. Similarly, when the protein encoded by a nucleic acid is isolated and expressed using recombinant means, the resulting protein is a recombinant protein. Recombinant proteins include those expressed by artificial recombinant means, as well as those that are, for example, in a cell, tissue, or subject in which they are expressed.

[0195] The term "protein" is understood to include a single polypeptide chain, i.e., a series of continuous amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., a polypeptide complex) linked to each other by covalent or non-covalent bonds. For example, the series of polypeptide chains can be covalently linked using appropriate chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0196] The term "polypeptide" or "polypeptide chain" is understood from the previous paragraph to mean a series of continuous amino acids linked by peptide bonds.

[0197] One of ordinary skill in the art will recognize that an "antibody" is generally considered to be a protein containing a variable region consisting of multiple polypeptide chains (e.g., a polypeptide containing a light chain variable region (V L ) and a polypeptide containing a heavy chain variable region (V H ). Antibodies generally also contain constant domains, and in the case of heavy chains, some of them can be arranged to form a constant region (which contains a constant fragment or crystallizable fragment (Fc)). V H and V LThey interact to form an Fv containing an antigen-binding region that can specifically bind to one or several closely related antigens. Generally, the light chain from a mammal is a κ light chain or a λ light chain, and the heavy chain from a mammal is α, δ, ε, γ, or μ. As an antibody, any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass is possible. The term "antibody" also includes humanized antibodies, primatized antibodies, human antibodies, synthetic humanized antibodies, and chimeric antibodies. The term "antibody" also includes variants in which the encoded C-terminal lysine residue is lost, deamidated variants, and / or glycosylated variants, and / or variants containing pyroglutamic acid at the N-terminus of a protein (e.g., an antibody), and / or variants lacking an N-terminal residue (e.g., the N-terminal glutamine in an antibody or V region), and / or variants containing all or part of a secretion signal. Deamidated variants of the encoded asparagine residue can generate isoaspartic acid isoforms and aspartic acid isoforms, and even become succinamide involving adjacent amino acid residues. Deamidated variants of the encoded glutamine residue can become glutamic acid. When referring to a specific amino acid sequence, it is assumed that the composition includes such a sequence and a heterogeneous mixture of variants.

[0198] In the context of the present disclosure, the term "half-antibody" means a protein containing a single antibody heavy chain and a single antibody light chain. The term "half-antibody" also includes a protein containing one antibody light chain and one antibody heavy chain, and the antibody heavy chain is mutated to prevent its association with another antibody heavy chain.

[0199] The terms "full-length antibody", "complete antibody", or "whole antibody" are used interchangeably and mean an antibody in a substantially complete form, as distinct from an antigen-binding fragment of an antibody. Specifically, whole antibodies include those having a heavy chain and a light chain that includes an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or its amino acid sequence.

[0200] As used herein, "variable region" means the portion of the light and / or heavy chain of an antibody as defined herein that specifically binds an antigen and includes, for example, the amino acid sequences of CDRs (i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) together with three CDRs. V H means the variable region of the heavy chain. V L means the variable region of the light chain.

[0201] As used herein, the term "complementary determining region" (synonym CDR; i.e., CDR1, CDR2, and CDR3) means the amino acid residues of the antibody variable region, the presence of which is a major contributor to specific antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. In one example, the amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). According to the Kabat numbering system, the FRs and CDRs of V H are at the following positions. That is, residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3), and 103-113 (FR4). According to the Kabat numbering system, V LThe FR and CDR are located at the following positions. That is, residues 1 to 23 (FR1), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3), and 98 to 107 (FR4).

[0202] The "framework region" (hereinafter referred to as FR) is the variable domain residue other than the CDR residue.

[0203] In this specification, the term "Fv" refers to any protein that forms a complex having an antigen-binding site (i.e., can specifically bind to an antigen) whether consisting of a number of polypeptides or a single polypeptide, in which V L and V H associate. The V H and V L forming the antigen-binding site can be in the form of a single polypeptide chain or a plurality of different polypeptide chains. Furthermore, the Fv of the present disclosure (as well as any protein of the present disclosure) can have a number of antigen-binding sites, which may or may not bind to the same antigen. This term is understood to include proteins corresponding to fragments directly derived from antibodies as well as such fragments produced using recombinant means. In some examples, V H is not linked to the heavy chain constant domain (C H )1, and / or V L is not linked to the light chain constant domain (C L ). Representative Fvs containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetra-bodies, or higher-order complexes, or their constant regions or constant domains (e.g., C H 2 domain or C HAny of the above (e.g., minibody) linked to a 3 - domain. A "Fab fragment" consists of a monovalent antigen - binding fragment of an antibody and can be generated by digesting the whole antibody with the enzyme papain to produce a fragment consisting of a complete light chain and part of the heavy chain, or can be generated using recombinant means. The "Fab' fragment" of an antibody can be obtained by treating the whole antibody with pepsin and then reducing it, resulting in a molecule consisting of a complete light chain and part of the heavy chain containing the V H and a single constant domain. Two Fab' fragments are obtained per antibody thus treated. Fab’ fragments can be produced by recombinant means. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments joined by two disulfide bonds and is obtained by treating the whole antibody molecule with the enzyme pepsin, without subsequent reduction. The "Fab2" fragment is a recombinant fragment containing two Fab fragments linked using, for example, a leucine zipper or a C H 3 - domain. A "single - chain Fv" or "scFv" is a recombinant molecule containing the variable - region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0204] The term "constant region" as used herein means the portion of an antibody heavy or light chain other than the variable region. In the heavy chain, the constant region generally includes a plurality of constant domains and one hinge region. For example, the IgG constant region includes the following linked elements, namely constant heavy (C H )1, linker, C H 2, and C H 3. In the heavy chain, the constant region includes the Fc. In the light chain, the constant region generally includes one constant domain (C L 1).

[0205] The terms "fragment crystallizable" or "Fc" or "Fc region" or "Fc portion" (used interchangeably herein) mean the region of an antibody that includes at least one constant region, is generally glycosylated (but not necessarily so), and can bind to one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of five isotypes: alpha, delta, epsilon, gamma, or mu. Further, since the heavy chains of various subclasses (such as the IgG subclasses of the heavy chain) are responsible for various effector functions, a protein having a desired effector function can be generated by selecting the desired heavy chain constant region. Representative heavy chain constant regions are gamma 1 (IgG1), gamma 2 (IgG2), and gamma 3 (IgG3), or hybrids thereof.

[0206] An "antigen-binding fragment" of an antibody includes one or more variable regions of a complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, half-antibodies, and multispecific antibodies formed from multiple antibody fragments.

[0207] The term "stabilized IgG4 constant region" is understood to mean an IgG4 constant region that has been modified such that Fab arm exchange is reduced, or the tendency to undergo Fab arm exchange or form half-antibodies is reduced. "Fab arm exchange" means a type of protein modification for human IgG4, in which the IgG4 heavy chain and the attached light chain (half-molecule) are exchanged for a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (become a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or a reducing agent (such as reduced glutathione).

[0208] As used herein, the term "monospecific" means a binding region that contains one or more antigen-binding sites, each having the same epitope specificity. Thus, a monospecific binding region can contain a single antigen-binding site (e.g., Fv, scFv, Fab, etc.), or it can contain several antigen-binding sites (e.g., diabodies or antibodies) that recognize the same epitope (e.g., are identical to each other). The condition that a binding region is "monospecific" does not mean that it binds to only one antigen. This is because multiple antigens can have shared epitopes, or epitopes that are very similar to each other, to which a single antigen-binding site can bind. A monospecific binding region that binds to only one antigen is said to "bind exclusively" to that antigen.

[0209] The term "multispecific" means a binding region that contains two or more antigen-binding sites, each binding to a different epitope (e.g., each binding to a different antigen). For example, a multispecific binding region can contain antigen-binding sites that recognize two or more different epitopes of the same protein, or it can recognize two or more different epitopes of different proteins (e.g., on the surface of BTN2A1 and BTN3 molecules (BTN3A1)). In one example, the binding region can be "bispecific". That is, the binding region contains two antigen-binding sites that specifically bind to two different epitopes. For example, a bispecific binding region specifically binds to two different epitopes on the same protein, or has specificity for two different epitopes on the same protein. In another example, a bispecific binding region specifically binds to two different epitopes on two different proteins (e.g., BTN2A1 and BTN3 molecules (BTN3A1)).

[0210] As used herein, the term "soluble T cell receptor" or "soluble TCR" refers to a TCR consisting of the chains of a full-length (e.g., membrane-bound) receptor, wherein at least the transmembrane region of the receptor chain is deleted or mutated such that the receptor does not associate with the membrane when expressed by a cell. Most typically, the soluble receptor consists only of the extracellular domains of the chains of the wild-type receptor (i.e., lacking the transmembrane and cytoplasmic domains). The soluble γδ TCRs of the present disclosure consist of heterodimers of γ and δ chains containing Vγ9 (referred to herein as "soluble Vγ9+ TCRs"). Particular specific combinations of γ and δ chains, particularly those corresponding to γδ TCR subsets known to exist in vivo, are preferred for use in the soluble γδ TCRs of the present invention, but it should be understood that soluble TCRs having substantially any combination of γ and δ chains containing Vγ9 are also contemplated for use in the present disclosure. The soluble γδ TCRs preferably contain γ and δ chains derived from the same animal species (e.g., mouse, human).

[0211] As used herein, the terms "disease", "disorder", or "condition" mean a disruption of normal function or an interference with normal function and are not limited to any particular condition, including diseases or disorders.

[0212] As used herein, a subject "at risk" of developing a disease or condition, or of recurrence of the disease or condition, may or may not have a detectable disease or symptoms of a disease, and may or may not have a detectable disease or symptoms of a disease prior to treatment according to the present disclosure. As is known in the art and / or as described herein, "at risk" means that the subject has one or more risk factors, which are measurable parameters that correlate with the development of the disease or condition.

[0213] As used herein, the terms "treating", "treat", or "treatment" include reducing or eliminating at least one symptom of a particular disease or condition, or delaying the progression of the disease or condition, by administering a protein described herein.

[0214] As used herein, the terms "preventing," "prevents," or "prevention" include providing prophylaxis against the occurrence or recurrence of a particular disease or condition. An individual may have a tendency or risk for the disease to progress or recur, but the disease or recurrence has not yet been diagnosed.

[0215] "Effective amount" means at least an amount effective to achieve the desired result at the time of administration and over the necessary period. For example, as the desired result, a therapeutic result or a prophylactic result is possible. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" means the amount necessary to achieve the treatment of a disease or condition described herein. In some examples of the present disclosure, the term "effective amount" means the amount necessary to effectuate or inhibit the activation of Vγ9+ TCR γδ T cells. In some examples of the present disclosure, the term "effective amount" means the amount necessary to effectuate or inhibit one or more of cytolytic function, cytokine production of one or more cytokines, or proliferation of γδ T cells. The effective amount can vary depending on the disease or condition being treated, or the factor being altered, and can also vary depending on body weight, age, ethnic background, gender, health and / or physical condition, and other factors related to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g., a "dosage" range), which can be determined by routine trials and experimentation by a medical practitioner. Thus, this term should not be construed as limiting the present disclosure to a particular amount (e.g., the weight or number of binding proteins). The effective amount can be administered as a single dose, or as a dose that is repeated one or more times over the course of treatment.

[0216] "An amount effective for treatment" is at least the minimum concentration necessary to effect a measurable improvement in a particular disease or condition. As used herein, the amount effective for treatment may vary depending on factors such as the disease state, age, sex, weight of the patient, and the ability of the antibody or antigen-binding fragment thereof to induce the desired response in that individual. An amount effective for treatment is an amount that does not exceed any toxic or detrimental effects of the antibody or antigen-binding fragment thereof.

[0217] As used herein, the expression "an amount effective for prevention" is understood to mean an amount of an agonist or antagonist of BTN2A1 sufficient to prevent, inhibit, or delay the appearance of one or more detectable symptoms of a disease or condition, or a complication thereof.

[0218] As used herein, the term "subject" is understood to mean any animal, including humans (e.g., mammals). Non-limiting examples of representative subjects include humans and non-human primates. For example, the subject is a human.

[0219] Antibody

[0220] In one example, an agonist or antagonist of BTN2A1 of the disclosure that is a protein comprising an antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

[0221] Immunization-based methods

[0222] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (eds.) Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, the protein, or its immunogenic fragment or epitope, or the cell expressing and presenting it (i.e., the immunogen) is optionally formulated with any suitable or desired vehicle, adjuvant, or pharmaceutically acceptable excipient and administered to a non-human animal (e.g., mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig). The immunogen can be administered by intranasal, intramuscular, subcutaneous, intravenous, intradermal, intraperitoneal, or other known routes.

[0223] The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various times after immunization. If it is necessary to achieve the desired antibody titer, the animal can be immunized one or more additional times. The process of boosting or titrating is repeated until an appropriate titer is achieved. When the desired level of immunogenicity is obtained, the immunized animal is bled, the serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0224] Monoclonal antibodies are one representative form of the antibodies contemplated in the present disclosure. The term "monoclonal antibody" or "mAb" means a homogeneous population of antibodies that can bind to the same antigen (e.g., the same epitope within the antigen). It is assumed that this term has no limitation regarding the source of the antigen or the method of producing the antigen.

[0225] Any one of a number of known techniques (e.g., the procedures exemplified in US4196265 or the above-mentioned literature of Harlow and Lane (1988)) can be utilized for the production of mAbs.

[0226] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody-producing cells. Representative animals are rodents such as rabbits, mice, and rats. Mice that have been genetically engineered to express human immunoglobulin proteins and do not express, for example, mouse immunoglobulin proteins (as described, for example, in WO2002066630) can also be used to generate the antibodies of the present disclosure.

[0227] After immunization, somatic cells having the ability to produce antibodies (e.g., B lymphocytes (B cells)) are selected for use in an mAb production protocol. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from peripheral blood samples. Next, B cells from the immunized animal are fused with cells of immortal myeloma cells generally derived from the same species as the animal immunized with the immunogen.

[0228] Hybrids are amplified by culturing in a selective medium containing an agent that blocks the de novo synthesis of nucleotides in tissue culture medium. Representative agents are aminopterin, methotrexate, and azaserine.

[0229] The amplified hybridomas are subjected to functional selection regarding antibody specificity and / or titer by, for example, flow cytometry and / or immunohistochemistry and / or immunoassay (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, etc.).

[0230] Alternatively, a cell line secreting mAb is generated using the ABL-MYC technology (NeoClone, Madison WI 53713, USA) (as described, for example, in Largaespada et al, J. Immunol. Methods. 197: 85-95, 1996).

[0231] Library-based methods

[0232] Also included in the present disclosure is the screening of libraries of antibodies or antigen-binding fragments thereof (e.g., those containing their variable regions).

[0233] Examples of libraries contemplated in the present disclosure include naive libraries (from unchallenged subjects), immunized libraries (from subjects immunized with an antigen), or synthetic libraries. Nucleic acids encoding antibodies or regions thereof (e.g., variable regions) are cloned by conventional techniques (e.g., as described in Sambrook and Russell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001), and used to encode and display proteins using methods known in the prior art. Other techniques for generating libraries of proteins are described, for example, in US6300064 (e.g., the HuCAL library of Morphosys AG); US5885793; US6204023; US6291158; or US6248516.

[0234] The antigen-binding fragments according to the present disclosure are soluble secreted proteins or can be presented as fusion proteins on the surface of cells or particles (such as phages or other viruses, ribosomes, or spores). Various forms of display libraries are known in the art. For example, the library can be an in vitro display library (such as a ribosome display library, a covalent display library, or an mRNA display library (such as described in US7270969)). In yet another example, the display library is a phage display library, and the protein containing the antigen-binding fragment of the antibody is expressed on the surface of the phage as described, for example, in US6300064; US5885793; US6204023; US6291158; or US6248516. Other phage display methods are known in the art and are contemplated by the present disclosure. Similarly, methods of cell display are contemplated by the present disclosure (such as bacterial display libraries (such as described in US5516637); yeast display libraries (such as described in US6423538) or mammalian display libraries).

[0235] Methods for screening display libraries are known in the art. In one example, the display libraries of the present disclosure are screened using affinity purification as described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). The method of affinity purification typically involves contacting the protein containing the antigen-binding fragment presented by the library with a target antigen (such as BTN2A1), followed by washing and eluting the domain that remains bound to the antigen.

[0236] Any variable region or scFv identified by screening can be readily modified into a full antibody if desired. Representative methods for modifying or reformatting a variable region or scFv into a full antibody are described, for example, in Jones et al., J Immunol Methods. 354:85-90, 2010; or Jostock et al., J Immunol Methods, 289: 65-80, 2004; or WO 2012040793. Alternatively, or in addition, standard cloning methods described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), and / or (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001) can be utilized.

[0237] Deimmunized antibody, chimeric antibody, humanized antibody, synthetic antibody, primatized antibody, and human antibody, or antigen-binding fragment

[0238] The antibodies or antigen-binding fragments of the present disclosure can be humanized.

[0239] The term "humanized antibody" is understood to mean a protein comprising a human-like variable region that contains CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted within the surface of the FRs from a human antibody (this type of antibody is also referred to as a "CDR-grafted antibody"). Humanized antibodies include antibodies in which one or more residues of the human protein have been modified by one or more amino acid substitutions and / or in which one or more FR residues of the human antibody have been replaced with the corresponding non-human residues. A humanized antibody can also include residues not found in either human antibodies or non-human antibodies. Any additional regions of the antibody (e.g., the Fc region) are generally human. Humanization can be carried out using methods known in the art (e.g., US5225539, US6054297, US7566771, or US5585089). The term "humanized antibody" also encompasses, for example, the hyperhumanized antibodies described in US7732578. A similar meaning is understood to apply to the term "humanized antigen-binding fragment".

[0240] As the antibody or antigen-binding fragment thereof of the present disclosure, a human antibody or its antigen-binding fragment is possible. The term "human antibody" as used herein means an antibody having a variable region found in a human (e.g., human germline or somatic cells), and optionally a constant antibody region, or an antibody from a library prepared using such regions. A "human" antibody can include amino acid residues not encoded by human sequences, such as mutations introduced by random or site-directed mutagenesis in vitro (in particular, mutations involving conserved substitutions, or mutations in a small number of residues of the protein (e.g., 1, 2, 3, 4, or 5 residues of the protein)). These "human antibodies" do not necessarily have to be generated as a result of a human immune response, but rather can be generated using recombinant means (e.g., screening of phage display libraries) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding the constant region and / or variable region of a human antibody and / or by using guided selection (e.g., as described in US5565332). The term also encompasses affinity-matured forms of such antibodies. For the purposes of the present disclosure, a protein containing an FR from a human antibody or a sequence from a consensus sequence of a human FR and having one or more of the CDRs being random or semi-random is also considered to be a human antibody (e.g., as described in US6300064 and / or US6248516). A similar meaning is understood to apply to the term "human antigen-binding fragment".

[0241] As the antibody or antigen-binding fragment thereof of the present disclosure, a synthetic humanized antibody or its antigen-binding fragment is possible. The term "synthetic humanized antibody" means an antibody prepared by the method described in WO2007019620. A synthetic humanized antibody includes, as the variable region of the antibody, an FR from a New World primate antibody variable region and a CDR from a non-New World primate antibody variable region.

[0242] The antibodies or antigen-binding fragments thereof of the present disclosure can be primatized. A "primatized antibody" includes variable regions from antibodies generated after immunizing non-human primates (e.g., cynomolgus monkeys). Optionally, the variable regions of the non-human primate antibodies are linked to human constant regions to generate primatized antibodies. Representative methods for generating primatized antibodies are described in US6113898.

[0243] In one example, the antibodies or antigen-binding fragments thereof of the present disclosure are chimeric antibody fragments. The terms "chimeric antibody" or "chimeric antigen-binding fragment" mean an antibody or fragment in which one or more of the variable domains are from a particular species (e.g., mouse (such as mouse or rat)) or belong to a particular class or subclass of antibodies, while the remainder of the antibody or fragment is from another species (e.g., human or non-human primate) or belongs to another class or subclass of antibodies. In one example, a chimeric antibody includes V H and / or V L from a non-human antibody (e.g., a mouse antibody), and the remaining region of the antibody is from a human antibody. The production of such chimeric antibodies and their antigen-binding fragments is known in the art and can be achieved by standard means (e.g., as described in US6331415; US5807715; US4816567, and US4816397).

[0244] In the present disclosure, deimmunized antibodies or antigen-binding fragments thereof, such as those described in WO2000034317 and WO2004108158, are also contemplated. Deimmunized antibodies and fragments have one or more epitopes (e.g., B cell epitopes or T cell epitopes) removed (i.e., mutated), thus reducing the likelihood that a subject will generate an immune response to the antibody or protein. For example, the antibodies of the present disclosure are analyzed to identify one or more B cell epitopes or T cell epitopes, and the immunogenicity of the antibody is reduced by mutating one or more amino acid residues among those epitopes.

[0245] Bispecific antibody

[0246] As an antibody or antigen-binding fragment of the present disclosure, a bispecific antibody or a fragment thereof is possible. A bispecific antibody is a molecule that includes two types of antibodies or antibody fragments (e.g., two half-antibodies) having specificities for different antigens or epitopes. A typical bispecific antibody binds to two different epitopes of the same protein. Alternatively, a bispecific antibody binds to two different epitopes of two different proteins.

[0247] A representative "key and hole" or "knob and hole" bispecific protein described in US5731168. In one example, a certain constant region (e.g., IgG4 constant region) includes a T366W mutation (i.e., knob), and a certain constant region (e.g., IgG4 constant region) includes T366S, L368A, and Y407V mutations (i.e., hole). In another example, the first constant region includes T350V, T366L, K392L, and T394W mutations (knob), and the second constant region includes T350V, L351Y, F405A, and Y407V mutations (hole).

[0248] Methods for generating bispecific antibodies are known in the art, and representative methods are described herein.

[0249] In one example, an IgG-type bispecific antibody is secreted by a hybrid hybridoma (quadroma) formed by fusing two types of hybridomas that produce IgG antibodies (Milstein C et al., Nature 1983, 305: 537-540). In another example, an antibody can be secreted by introducing into cells the genes of the L chains and H chains that constitute two co-expressed IgGs of interest (Ridgway, JB et al. Protein Engineering 1996, 9: 617-621;Merchant, AM et al. Nature Biotechnology 1998, 16: 677-681).

[0250] In one example, bispecific antibody fragments are prepared by chemically crosslinking Fabs derived from different antibodies (Keler T et al. Cancer Research 1997, 57: 4008-4014).

[0251] In one example, leucine zippers derived from, for example, Fos and Jun are used to form bispecific antibody fragments (Kostelny SA et al. J. of Immunology, 1992, 148: 1547-53).

[0252] In one example, bispecific antibody fragments are prepared in the form of diabodies containing two crossover scFv fragments (Holliger P et al. Proc. of the National Academy of Sciences of the USA 1993, 90: 6444-6448).

[0253] Antibody fragment

[0254] Single domain antibody

[0255] In some examples, the antigen-binding fragments of the antibodies of the present disclosure are single domain antibodies (used interchangeably with the terms "domain antibody" or "dAb") or include single domain antibodies. A single domain antibody is a single polypeptide chain that includes all or part of the heavy chain variable domain of an antibody. For example, a single domain antibody is a nanobody.

[0256] Diabody, triabody, tetrabody

[0257] In some examples, the antigen-binding fragments of the present disclosure are diabodies, triabodies, tetra bodies, or higher-order protein complexes as described in WO98 / 044001 and / or WO94 / 007921, or include these.

[0258] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain having a structure V L -X-V H or V H -X-V L (wherein X is a linker that contains residues insufficient for V H and V L within a single polypeptide chain to associate (or form an Fv), and the V H of one polypeptide chain binds to the V L of the other polypeptide chain to form an antigen-binding site that can specifically bind to one or more antigens, i.e., an Fv molecule). To form a bispecific diabody (i.e., one containing two Fvs with different specificities), it is possible for V L and V H to be the same within each polypeptide chain, or for V L and V H to be different within each polypeptide chain.

[0259] Single-chain Fv (scFv) fragment

[0260] One skilled in the art will appreciate that an scFv contains a V H region and a V L region within a single polypeptide chain, and a polypeptide linker between V H and V L such that the linker enables the scFv to form a structure desirable for antigen binding (i.e., the V H and V L of the single polypeptide chain associate with each other to form an Fv). For example, the linker contains 12 excess amino acid residues, and (Gly4Ser)3 is one of the most preferred linkers for scFv.

[0261] In one example, the linker contains the sequence SGGGGSGGGGSGGGGS.

[0262] In the present disclosure, Fv (or diFv or dsFv) stabilized by disulfide is also considered. Here, a single cysteine residue is introduced into the FR of V H and the FR of V L and these cysteine residues are linked by disulfide bonds to produce a stable Fv.

[0263] Alternatively, or in addition thereto, the present disclosure includes a dimeric scFv, i.e., a protein comprising two scFv molecules bound non-covalently or covalently, for example, by a leucine zipper domain (derived from, for example, Fos or Jun). Alternatively, as described, for example, in US20060263367, two scFvs are linked by a peptide linker of sufficient length such that both scFvs are formed and capable of binding to an antigen.

[0264] Half-antibody

[0265] In some examples, the antigen-binding fragment of the present disclosure is a half-antibody or a half-molecule. Those skilled in the art will recognize that a half-antibody means a protein comprising a single heavy chain and a single light chain. The term "half-antibody" also includes a protein comprising one antibody light chain and one antibody heavy chain, wherein the antibody heavy chain is mutated to prevent association with another antibody heavy chain. In one example, a half-antibody is formed when one antibody dissociates to form two molecules, each of which contains a single heavy chain and a single light chain.

[0266] Methods for generating half-antibodies are known in the art, and representative methods are described herein.

[0267] In one example, the half-antibody can be secreted by introducing it into the cell genes of a single heavy chain and a single light chain that make up the IgG of interest for expression. In one example, the constant region (e.g., IgG4 constant region) contains a "key or hole" (or "knob or hole") mutation that prevents the formation of heterodimers. In one example, the constant region (e.g., IgG4 constant region) contains the T366W mutation (i.e., knob). In another example, the constant region (e.g., IgG4 constant region) contains the T366S, L368A, and Y407V mutations (i.e., hole). In another example, the constant region contains the T350V, T366L, K392L, and T394W mutations (knob). In another example, the constant region contains the T350V, L351Y, F405A, and Y407V mutations (hole). Representative constant region amino acid substitutions are numbered according to the EU numbering system.

[0268] Other antibodies and antibody fragments

[0269] The present disclosure also contemplates other antibodies and antibody fragments, such as, for example: (i) minibodies (e.g., as described in US5837821); (ii) heteroconjugate proteins (e.g., as described in US4676980); (iii) heteroconjugate proteins made using chemical cross-linking agents (e.g., as described in US4676980); and (iv) Fab3 (e.g., as described in EP19930302894).

[0270] Stabilized protein

[0271] The antigen-binding protein of the present disclosure can include an IgG4 constant region or a stabilized IgG4 constant region. The expression "stabilized IgG4 constant region" is understood to mean an IgG4 constant region modified to reduce the tendency to undergo Fab arm exchange, or the tendency to receive Fab arm exchange, or the formation of half antibodies, or the tendency to form half antibodies. "Fab arm exchange" means a kind of protein modification for human IgG4, in which the IgG4 heavy chain and the attached light chain (half molecule) are exchanged with a heavy-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (become a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or a reducing agent (such as reduced glutathione).

[0272] In one example, the stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally serine. After serine is substituted with proline, the IgG4 hinge region contains the sequence CPPC. In this regard, the "hinge region" is the proline-rich portion of the antibody heavy chain constant region that is linked to the Fc region and the Fab region, and it will be understood by those skilled in the art that it confers mobility to the two Fab arms of the antibody. This hinge region contains cysteine residues involved in inter-heavy chain disulfide bonds. It is generally defined as the portion from Glu226 to Pro243 of human IgG1 according to the Kabat numbering system. The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues that form the inter-heavy chain disulfide (S-S) bond at the same positions (see, for example, WO2010080538).

[0273] Immunoglobulin and immunoglobulin fragment

[0274] One example of the antigen-binding protein of the present disclosure is a protein that includes the variable region of an immunoglobulin (TCR or heavy chain immunoglobulin (e.g., IgNAR, camel antibody)).

[0275] Heavy chain immunoglobulin

[0276] Heavy-chain immunoglobulins differ in structure from many other forms of immunoglobulins (such as antibodies) in that they contain heavy chains but no light chains. Therefore, these immunoglobulins are also called "antibodies consisting only of heavy chains". Heavy-chain immunoglobulins are found, for example, in camels and cartilaginous fish (also called IgNAR).

[0277] The variable regions present in native heavy-chain immunoglobulins are distinguished from the heavy-chain variable regions present in normal four-chain antibodies (referred to as "V H domains") and from the light-chain variable regions present in normal four-chain antibodies (referred to as "V L domains"). Therefore, in camel Ig, they are generally referred to as "V HH domains", and in IgNAR, they are called V-NAR.

[0278] Heavy-chain immunoglobulins do not require the presence of light chains to bind to the relevant antigen with high affinity and high specificity. This means that single-domain binding fragments can be derived from heavy-chain immunoglobulins, which are easy to express and are generally stable and soluble.

[0279] General descriptions of heavy-chain immunoglobulins from camels and their variable regions, and methods for their production and / or isolation and / or use can be found, inter alia, in the following references WO94 / 04678, WO97 / 49805, and WO 97 / 49805.

[0280] General descriptions of heavy-chain immunoglobulins from cartilaginous fish and their variable regions, and methods for their production and / or isolation and / or use can be found, inter alia, in WO2005118629.

[0281] V-like proteins

[0282] In one example, the antigen-binding protein of the present disclosure comprises a TCR. The T cell receptor has two V domains incorporated into a structure similar to the Fv module of an antibody. Novotny et al., Proc Natl Acad Sci USA 88: 8646-8650, 1991 describes how the two V-domains of the T cell receptor (referred to as alpha and beta) can be fused and expressed as a single-chain polypeptide, and further how surface residues can be altered to reduce hydrophobicity, similar to an antibody scFv. Other publications describing the production of single-chain T cell receptors or multimeric TCRs containing two of the V-alpha and V-beta domains include WO1999045110 or WO2011107595.

[0283] Other non-antibody proteins containing antigen-binding domains generally are proteins having V-like domains that are monomeric. Examples of proteins containing such V-like domains include CTLA-4, CD28, and ICOS. Further disclosure of proteins containing such V-like domains is included in WO1999045110.

[0284] Adnectin

[0285] In one example, the antigen-binding protein of the present disclosure comprises an adnectin. Adnectin is based on the tenth fibronectin type III ( 10 Fn3) domain of human fibronectin, with loop regions therein altered to bind an antigen. For example 10 Three loops at one end of the β-sandwich of the Fn3 domain can be engineered to enable adnectin to specifically recognize an antigen. See US20080139791 or WO2005056764 for further details.

[0286] Anticalin

[0287] In yet another example, the antigen-binding protein of the present disclosure includes an anti-carin. Anti-carins are derived from lipocalins, a family of extracellular proteins that transport small hydrophobic molecules (such as steroids, bilins, retinoids, and lipids). Lipocalins have a rigid β-sheet secondary structure with multiple loops at the open end of a conical structure that can be engineered to bind to an antigen. Such engineered lipocalins are known as anti-carins. For further details, see US7250297 or US20070224633.

[0288] Affibody

[0289] In yet another example, the antigen-binding protein of the present disclosure includes an affibody. Affibodies are scaffolds derived from the Z domain (antigen-binding domain) of staphylococcal protein A that can be engineered to bind to an antigen. The Z domain consists of a three-helix bundle of approximately 58 amino acids. A library has been created by randomization of surface residues. For further details, see EP1641818.

[0290] Avimer

[0291] In yet another example, the antigen-binding protein of the present disclosure includes an avimer. Avimers are multi-domain proteins derived from the A domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by shuffling the natural variations presented by the A domain family. For further details, see WO2002088171.

[0292] DARPin

[0293] In yet another example, the antigen-binding protein of the present disclosure includes a designed ankyrin repeat protein (DARPin). DARPin is derived from ankyrin, which is one family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. One ankyrin repeat is a 33-residue motif consisting of two α-helices and one β-turn. By randomizing the residues in the first α-helix and the β-turn of each repeat, DARPin can be engineered to bind different target antigens. Its binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see US20040132028.

[0294] Annexin

[0295] In one example, the antigen-binding protein of the present disclosure includes annexin.

[0296] Annexin, also known as lipocortin, forms one family of soluble proteins that bind in a Ca2+-dependent manner to membranes that are exposed to negatively charged phospholipids, particularly phosphatidylserine (PS). Annexin is formed by four (exceptionally eight) repeats of a highly conserved 70-amino acid domain and a variable amino (N)-terminal domain (thought to be responsible for functional specificity). Annexin is important in various cellular and physiological processes, for example providing membrane scaffolds involved in changes in cell shape. Annexin has also been shown to be involved in vesicle trafficking and organization, exocytosis, endocytosis, and the formation of calcium ion channels.

[0297] Annexin species II, V, and XI are known to be located within the cell membrane. Annexin A5 is the most abundant membrane-bound annexin scaffold. Annexin A5 forms a two-dimensional network when binding to phosphatidylserine units of the membrane. Annexin A5 is effective in stabilizing changes in cell shape, among other cell membrane processes, between endocytosis and exocytosis.

[0298] Annexin species I (or Annexin A1) preferably localizes to the cytoplasmic face of the plasma membrane and binds to the phosphatidylserine units of the membrane. Annexin A1 does not form a two-dimensional network on the surface of the activated membrane.

[0299] In one example, this annexin species is an annexin derivative or a variant thereof. Annexin derivatives or variants thereof are known in the art and representative derivatives or variants are disclosed herein. For example, annexin variants / derivatives are disclosed in WO199219279, WO2002067857, WO2007069895, WO2010140886, WO2012126157, Schutters et al., Cell Death and Differentiation 20: 49-56, 2013, or Ungethum et al., J Biol Chem., 286(3):1903-10, 2011.

[0300] For example, an annexin derivative can contain amino acids that are shortened (e.g., containing one or more domains or fewer amino acid residues compared to the native protein) or substituted. In one example, the annexin derivative is truncated annexin 1. For example, truncated annexin 1 does not contain an N-terminal autocleavage site (e.g., 41 N-terminal amino acids are deleted). In one example, the modified annexin can have an N-terminal chelate site containing an amino acid extension (e.g., X1-Gly-X2 where X1 and X2 are selected from Gly and Cys). In one example, the annexin derivative or modified annexin binds to phosphatidylserine. In one example, the annexin derivative or modified annexin binds to phosphatidylserine at a level equivalent to that of wild-type annexin. For example, the annexin derivative or modified annexin binds to phosphatidylserine at the same level as wild-type annexin.

[0301] In one example, the antigen-binding protein of the present disclosure comprises annexin A5. For naming purposes only, and without limitation, the amino acid sequence of annexin A5 is taught in Gene Accession ID 308, NCBI reference sequence NP_001145, and / or SEQ ID NO: 5. For naming purposes only, and without limitation, the amino acid sequence of annexin A1 is taught in NCBI reference sequence NP_000691.1 and / or SEQ ID NO: 7.

[0302] Gamma-carboxyglutamic acid-rich (GLA) domain

[0303] In one example, the antigen-binding protein of the present disclosure comprises a gamma-carboxyglutamic acid-rich (GLA) domain or a variant thereof.

[0304] The GLA domain contains glutamic acid residues that are post-translationally modified by vitamin K-dependent carboxylation, which forms gamma-carboxyglutamic acid (Gla).

[0305] Proteins known to contain the GLA domain are known in the art and non-limiting examples thereof include vitamin K-dependent proteins S and Z, prothrombin, transthyretin, osteocalcin, matrix Gla protein, inter-alpha-trypsin inhibitor heavy chain H2, and growth arrest-specific protein 6.

[0306] Lactadherin domain

[0307] In one example, the antigen-binding protein of the present disclosure comprises a lactadherin domain.

[0308] Lactadherin is a glycoprotein secreted by various types of cells and has two C domains (C1C2 and C2) with sequence homology to the C1 and C2 domains of blood coagulation factors V and VIII and two EGF domains. Similar to these coagulation factors, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity.

[0309] In one example, the lactadherin domain is a C1C2 domain (e.g., as shown in SEQ ID NO: 27). In another example, the lactadherin domain is a C2 domain.

[0310] Protein kinase domain

[0311] In one example, the present disclosure provides an antigen-binding protein comprising a protein kinase C domain.

[0312] Protein kinase (PKC) is one family of protein kinase enzymes, or a member of this family, involved in controlling the function of other proteins through phosphorylation of the hydroxyl groups of the amino acid residues serine and threonine on these proteins.

[0313] The structure of PKC is known in the art and consists of a regulatory domain and a catalytic domain connected to each other by a hinge region. The regulatory domain contains a C1 domain and a C2 domain, which bind to DAG and Ca 2+ respectively, and recruit PKC to the plasma membrane.

[0314] In one example, the protein kinase C domain is a C1 domain. In another example, the protein kinase C domain is a C2 domain.

[0315] Pleckstrin homology domain

[0316] In one example, the present disclosure provides an antigen-binding protein comprising a pleckstrin homology (PH) domain.

[0317] PH domains are well known in the art and are small modular domains that occur in a wide range of proteins involved in intracellular signaling or as components of the cytoskeleton. The PH domain contains approximately 120 amino acids. This domain can bind to phosphatidylinositol in biological membranes and proteins (such as the beta / gamma subunits of heterotrimeric G proteins). Through these interactions, the PH domain plays a role in recruiting proteins to different membranes, directing those proteins to appropriate cell compartments, or enabling those proteins to interact with other components of the signaling pathway.

[0318] Peptide that interacts with phosphatidylserine

[0319] In one example, the present disclosure provides an antigen-binding protein comprising a peptide that interacts with phosphatidylserine. Suitable peptides are known in the art and include, for example, PSP1 described in Thapa et al., J. Cell. Mol. Med. 12. 1649-1660, 2008 and Kim et al., PLOS One, 10(3): e0121171. PSP1 contains the sequence CLSYYPSYC (SEQ ID NO: 28). Variants of PSP1 that retain the ability to bind to phosphatidylserine are also contemplated in the present disclosure.

[0320] Soluble T cell receptor

[0321] In one example, the BTN2A1 antagonist of the present disclosure is a soluble Vγ9+ TCR.

[0322] Soluble Vγ9+ TCRs useful in the present disclosure are typically heterodimers comprising a γ chain containing a Vγ9+ γ chain and a δ chain, but multimers (such as tetramers) comprising two different γδ heterodimers or two identical γδ heterodimers are also contemplated for use in the present disclosure.

[0323] The soluble Vγ9+ TCRs of the present disclosure can be generated by any suitable method known to those of skill in the art, most typically by recombination. According to the present disclosure, recombinant nucleic acid molecules useful for generating soluble γδ TCRs typically comprise a recombinant vector and a nucleic acid sequence encoding one or more compartments (e.g., chains) of the γδ TCR. According to the present disclosure, a recombinant vector is an engineered (i.e., artificially created) nucleic acid molecule used as a tool for manipulating a selected nucleic acid sequence and / or introducing such a nucleic acid sequence into a host cell. Thus, a recombinant vector is suitable for cloning, sequencing, and / or other manipulation of a selected nucleic acid sequence (e.g., expressing the selected nucleic acid sequence and / or forming recombinant cells by delivering it into a host cell). Such vectors typically contain a heterologous nucleic acid sequence, i.e., a nucleic acid sequence not naturally found adjacent to the nucleic acid sequence being cloned or delivered, although the vector can also contain a nucleic acid sequence encoding a protein of interest (e.g., a TCR chain) or regulatory nucleic acid sequences (e.g., promoters, untranslated regions) naturally found adjacent to nucleic acid sequences useful for the expression of nucleic acid molecules. The vector can be RNA or DNA, prokaryotic or eukaryotic, and is typically a plasmid.

[0324] Typically, a recombinant nucleic acid molecule comprises at least one nucleic acid molecule of the invention operably linked to one or more transcriptional control sequences. As used herein, the terms "recombinant molecule" or "recombinant nucleic acid molecule" primarily mean a nucleic acid molecule or nucleic acid sequence operably linked to a transcriptional control sequence, although the term "nucleic acid molecule" can be used interchangeably therewith when such nucleic acid molecule is a recombinant molecule as described herein. According to the present disclosure, the expression "operably linked" means linking a nucleic acid molecule to a transcriptional control sequence such that the molecule can be expressed when transfected (i.e., transformed, transduced, transfected, conjugated, or introduced) into a host cell. A transcriptional control sequence is a sequence that controls the initiation, elongation, or termination of transcription. Particularly important transcriptional control sequences are those that control the initiation of transcription (such as promoter sequences, enhancer sequences, operator sequences, and repressor sequences). Suitable transcriptional control sequences include any transcriptional control sequence that can function in the host cell or host organism into which the recombinant nucleic acid molecule is introduced.

[0325] One or more recombinant molecules of the invention can be used to generate the encoded products of the disclosure (e.g., soluble γδ TCR). In one embodiment, the encoded product is produced by expressing the nucleic acid molecules described herein under conditions effective for protein production. One preferred method for producing the encoded protein is to transfect one or more recombinant molecules into a host cell to form a recombinant cell. Non-limiting examples of host cells suitable for transfection include any bacterial, fungal (e.g., yeast), insect, plant, or animal cell capable of being transfected. As the host cell, it is possible to use a non-transfected cell or a cell that has already been transfected with at least one other recombinant nucleic acid molecule. The resulting protein of the invention may remain within the recombinant cell; it may be secreted into the medium; it may be secreted into the space between two cell membranes; or it may be retained on the outer surface of the cell membrane. The expression "recovering the protein" means recovering the entire medium containing the protein and does not necessarily include additional steps of separation or purification. The proteins produced according to the disclosure can be purified using a variety of standard protein purification techniques, non-limiting examples of which are affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing, and differential solubilization. The proteins produced according to the disclosure are preferably recovered in a "substantially pure" form. As used herein, "substantially pure" means a purity at which the soluble γδ TCR can be effectively utilized in the compositions and methods of the disclosure.

[0326] For example, a recombinant construct containing the relevant γ and δ genes (e.g., nucleic acid sequences encoding the desired portions of the γ and δ chains of γδ TCR) can be newly synthesized or generated by PCR of TCR cDNA from a source of γδ T cells expressing the desired receptor (e.g., hybridoma, clone, transgenic cell). PCR amplification of the desired γ and δ genes can be designed such that the transmembrane and cytoplasmic domains of these chains are removed (i.e., to generate a soluble receptor). The portions that form interchain disulfide bonds with these genes are retained, and it is preferred that the formation of the γδ heterodimer is maintained. In addition, if desired, a sequence encoding a selectable marker for purifying or labeling the product or construct can be added to the construct. Subsequently, the amplified pair of γ cDNA and δ cDNA is cloned, the sequence is verified, and transferred into an appropriate vector (e.g., a baculovirus vector containing a dual baculovirus promoter (e.g., pAcUW51, Pharmingen Corp., San Diego, Calif.)).

[0327] Subsequently, when the soluble γδ TCR DNA construct is co-transfected into an appropriate host cell (e.g., an appropriate insect host cell in the case of a baculovirus vector, or an appropriate mammalian host cell in the case of a mammalian expression vector), the recombinant receptor is expressed and secreted, for example, into the supernatant. Subsequently, the culture supernatant containing the soluble γδ TCR can be purified using various affinity columns (such as nickel nitrilotriacetic acid affinity column). The product can be concentrated and stored. It will be apparent to those skilled in the art that it is possible to generate soluble TCRs for use in the present disclosure using other methods and protocols, and such methods are explicitly contemplated for use herein.

[0328] Pharmaceutical composition

[0329] In a composition or method for administering an agonist or antagonist of BTN2A1 to a subject, the agonist or antagonist of BTN2A1 is suitably combined with a pharmaceutically acceptable carrier, as understood in the art. Thus, by way of example of the present disclosure, there is provided a composition (e.g., a pharmaceutical composition) comprising the agonist or antagonist of BTN2A1 of the present disclosure in combination with a pharmaceutically acceptable carrier.

[0330] Generally, the term "carrier" means a solid or liquid filler, binder, diluent, encapsulating material, emulsifying agent, wetting agent, solvent, suspending agent, coating, or lubricant that can be safely administered to any subject (e.g., a human). As described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), a variety of acceptable carriers can be used depending on the specific route of administration.

[0331] The invention of the agonist or antagonist of BTN2A1 is useful for parenteral administration, topical administration, oral administration, or local administration, aerosol administration, or transdermal administration for prevention or treatment. In one example, the agonist or antagonist of BTN2A1 is administered parenterally (subcutaneously or intravenously). For example, the agonist or antagonist of BTN2A1 is administered intravenously.

[0332] The formulation of the agonist or antagonist of BTN2A1 to be administered will vary depending on the route of administration and the formulation (e.g., solution, emulsion, capsule) selected. Suitable pharmaceutical compositions containing the agonist or antagonist of BTN2A1 to be administered can be prepared in a physiologically acceptable base. For solutions or emulsions, suitable bases include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, which contain physiological saline and a buffering medium. Parenteral vehicles can contain sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. A variety of suitable aqueous bases are known to those skilled in the art and include water, buffered water, buffered physiological saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles can contain various additives, preservatives, or fluids, nutrient or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The composition can optionally contain pharmaceutically acceptable auxiliary substances (such as pH regulators and buffers, and toxicity regulators, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate) to equilibrate physiological conditions as necessary. The agonist or antagonist of BTN2A1 can be stored in the liquid phase or lyophilized for storage and reconstituted in a suitable base prior to use according to known lyophilization and reconstitution techniques.

[0333] Functional measurement of γδ T cell immune response

[0334] The present disclosure also relates to agonists or antagonists of BTN2A1 that can activate or inhibit cell lysis function, cytokine production of one or more cytokines, and / or proliferation of γδ T cells. The number and function of T cells can be monitored by assays that detect T cells by activity (such as cytokine production, proliferation, or cytotoxicity). Such activity may correlate with clinical outcomes. For example, activation of cytotoxic activity may cause tumor targets or infected cells to lyse after treatment with an agonist or antagonist of BTN2A1. Activation and increased cytokine production may lead to induction of cell death of tumors or other targets by cytokines.

[0335] Activating the cytolytic function of γδ T cells means an increase in the cytotoxicity of γδ T cells, i.e., an increase in the specific lysis of target cells by γδ T cells. Inhibiting the cytolytic function of γδ T cells means a decrease in the cytotoxicity of γδ T cells, i.e., a decrease in the specific lysis of target cells by γδ T cells. The cytolytic function of γδ T cells can be measured, for example, by a direct cytotoxicity assay. A cytotoxicity assay typically involves mixing a sample containing T cells or PBMCs with a target loaded with 51 Cr or europium, and measuring the release of chromium or europium after lysis of the target cells. Surrogate targets, such as tumor cell lines, are often used. The target can be loaded with an antigen (such as pAg). The sample and the target are incubated in the presence or absence of an agonist or antagonist of BTN2A1. The lysis rate of the target after incubation for about 4 hours is calculated by comparing it with the maximum achievable lysis rate of the target. The cytotoxicity assay can be used to monitor the activity of passively delivered T cells and active immunotherapy approaches.

[0336] Activating or inhibiting cytokine production of one or more cytokines by γδ T cells means increasing or decreasing, respectively, the total cytokine production of one or more specific cytokines (e.g., IFN-γ, TNF-α, GM-CSF, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, or IL-17A) by γδ T cells. Cytokine secretion by T cells can be detected by measurement of bulk cytokine production (by ELISA), by bead-based assays (e.g., Luminex), or by counting individual cytokine-producing T cells (by ELISPOT assay).

[0337] In an ELISA assay, PBMC samples are incubated with or without added cells expressing BTN2A1, in the presence or absence of an agonist or antagonist of BTN2A1, for a defined period of time. After the defined time has elapsed, the supernatant is recovered from the culture and added to a microtiter plate coated with an antibody for the cytokine of interest. An antibody conjugated to a detectable label or reporter molecule is added, and the plate is washed and read. Typically, a single cytokine is measured in each well, but up to 15 cytokines can be measured in a single sample. The antibody to the cytokine of interest can be covalently attached to microspheres together with a uniform and distinguishable proportion of a fluorescent dye. Subsequently, a detection antibody conjugated to a fluorescent reporter dye is added, and flow cytometry is performed. By gating on the specific fluorescence indicating the specific cytokine of interest, it is possible to quantify the amount of cytokine proportional to the amount of fluorescence of the reporter.

[0338] In bead-based assays such as Luminex, the sample is typically added to a mixture of color-coded beads pre-coated with an analyte-specific capture antibody. The antibody binds to the analyte of interest. A biotinylated detection antibody specific for the analyte of interest is added to form an antibody-antigen sandwich. When streptavidin conjugated to a fluorescent molecule is added, it binds to the biotinylated detection antibody. The beads are read in a flow-based detection device. One laser classifies the beads and reveals the analyte being detected. When the signal magnitude from the fluorescent molecule is determined with a second laser, it is directly proportional to the amount of analyte bound.

[0339] The ELISPOT assay typically involves coating a 96-well microtiter plate with a purified cytokine-specific antibody; blocking the plate to prevent non-specific absorption of random proteins; incubating cytokine-secreting T cells with stimulator cells in the presence or absence of an agonist or antagonist of BTN2A1 at several different dilutions; lysing the cells with a detergent; adding a labeled secondary antibody; and detecting the antibody-cytokine complex. The end product of the final step is usually an enzyme / substrate reaction that produces a colored product, which can be quantified microscopically, visually, or electronically. Each spot represents a single cell secreting the cytokine of interest.

[0340] The cytokine production of one or more cytokines by γδ T cells can also be detected by multiparameter flow cytometry. Here, cytokine secretion is blocked in γδ T cells for 4 - 24 hours with brefeldin A or monensin (both are protein transport inhibitors that act on the Golgi apparatus in different ways, depending on the cytokine being investigated). After that, these cells are surface stained for markers of interest, then fixed and permeabilized, and then intracellularly stained with antibodies that are conjugated to fluorescent molecules and target the cytokines of interest. Thereafter, these cells can be analyzed by flow cytometry. It is possible to monitor the immune response in humans by characterizing the cytokine secretion patterns of T cells in peripheral blood, lymph nodes, or tissues by flow cytometry. This can be carried out in vitro without treatment with BFA or monensin.

[0341] Activating or inhibiting the proliferation of γδ T cells means increasing or decreasing the number of γδ T cells, respectively. Proliferation can be measured using a lymphocyte proliferation assay. A sample of purified T cells or PBMCs is mixed with stimulator cells at various dilutions in the presence or absence of an agonist or antagonist of BTN2A1. After 72 - 120 hours, 3 When [³H] thymidine is added, DNA synthesis (as one indicator of proliferation) can be quantified using a gamma counter, and the amount of radio-labeled thymidine incorporated into DNA can be measured. Using the proliferation assay, the γδ T cell response before and after administration of an agonist or antagonist of BTN2A1 can be compared.

[0342] The present disclosure also relates to agonists or antagonists of BTN2A1 that can activate or inhibit the activity and / or survival of cells expressing BTN2A1 (e.g., monocytes, macrophages, and / or dendritic cells). Activating or inhibiting the activity of cells expressing BTN2A1 means that an agonist or antagonist of BTN2A1 increases or decreases, respectively, the expression of costimulatory molecules (such as CD86, CD80, and HLA-DR) on the cell surface, and / or increases the pro-inflammatory response induced by Toll-like receptor (TLR) ligands in these cells, and / or changes the expression of immune checkpoint molecules (such as PD-L1, PD-L2). The activity and / or survival of cells expressing BTN2A1 can be measured by an antigen presentation assay. Briefly, CD14+ cells can be isolated from PBMCs and cultured in a medium containing GM-CSF and IL-4 for 5 days to produce MODCs. As previously described, an antibody-protein complex can be added to these, HLA-matched T cells capable of recognizing the protein added to the MODCs can be added, and subsequently, the presentation ability can be measured by performing ICS on these T cells.

[0343] Adaptation

[0344] The present disclosure also relates to agonists or antagonists of BTN2A1 that can be used to prevent, treat, delay the progression of, prevent the recurrence of, or alleviate the symptoms of a disease or condition. The agonists or antagonists of BTN2A1 can be administered directly to a subject in need thereof or used for ex vivo stimulation and adoptive transfer of cells (including γδ T cells).

[0345] One of ordinary skill in the art will recognize that whether the use of an agonist or antagonist of BTN2A1 is desired to enhance or suppress one or more γδ T cell immune responses depends on whether the population of γδ T cells targeted is immunosuppressive or immunostimulatory. In one embodiment, the function of γδ T cells is manipulated to promote, for example, cytotoxicity against tumor cells or infected cells, thereby promoting, for example, the anti-tumor activity or anti-pathogen activity of γδ T cells. In another embodiment, the function of γδ T cells is manipulated to promote the immunosuppressive activity and / or regulatory activity of γδ T cells during an immune response.

[0346] The symptoms of cancer can be prevented, treated, the progression delayed, recurrence prevented, or alleviated using an agonist or antagonist of BTN2A1.

[0347] The symptoms of an infectious disease can be prevented, treated, the progression delayed, recurrence prevented, or alleviated using an agonist or antagonist of BTN2A1.

[0348] An agonist or antagonist of BTN2A1 can be used as a vaccine adjuvant for treating cancer or an infectious disease.

[0349] An agonist or antagonist of BTN2A1 can also be used to prevent, treat, delay the progression of, prevent recurrence of, or alleviate the symptoms of an autoimmune disease.

[0350] A BTN2A1 antagonist can be used in combination with other immunosuppressive agents and chemotherapeutic agents, non-limiting examples of which are prednisone, azathioprine, cyclosporine, methotrexate, and cyclophosphamide.

[0351] Example 1: Materials and Methods

[0352] Human Samples

[0353] Human peripheral blood mononuclear cells (PBMCs) derived from the blood of healthy donors were obtained from the Australian Red Cross Blood Service under ethical approval 17-08VIC-16 or 16-12VIC-03 in addition to ethical approval from the University of Melbourne Human Ethics Sub-Committee (1035100) or the Olivia Newton John Cancer Research Institute (ONJCRI) Austin Health Human Research Ethics Committee (H2012-04446), and isolated through density gradient centrifugation (Ficoll-Paque PLUS GE Healthcare) and erythrocyte lysis (ACK buffer, homemade). The established cell lines were confirmed to be mycoplasma negative by a standard method using the MycoAlert test (Lonza), and cross-contamination was excluded by STR profiling.

[0354] Flow cytometry

[0355] Human cells were pelleted (400×g), washed, and incubated at 4°C with PBS / 2% fetal bovine serum (FBS) containing human Fc receptor block (Miltenyi Biotec). Mouse NIH-3T3 cells were incubated with anti-CD16 / CD32 (clone 2.4G2, self-made). Next, the cells were incubated with antibodies in addition to 7-aminoactinomycin D (7-AAD, Sigma) or LIVE / DEAD® viability marker (ThermoFisher) (Table 1). BTN2A1 and BTN3A were detected using self-made monoclonal antibodies (see below). Anti-BTN2A1 mAb or a matched isotype control (clone BM4, self-made) was conjugated to Alexa Fluor®-647 (Thermo Fisher) by amine coupling, and anti-BTN3A (clone 103.2) was conjugated to R-phycoerythrin (Prozyme) using a sulfo-SMCC heterobifunctional crosslinker. In some experiments, unlabeled anti-BTN2A1 mAb was detected using goat anti-mouse polyclonal secondary antibody PE (BD-Pharmingen), followed by a blocking step (5% normal mouse serum). The cells were tetramer Vγ9Vδ2 + γδTCR, BTN2A1, or mouse CD1d-α-GalCer ectodomain (self-made, see below), or an equivalent amount of streptavidin conjugate alone (BD) were also stained. Each reagent was titrated to determine the optimal dilution factor. All data were acquired on an LSRFortessa™ II (BD) and analyzed with FACSDiva and FlowJo (BD) software. All samples were gated using the parameters time, forward scatter area vs height, and viability dye, respectively, to exclude unstable events, doublets, and dead cells.

[0356]

Table 1-1

Table 1-2

[0357] Isolation and proliferation of γδ T cells

[0358] In some experiments, γδ T cells were enriched by MACS using anti-phycoerythrin-mediated magnetic bead purification after anti-γδTCR-PECy7, or using a γδ T cell isolation kit (Miltenyi Biotec). After enrichment, CD3 + Vδ2 + γδ T cells were further purified. Enriched γδ T cells were stimulated in vitro for 48 hours using plate-bound anti-CD3ε (OKT3, 10 μg / ml, Bio-X-Cell), soluble anti-CD28 (CD28.2, 1 μg / ml, BD Pharmingen), phytohemagglutinin (0.5 μg / ml, Sigma), and recombinant human IL-2 (100 U / ml, PeproTech), and then maintained for 14 - 21 days using IL-2. Cell culture was performed in a complete medium consisting of a 50:50 (v / v) mixture of RPMI-1640 and AIM-V (Invitrogen) supplemented with 10% (v / v) FCS (JRH Biosciences), penicillin (100 U / ml), streptomycin (100 μg / ml), Glutamax (2 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), and HEPES buffer (15 mM), pH 7.2 - 7.5 (all from Invitrogen Life Technologies) plus 50 μM 2-mercaptoethanol (Sigma-Aldrich).

[0359] Transfection

[0360] BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL3, and BTNL8 (all isoform 1) were cloned into the pMIG II mammalian expression vector (gift from D. Vignali (Addgene plasmid # 52107) (J. Holst et al. (2006))) and verified by Sanger sequencing. Mouse NIH-3T3 cells, hamster CHO-K1 cells, and human LM-MEL-62 cells were seeded the day before and transfected with FuGene HD® or Viafect™ in OptiMEM according to the manufacturer's instructions. After 48 h (72 h for LM-MEL-62 cells) to allow gene expression, cells were examined for GFP and gene expression and then used in phenotypic or functional assays.

[0361] Functional assay of γδ T cells

[0362] Fresh PBMC (2×10 6 cells) were cultured in 24-well plates ± zoledronate (4 μM, Sigma), and mAbs against BTN2A1, BTN3A1, or isotype control IgG1κ (MOPC-21, BioLegend) (10 μg / ml) were purified. After 24 h, CD3ε + γδTCR + Vδ2 + / -The activation of γδ T cells was evaluated by flow cytometry, and cytokine production was examined using cytometry beads according to the manufacturer's instructions (BD). For the assay of Figure 14, PBMCs were cultured in 24-well plates and blocked for 30 minutes with mAbs against BTN2A1, BTN3A1, or isotype control (10 μg / ml). The cells were then added to IL-2 (25 U / ml, Miltenyi) and the Golgiplug protein transport inhibitor (BD Biosciences), and stimulated for 18 hours using a combination of HMBPP (0.5 ng / ml, Sigma), zoledronate (4 μM, Sigma), and CEF (1 μg / ml, Miltenyi). After surface staining of the cells, they were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer's protocol, and then stained with anti-IFN-γ (Biolegend). For the co-culture assay, purified and in vitro-expanded γδ T cells (5×10 5 cells) were cultured in 96-well plates with APCs (3×10 5 cells) ± zoledronate (4 μM) for 24 hours, and the activation of γδ T cells was revealed by flow cytometry as described above. (In Figure 3C) Instead, 4×10 4 primary γδ T cells purified from PBMC donors using the γδ T cell magnetic bead isolation kit (Miltenyi) were cultured at a 2:1 ratio with LM-MEL-62 WT or BTN2A1 null1 APCs in the presence of 1 μM zoledronate for 2 days. The non-adherent T cells were then washed and added to the medium in a new plate and cultured for an additional 7 days without APCs in 100 U / ml IL-2. Subsequently, Vδ2 + γδ T cells were counted by flow cytometry.

[0363] FRET assay

[0364] To detect FRET between the extracellular domains of BTN2A1 and BTN3A1, cells were stained with PE-conjugated anti-BTN3A1 (donor) and Alexa 647-conjugated BTN2A1 (acceptor). FRET was detected in the compensated yellow 670 / 30 channel. CFP (mTurquoise2, donor) constructs and YFP (mVenus, acceptor) constructs containing either long (used for BTN3A1 and BTNL3) or short (used for BTN2A1 and BTNL8) flexible N-terminal linkers (Figure 19B) were synthesized (ThermoFisher) and cloned between the in-frame MfeI site introduced by site-directed mutagenesis in the C-terminus of the butyrophilin construct and the 3′ SalI site that also removed the pMIG IRES-GFP motif. CFP was detected in the violet 450 / 50 channel, YFP was detected using yellow 585 / 15, and FRET was detected using the violet 530 / 30 channel where bleed-through of CFP and YFP had been removed by compensation. FRET + The frequency of cells identified as such was gated for CFP for double transfectants + YFP + in NIH-3T3 cells for CFP for single transfectants + NIH-3T3 cells or YFP + in NIH-3T3 cells and examined.

[0365] Tumor survival assay

[0366] Tumors (10 4 ) cells were seeded into RF-10 in 96-well plates. The next day, 2 × 10 4 γδ T cells were added along with 100 U / ml of IL-2 (Miltenyi) ± 1 μM of zoledronate (Sigma). After 1 or 3 days of incubation, the survival rate was evaluated at each time point by the MTS assay, the absorbance was measured at 490 nm using a SpectroStar Nano plate reader (BMG Labtech), corrected for background, and normalized to wells containing only APC.

[0367] Single-cell γδ TCR sequencing

[0368] CD3ε derived from healthy PBMC donors + γδ TCR + Vδ2 + γδ T cells were sorted individually. Next, γδ TCR was amplified using the primers listed in Supplementary Table 2. Next, the PCR amplicons were cloned into pHL-sec containing the ectodomain of the γ- or δ-chain for expression (Figure 8C).

[0369] Genome-wide CRISPR / Cas9 knockdown screen

[0370] The CRISPR / Cas9 knockdown screen was performed essentially as described by J. Young et al. (2017). Briefly, Endura (trademark) ElectroCompetent cells (Lucigen) were transformed with a pooled lentiviral human gRNA knockout library (GeCKOv2, a gift from Feng Zhang, Addgene #1000000048) containing n = 6 gRNAs per gene at a coverage of >500× and grown at 37 °C for 16 h in 1 L of liquid Luria Broth culture. Plasmid DNA was purified (PureLink (trademark) gigaprep, ThermoFisher), and the amount of gRNA in the library before and after amplification was determined by sequencing the PCR-amplified library (Illumina HiSeq, 60×10 per sample 6Verification by back-lead showed that the dropout of gRNA was less than 0.2%. In addition to the gRNA library DNA, lentiviral particles were generated by transiently transfecting HEK-293T cells with the packaging plasmid using FuGENE® (Promega). The culture supernatant was titrated against LM-MEL-62 cells using puromycin (1 μg / ml, ThermoFisher) to determine the virus titer. Four biological replicates of LM-MEL-62 cells (2×10 8 each) were transduced with the lentiviral library at a multiplicity of infection of approximately 0.3. Next, the transformed cells were selected with puromycin for an additional 5 days, after which Vγ9Vδ2 + γδTCR tetramer #6 low cells were sorted from half of each replicate (approximately 6×10 7 each), and the other half was used as an unsorted control. The sorted cells were regrown for approximately 2 weeks and then sorted again. This was repeated two more times to enrich the LM-MEL-62 cells for a distinct Vγ9Vδ2 + γδTCR tetramer #6 low population (Figure 9A). Genomic DNA was then extracted as previously described by S. Chen et al. (2015), including an additional phenol-chloroform purification step. Approximately 6×10 7 unsorted cells and approximately 3×10 7gRNAs from individual sorted cells were amplified from genomic DNA using PCR (33 cycles) with a Pfu-based DNA polymerase (Herculase II Fusion, Agilent Technologies) and a one-step primer (IDT Ultramer oligos) containing the previously reported (J. Young et al (2017)) index and adapter sequences. Amplicons were gel extracted after electrophoresis (Wizard® SV Gel Clean-Up System, Promega), quantified with PicoGreen® (ThermoFisher), and sequenced using NovaSeq (Illumina). Cutadapt (M. Martin et al (2011)) was utilized to demultiplex the sample data using a combination of forward primer stagger motif and reverse octamer barcode, and analyzed using the EdgeR software package (M.D. Robinson et al. (2010)) in R studio. The processAmplicons function was used to count the guides. At that time, single base pair mismatches, or shifted guide positions were allowed. Guides with less than 0.5 counts / 10 in at least 5 samples were excluded from the analysis. After variance estimation, the difference in gRNA expression between unsorted and sorted samples was determined using the exactTest function, and a false discovery rate (FDR) of less than 0.05 was considered statistically significant. 6 After variance estimation, the difference in gRNA expression between unsorted and sorted samples was determined using the exactTest function, and a false discovery rate (FDR) of less than 0.05 was considered statistically significant.

[0371] Production of soluble proteins

[0372] The pHL-sec vector DNA (A.R. Aricescu et al. (2006)) encoding a construct with C-terminal biotin ligase (AviTag™) and His6 tag was transiently transfected into mammalian Expi293F cells or GNTI-deficient HEK-293S cells using ExpiFectamine or PEI, respectively, to express soluble human γδTCR, butyrophilin 2A1, and mouse CD1d ectodomain. MR1-5-OP-RU tetramers were generated as previously described (H.F. Koay et al. (2019)). Proteins were purified from the culture supernatant using immobilized metal affinity chromatography (IMAC) and gel filtration and enzymatically biotinylated using BirA (self-made). Proteins were re-purified by size exclusion chromatography and stored at -80 °C. Biotinylated proteins were tetramerized with streptavidin-PE (BD) at a 4:1 molar ratio. A DNA construct encoding the C-terminal His6-tagged butyrophilin B30.2 intracellular domain was newly synthesized (ThermoFisher) and cloned into the pET-30 bacterial expression vector. BL21 DE3(pLysS) Escherichia coli was used and expressed overnight at 30 °C after induction with IPTG (1 mM). Cell pellets were washed and lysed in PBS / 1 mM DTT using a sonicator, and the B30.2 protein was purified from the clear lysate using IMAC and gel filtration.

[0373] Generation of anti-BTN2A1 monoclonal antibody

[0374] Using a human antibody phage display library, antibody clones specific for BTN2A1 were screened. The screening consisted of three rounds of selection for binding to 50 nM of recombinant soluble C-terminal His-tagged BTN2A1 ectodomain immobilized on paramagnetic beads (Dynal) coated with streptavidin, with non-specific binding substances pre-adsorbed on the surface of a His-tagged protein, which was also immobilized on beads coated with streptavidin and served as an irrelevant control. After thorough washing, the bound phages were eluted and amplified overnight by infecting exponentially growing bacterial cultures (TG1; Stratagene). The purified phages were then used in subsequent rounds of panning. After three rounds, the bound phages were eluted, 190 clones were randomly picked, and their binding to BTN2A1 immobilized on microplates was examined by ELISA. Sequencing of the positive clones revealed a total of 52 individual antibody clones, 45 of which were subcloned into mammalian expression vectors for expression in Expi293F™ cells (ThermoFisher) and purified as full-length IgG molecules containing the human IgG4 Fab region and the mouse IgG2a Fc region on MabSelect SuRe resin (GE Lifesciences). The isotype control clone BM4 contained the same Fc region except for the mouse Fab region with an irrelevant specificity.

[0375]

Table 2-1

Table 2-2

[0376] Generation of anti-BTN3A antibodies

[0377] A DNA construct encoding the anti-BTN3A antibody variable domains (clone 20.1 and 103.2; described in Palakodeti et al. (2012)) was synthesized (ThermoFisher) and cloned into a mammalian expression vector containing the murine IGHV signal peptide and IgG1 constant region. The antibody was expressed in Expi293F™ cells as described above, purified using protein G column chromatography (GE), and then placed in PBS by buffer exchange.

[0378] Enzyme-linked immunosorbent assay

[0379] Purified recombinant protein (0.2 - 20 μg / ml) was immobilized overnight at 4°C in PBS buffer in the wells of a microplate. Nonspecific binding was blocked by incubating in PBS containing 5% skim milk powder or 0.5% (w / v) bovine serum albumin (BSA) in addition to 0.05% Tween 20. The wells were then incubated for 60 minutes at room temperature in PBS / 0.05% Tween-20 / 2% skim milk powder or 0.5% BSA in the presence of 2 - 5 μg / mL of the antibody, and then washed in PBS / 0.05% Tween-20. The plate was then incubated with a goat anti-mouse IgG secondary antibody labeled with HRP (Chemicon) or a donkey anti-mouse IgG secondary antibody (Millipore), detected using 3,3',5,5'-tetramethylbenzidine substrate (Sigma), and the absorbance was measured at 450 nm using a plate reader.

[0380] Generation of CRISPR / Cas9-mediated knockdown cell lines

[0381] For the BTN2A1 knockout line, two gRNAs (BTN2A1 null : 5'-TCACAAAGGTGGTTCTTCCT-3' (SEQ ID NO: 55) and BTN2A1 null2: 5'-CAATAGATGCATACGGCAAT-3' (SEQ ID NO: 57) was cloned into the GeneArt® CRISPR Nuclease Vector Kit (Life Technologies) according to the manufacturer's protocol, and the sequence was verified by Sanger sequencing. Cells were transfected using Lipofectamine 2000 and sorted based on orange fluorescent protein expression 48 hours later. Cells were cultured, stained with anti-BTN2A1 (clone Hu34C), and the negative fraction was sorted. For the BTN3A1 knockout system, the BTN3A1 CRISPR / Cas9 KO Plasmid Kit (Santa Cruz Biotechnology) containing three specific gRNA sequences (5'-GGCACTTACGAGATGCATAC-3' (SEQ ID NO: 59), 5'-GAGAGACATTCAGCCTATAA-3' (SEQ ID NO: 60), 5'-ACCATCAGAAGTTCCCTCCT-3' (SEQ ID NO: 61)) was used. Cells were transfected using Lipofectamine 3000 (ThermoFisher) and sorted based on green fluorescent protein 48 hours later. The sorted cells were cultured, stained with anti-BTN3A1 (clone 103.2), and the negative fraction was sorted and cultured.

[0382] Jurkat assay

[0383] APC of LM-MEL-62 or LM-MEL-75 was seeded at 2.5×10 4 cells / well in a 96-well plate and incubated overnight. Then, 2×10 4 J.RT3-T3.5 (ATCC® TIB-153™) (Jurkat) cells expressing the G115 mutant γδ TCR ± zoledronate, HMBPP, or IPP were added over 20 hours. The expression of CD69 was measured by flow cytometry on the surface of GFP + Jurkat cells. Nineteen single-residue alanine (Ala) mutants, each with Vγ9Vδ2 +(which is in the Vγ9 domain or Vδ2 domain of the G115 TCR) was generated by site-directed mutagenesis using the primers listed in Table 2. After phosphorylating the primers (IDT) (PNK, NEB), WT G115 in pMIG was used as a template, and 25 cycles of PCR were performed using KAPA HiFi master mix (KAPA Biosystems). The PCR products were digested with DpnI (NEB) and ligated using T4 DNA ligase (NEB). Subsequently, the sequence of the construct was verified by Sanger sequencing and then transfected. To examine the ability of the G115 TCR mutants to bind to the BTN2A1 tetramer, HEK-293T cells were transfected in a 1:3 ratio with a pMIG construct encoding human CD3γδεζ linked with 2A, in addition to each of the corresponding WT δ-chain or γ-chain for each individual γ-chain or δ-chain mutant, using FuGENE® HD (Promega) in OptiMEM™ (Gibco, Thermo-Fisher). 48 hours after transfection, the HEK293T cells were resuspended by pipetting and stained to detect CD3ε expression and binding of the PE-labeled BTN2A1 tetramer or the control, streptavidin conjugated with PE. The median fluorescence intensity (MFI) of the BTN2A1 tetramer that interacts with the mutant G115 TCR was determined by flow cytometry for gated CD3 + GFP + examined for HEK293T cells.

[0384] The ability of the G115 variant to respond to pAg stimulation was examined by transducing the G115 variant TCR into J.RT3-T3.5 Jurkat cells. HEK-293T cells were mixed with the corresponding wild-type δ-chain or γ-chain, respectively, in addition to the variant of each specific γ-chain or δ-chain, human CD3, pVSV(-G), and pEQ-Pam3(-E) in a ratio of 1:3 and transfected using FuGENE® HD in OptiMEM™. After 24 hours, the viral supernatant was collected, filtered through a 0.45 μm CA syringe filter, and then incubated with JRT3-T3.5 JurakT cells for 12 hours. This process was repeated twice a day for 4 days. CD3 + GFP + Jurkat cells were purified by FACS (BD FACSAria™ III), and their ability to respond to pAg presented by wild-type LM-MEL-75 APCs was examined as described above.

[0385] To measure the reactivity of Jurkat cells expressing the G115 γδTCR against the anti-BTN3A1 (clone 20.1) mAb, 2.5×10 4 LM-MEL-75 APC cells were pre-incubated with functional grade 20.1 (10 μg / ml, Biolegend) or a matched isotype control for 30 minutes at room temperature and then seeded in a flat-bottom 96-well plate. When the APCs adhered, 2.5×10 4 Jurkat cells were added to bring the final concentration of the antibody to 5 μg / ml. After 24 hours of co-culture, CD3 + GFP + The level of CD69 on the surface of Jurkat cells was revealed by flow cytometry.

[0386] Surface plasmon resonance

[0387] The SPR experiment was performed at 25 °C using a Proteon XPR36 instrument (Bio-Rad) with 10 mM HEPES-HCl (pH 7.4), 300 mM NaCl, and 0.005% Tween-20 buffer. The γδ TCR ectodomain was immobilized directly onto the surface of a streptavidin pre-immobilized Biacore sensor chip SA up to 260 resonance units (RU). Soluble butyrophilin was serially diluted (200–3.1 μM) and simultaneously injected at a rate of 30 μl / min onto the test and reference surfaces. After subtracting data from the reference flow cell (streptavidin only) injection and blank injection, the interaction was analyzed using Biacore T200 evaluation software (GE Healthcare) and Prism version 8 (GraphPad), and the equilibrium dissociation constant (K D ) was derived at equilibrium.

[0388] Isothermal titration calorimetry

[0389] The ITC experiment was performed at 25 °C using a MicroCal ITC200 instrument (GE Healthcare). The B30.2 domain of BTN2A1 or BTN3A1 was buffer-exchanged into PBS and adjusted to a final concentration of 100 μM. HMBPP (Cayman Chemical) or IPP was adjusted to a final concentration of 2 mM, and after injecting the first 0.4 μl (excluded from the analysis) into the cell, subsequent injections were made in 2 μl increments. The data were analyzed using Microcal Origin software.

[0390] Confocal microscopy

[0391] LM-MEL-75 WT cells, BTN2A1 null cells, BTN3A1 nullCells were cultured overnight in RPMI-1640 (Thermo-Fisher) supplemented with 10% (v / v) FCS (JRH Biosciences), penicillin (100 U / ml), streptomycin (100 μg / ml), Glutamax (2 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), and HEPES buffer (15 mM), pH 7.2 - 7.5 (all from Invitrogen Life Technologies) with the addition of 50 μM 2-mercaptoethanol (Sigma-Aldrich), and then attached to chamber well slides (Lab-Tek, Thermo-Fisher). The next day, the cells were washed and incubated for 20 minutes with human Fc receptor block (Miltenyi Biotec) diluted in OptiMEM™ (Thermo-Fisher) on ice. The cells were washed and stained for 20 minutes with anti-BTN2A1-AF647 (clone 259), anti-BTN3A-PE (clone 103.2), and anti-pan HLA class I-AF488 (clone W6 / 32, BioLegend) diluted in OptiMEM™ on ice. The cells were fixed for 20 minutes with PBS containing 1% paraformaldehyde (Electron Microscopy Sciences), then mounted with ProLong Gold AntiFade (Thermo-Fisher) and covered with a #1 coverslip (Menzel-Glaser) overnight. Each reagent was titrated to determine the optimal dilution factor. Single tile images of a Z-stack with a voxel size of 76.9 nm in the lateral direction and 400 nm in the axial direction and a voxel density of 1024×1024 were acquired with an LSM780 laser scanning confocal microscope with an inverted 20× (0.8 NA) objective lens and Zen software (Zeiss). The fluorescent dyes were excited with 488-nm, 561-nm, and 633-nm laser beams. The images were deconvolved with Huygens Professional (Scientific Volume Imaging) and analyzed with Imaris (Oxford Instruments) software.An interesting region defining the range of the photographed cells was defined based on the bright-field channel, and the Pearson's correlation coefficient of the voxels was calculated using the Imaris Coloc module. The intensity threshold was set for each channel analyzed based on the negative control for each staining.

[0392] BTN2A1 is Vγ9 + a ligand for γδ TCR

[0393] The inventors generated soluble Vγ9Vδ2 + TCR tetramers derived from pAg-reactive γδ T cells to identify candidate ligands for γδ TCR, and used them to stain a diverse group of human cell lines. It was then revealed that several lines including HEK-293T were clearly stained, while other lines including the B cell line C1R were not stained (Figure 1A). In particular, the melanoma cell line LM-MEL-62 was strongly stained (A. Behran et al. (2013)) (Figure 1A). Using a genome-wide knockdown screen (Figure 9), the most important guide RNA (gRNA) responsible for the reactivity of the Vγ9Vδ2 + TCR-tetramer was BTN2A1, which was enriched more than 13-fold compared to the control (Figures 1A and 9). BTN2A1 is a member of the butyrophilin family that has been poorly characterized, found in humans but not in mice. Similar to BTN3A1, it consists of two extracellular domains (IgV and IgC) and one intracellular B30.2 domain. With the exception of one study (G. Malcherek et al (2007)) suggesting that BTN2A1 may interact with the C-type lectin receptor CD209 (DC-SIGN) in a glycosylation-dependent manner, it is generally regarded as an orphan receptor. To further investigate the significance of this finding, the inventors generated two independent LM-MEL-62 BTN2A1 mutant lines (BTN2A1 + and BTN2A1 null1 and BTN2A1 null2 ) in which Vγ9Vδ2 +Loss of reactivity to TCR tetramers was confirmed and was similar in different LM-MEL-75 BTN2A1 mutant cell lines (Figure 1C and Figure 10). This was independent of BTN3A1 expression and essentially unchanged between the parental LM-MEL-62 line and the BTN2A1 null lines (Figure 1C and Figure 10A). In addition, the reactivity of the BTN3A1 null lines to Vγ9Vδ2 TCR tetramers was equivalent to that of the parental line (Figure 10B). Reintroduction of BTN2A1 into LM-MEL-62 BTN2A1 null1 cells or BTN2A1 null2 cells restored reactivity to Vγ9Vδ2 TCR tetramers, whereas transfection of BTN3A1 had no effect (Figure 1D). Thus, expression of BTN2A1 is essential for reactivity to Vγ9Vδ2 + TCR tetramers.

[0394] The inventors next generated a panel of BTN2A1-reactive mAbs. These showed various degrees of cross-reactivity to BTN2A2 (87% ectodomain homology), but no cross-reactivity to BTN3A2 (45% ectodomain homology) (Figure 11A-C). These mAbs stained parental LM-MEL-62 but could not bind to the LM-MEL-62 BTN2A1 null lines, thus confirming the reactivity of these mAbs to BTN2A1 (Figure 11D-E). The majority of anti-BTN2A1 clones blocked or partially blocked Vγ9Vδ2 TCR tetramer staining on the surface of LM-MEL-62 cells, LM-MEL-75 cells, and 293T cells (Figure 1E). This suggests that BTN2A1 is a ligand for Vγ9Vδ2 + γδ TCR.

[0395] To examine whether BTN2A1 selectively binds to Vγ9Vδ2 + γδ T cells, the inventors generated fluorescent BTN2A1 ectodomain tetramers (Figure 12). These were CD3 in PBMC +A subset of T cells was stained, while other types of cells were not stained (Figure 2A). The BTN2A1 tetramer + cells were γδTCR + but not αβTCR + (Figure 2A). The BTN2A1 tetramer labeled almost all Vγ9 + Vδ2 + γδ T cells and Vγ9 + Vδ1 + γδ T cells, but not Vγ9 - Vδ1 + γδ T cells, suggesting that the Vγ9 domain of the TCR γ-chain is involved in reactivity (Figure 2B). Furthermore, Förster resonance energy transfer (FRET) (P. Batard et al (2002)) between the fluorescent BTN2A1 tetramer and anti-CD3ε mAb indicated that the BTN2A1 tetramer was bound within approximately 10 nm of the surface of the γδTCR (Figure 2C). To directly assess whether BTN2A1 binds to the Vγ9 + γδTCR, the inventors performed surface plasmon resonance (SPR) to measure the interaction between soluble BTN2A1 and the γδTCR ectodomain. Soluble BTN2A1 TCR #6 (Vγ9Vδ2 + ) had an affinity of K D = 40 μM, similar to that observed in classical αβ T cells (M.E. Birnbaum et al (2014)). It also bound to a "hybrid" γδTCR that co-expressed the TCR #6 γ-chain paired with an irrelevant Vδ1 + γ-chain with an equivalent affinity (50 μM). However, BTN2A1 did not bind to Vγ5 + paired with the Vδ1 +did not bind to γδ TCRs containing the γ-chain (Figure 2D). Finally, the inventors examined whether other members of the butyrophilin family could bind to Vγ9Vδ2 TCRs. BTN2A2 showed only very weak binding, and cells transfected with BTN3A1+BTN3A2 and BTNL3+BTNL8 did not bind to Vγ9Vδ2 TCR tetramers (Figure 13). Thus, BTN2A1 is a ligand for Vγ9 + γδ TCRs.

[0396] BTN2A1 is important for γδ T cell responses to pAg

[0397] The inventors next examined whether BTN2A1 is important in pAg-mediated γδ T cell responses. As expected, culturing PBMC with the aminobisphosphonate compound zoledronate (A.J. Roelofs et al. (2009)), which induces the accumulation of pAg IPP, led to CD25 induction and downregulation of surface CD3 (Figure 3A) and production of IFN-γ and TNF (Figure 3B) in Vδ2 + γδ T cells rather than Vδ1 + γδ T cells. These indicators of TCR-dependent activation were significantly inhibited by the anti-BTN2A1 mAb clone Hu34 compared to samples treated with an isotype control mAb, and the degree of inhibition by clones 259 and 267 was less. Next, purified and pre-expanded Vγ9Vδ2 + T cells were cultured with parental LM-MEL-62 cells as APCs or BTN2A1 null LM-MEL-62 cells. A robust Vδ2 + T cell response to zoledronate with respect to upregulation of CD25 and downregulation of CD3 was observed in the presence of parental LM-MEL-62 APCs. However, neither BTN2A1 null1 nor BTN2A1 null2 APCs could promote γδ T cell activation beyond that of the APC-free control cultures (Figure 3C). Similarly, the proliferation of Vδ2+ γδ cells was not affected by BTN2A1 null1Decreased when using APC (Figure 3D). The inventors observed that the killing of parental LM-MEL-62 tumor cells mediated by γδ T cells occurs in a zoledronate-dependent manner, but does not occur in BTN2A1 null1 cells, suggesting that BTN2A1 is important for the cytotoxicity of tumor-targeting Vγ9Vδ2 + T cells (Figure 3D). These data indicate that BTN2A1 is important for the γδ T cell response to the endogenous form of pAg.

[0398] Vγ9Vδ2 + γδ T cells can self-present the high-affinity foreign form of pAg (such as microbial HMBPP) in the absence of APC (C.T. Morita et al. (1995)). BTN2A1 was also essential in this setting. This is because purified and pre-proliferated Vδ2 + T cells were unable to upregulate CD25 and produce IFN-γ in the presence of neutralizing anti-BTN2A1 mAbs (clones Hu34C, 227, 236, and 266) (Figure 3E). Clone 267 was only a partial inhibitor of HMBPP-induced activation (Figure 3E). Importantly, these mAbs did not inhibit activation mediated by anti-CD3 and anti-CD28 (Figure 3E), nor did they block the activation of primary CD8 + αβ T cells mediated by a mixture of viral peptides (「CEF」 peptides) derived from cytomegalovirus, Epstein-Barr virus, and influenza epitopes (Figure 14). Therefore, these BTN2A1 mAbs are specific antagonists of both the self and foreign forms of T cell immunity driven by pAg. Collectively, BTN2A1 plays an important role in pAg-mediated cytokine production, activation, proliferation, and tumor cytotoxicity by human Vγ9Vδ2 + γδ T cells.

[0399] BTN2A1 collaborates with BTN3A1 to induce the pAg response by γδ T cells

[0400] We next addressed whether BTN2A1-dependent pAg responses are specifically mediated through γδ TCR signaling. + J.RT3-T3.5 (Jurkat) T cells expressing the TCR clonotype (TJ Allison et al. (2001)) upregulated CD69 in response to zoledronate after culture with parental LM-MEL-75 or parental LM-MEL-62 APCs; however, BTN2A1 null and BTN3A1 null APCs from 100% were barely able to induce pAg reactivity (Figure 4A). Non-transduced (parental) Jurkat cells or Jurkat cells expressing an irrelevant γδ TCR (clone 9C2; AP Uldrich et al. (2013)) also failed to respond to pAg. Similar results were obtained using HMBPP and IPP (Figures S15A-C), which suggested that Vγ9Vδ2 + We show that both BTN2A1 and BTN3A1 are required to specifically mediate pAg responses in a γδ TCR-dependent manner.

[0401] BTN3A1 is essential for pAg-mediated responses, but forced overexpression of BTN3A1 cannot confer pAg-driven γδ T cell-stimulating ability to hamster and mouse APCs, indicating that other factors are required (A. Sandstrom et al. (2014); F. Riano et al. (2014)). The inventors found that both hamster and mouse APCs transfected with BTN2A1 and BTN3A1 in combination, rather than alone, are capable of activating γδ T cells in a pAg-dependent manner (Figures 4B and 16A - B). Another butyrophilin molecule, BTN3A2, was not required for this response, but when combined with BTN2A1 and BTN3A1, it moderately enhanced γδ T cell activation, consistent with its potential role in increasing BTN3A1 activity (P. Vantourout et al. (2018)). A modified BTN2A1 construct (referred to as BTN2A1ΔB30) with unrelated transmembrane and intracellular domains derived from mouse paired immunoglobulin-like type 2 receptor beta was also examined. This still expressed on the cell surface and bound to the Vγ9Vδ2 + TCR tetramer (Figure 16C), but did not confer pAg-presenting ability (Figure 4C). Thus, the intracellular or transmembrane domain of BTN2A1, in addition to the role of the extracellular domain in binding to the Vγ9 + γδ TCR, may also be important for pAg-mediated activation of Vγ9Vδ2 + γδ T cells. This did not appear to be due to the intracellular B30.2 domain of BTN2A1 directly binding to purified pAg (HMBPP or IPP). Because, as expected, in contrast to the clear interaction between the BTN3A1 B30.2 domain and pAg (A. Sandstrom et al. (2014), S. Gu et al., (2017), M. Salim et al (2017)), no clear interaction between these molecules was detected using isothermal titration calorimetry (Figure 17).

[0402] Finally, the inventors examined whether BTN2A1 and BTN3A1 induced pAg-mediated activation when expressed on the surface of the same cell (in cis) or on the surface of separate cells (in trans). BTN3A1 + APC and BTN3A1 + BTN3A2 + BTN2A1 mixed with either of the APCs + The APCs were unable to induce a γδ T cell response to pAg (Figure 4D), suggesting that these molecules must be expressed on the surface of the same APC to mediate the activation of γδ T cells induced by pAg.

[0403] BTN2A1 associates with BTN3A molecules on the cell surface

[0404] The requirement for co-expression of BTN2A1 and BTN3A1 in cis raised the possibility that they associate with each other. Parent LM-MEL-75 cells stained with anti-BTN2A1 mAb and anti-BTN3A1 / 3A2 / 3A3 (“BTN3A molecules”) mAb showed a staining pattern similar to that of BTN2A1 and BTN3A molecules on the cell surface (Figs. 5A–C). The Pearson correlation coefficient showed significant overlap between the staining of BTN2A1 and BTN3A molecules compared to overlap with any of the irrelevant controls (HLA-A, B, C). Thus, BTN2A1 and BTN3A molecules appear to associate on the surface of the plasma membrane (Fig. 5B). Furthermore, co-staining of LM-MEL-75 cells with anti-BTN2A1 (clone 259) and anti-BTN3A (clone 103.2) yielded a distinct FRET signal (Fig. 5C). This indicates co-localization on the cell surface (Fig. 5C). Co-staining with anti-BTN3A (clone 20.1) did not produce FRET, and similarly, several other anti-BTN2A1 clones (Hu34C and 267) resulted in only weak FRET. This may be because some combinations of mAbs produced donor and acceptor fluorophores that were spatially separated beyond the detection limit of FRET of 10 nm. Similar results were obtained in mouse NIH-3T3 fibroblasts transfected with different combinations of BTN molecules (Fig. 18). Interestingly, co-staining with anti-BTN2A1 and anti-BTN3A of BTN2A1ΔB30 + BTN3A1 + NIH-3T3 cells or BTN2A1ΔB30 + BTN3A2 + Staining of NIH-3T3 cells also produced distinct FRET. The latter finding suggests that the association between these molecules is independent of the B30.2 domain, as BTN3A2 also lacks the B30.2 domain (Fig. 18).

[0405] The inventors next determined whether the intracellular domains of BTN2A1 and BTN3A1 also associate, by generating butyrophilin constructs conjugated to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) (Figure 19). Co-transfection of mouse NIH-3T3 fibroblasts with BTN2A1CFP + BTN3A1 YFP or BTN2A1 YFP + BTN3A1 CFP yielded a distinct FRET signal similar to that of a positive control (butyrophilin-like molecule 3 (BTNL3) CFP + BTNL8 YFP ) known to associate. Similarly, co-transfection of BTN3A1 CFP + BTNL8 YFP or BTNL3 CFP + BTN2A1 YFP or single transfectant controls yielded little or no FRET (Figures 5D and 20A). The inventors also examined whether pAg altered the FRET signal between BTN2A1 and BTN3A1 and detected no significant change (Figures 20B and 20C); however, all antagonist-active anti-BTN2A1 mAb clones (from Figure 3D) disrupted their association (Figure 21). Thus, both the extracellular and intracellular domains of BTN2A1 and BTN3A1 are closely associated.

[0406] Vγ9Vδ2 + γδ TCRs recognize at least two ligands simultaneously

[0407] BTN2A1 binds to all Vγ9 + γδ TCRs, yet only Vγ9 + Vδ2 + T cells are pAg-responsive; thus, the inventors hypothesized that Vδ2 also participates in the interaction. One necessary consequence of this hypothesis is that Vγ9Vδ2 +Multiple binding domains are separately present on the γδ TCR, one responsible for binding to BTN2A1 is located within the region encoded by the germline of Vγ9, and another responsible for pAg reactivity may incorporate Vδ2 specificity. Mutations from Arg20, Glu70, and His85 (to a lesser extent Glu22) of Vγ9 to Ala all completely abolished reactivity to the BTN2A1 tetramer, whereas none of the Vδ2 mutations affected this (Figure 6A). The side chains of these Vγ9-sensitive residues are close to each other (the distance between Glu70-His85 is 2.8 Å; the distance between His85-Arg20 is 5.1 Å), and are located on the outer surfaces of the B, D, and E strands of the ABED anti-parallel β-sheet of Vγ9 respectively. Together, these form a polar triad within the framework region of Vγ9 (Figure 6B). This is consistent with BTN2A1 binding to the majority of Vγ9 + T cells (Figure 2B). Thus, BTN2A1 appears to bind to the side of Vγ9, typically distal to the δ-chain, rather than in the vicinity of the complementarity-determining region (CDR) loops typically associated with Ag recognition.

[0408] The inventors next examined which residues are important for mediating the functional response to pAg. Jurkat cells transfected with γδ TCR variants expressed similar levels of the CD3 / γδ TCR complex on their surface and responded similarly to immobilized anti-CD3 mAb (Figure 22), but mutations to each of the BTN2A1-binding triads of the γ-chain variants also abolished pAg-mediated activation of Jurkat cells (Figure 6B). However, mutations to two additional residues, namely Arg51 of the CDR2 loop encoded by Vδ2 and Lys108 of the CDR3 loop of the TCR γ-chain, also abolished pAg-mediated activation (Figure 6C and (H. Wang et al (2010)). These residues had no effect on BTN2A1 binding (Figure 6B) and were located on the opposite side of the TCR from the putative BTN2A1 footprint (a separation of approximately 30-40 Å). However, they are close to each other (approximately 11 Å) (Figure 6D), and thereby, rather than BTN2A1 binding, Vγ9Vδ2 +It may be another binding interface necessary for pAg-mediated activation by γδTCR. This second binding interface is (i) Vδ2 through the involvement of residues encoded by the germline + explains the importance of the TCR δ-chain and (ii) the importance of the invariant nature of the CDR3γ motif among pAg-reactive γδ T cells through the involvement of specific residues within this loop.

[0409] Finally, the inventors examined activation mediated by the agonist BTN3A1 mAb (clone 20.1). This activation is thought to mimic pAg-mediated signaling by conformational change or cross-linking of BTN3A1 (C. Harly et al. (2012)). Agonist BTN3A1 mAb-pulsed parental APCs activated Vγ9Vδ2 γδTCR + Jurkat cells (Figure 7), but this did not occur in BTN2A1 null APCs. This suggests that BTN2A1 is extremely important for BTN3A1-mediated γδ T cell activation. Furthermore, all Jurkat cells expressing TCR γ-chain Ala mutants of the BTN2A1-binding residues His85, Arg20, and Glu70 and mutants of Arg51 (δ-chain) and Lys108 (γ-chain) independent of BTN2A1 did not respond to parental APCs pulsed with the agonist anti-BTN3A1 mAb (Figure 7). Therefore, the interaction between BTN2A1 and Vγ9 + TCRγ-chain is essential but not sufficient for the γδ T cell response driven by BTN3A1. This fact may explain why, in initial studies, the agonist anti-BTN3A1 mAb was unable to induce γδ T cell activation in co-cultures with mouse-derived APCs transfected only with human BTN3A1 (A. Sandstorm et al. (2014)). Because mice do not express BTN2A1.

[0410] Therefore, the study of these mutants is related to Vγ9Vδ2 necessary for pAg-mediated activation and BTN3A1-mediated activation+ The existence of two distinct interaction sites on the γδ TCR was revealed. One site on the Vγ9 side is essential for both the binding and activation of BTN2A1, whereas the other site incorporates both the Vδ2 CDR2 and the γ-chain CDR3 loop and is required for pAg-mediated activation and BTN3A1-mediated activation. Thus, Vγ9Vδ2 + T cells are selectively activated by pAg through distinct dual-ligand interactions, whereby BTN2A1 binds to the Vγ9 domain and another ligand (potentially BTN3A1) appears to bind to another interface incorporating both the Vγ9 domain and the Vδ2 domain.

[0411] Conclusion

[0412] These findings support a model in which BTN2A1 and BTN3 associate at the cell surface and both are required for pAg-mediated γδ T cell activation. This model suggests that after pAg binds to BTN3 (e.g., BTN3A1) through its intracellular B30.2 domain, the BTN2A1 / BTN3 complex binds to the γδ TCR through two different binding sites, i.e., BTN2A1 binds to the Vγ9 framework region, whereas another ligand, presumably BTN3 (e.g., BTN3A1), binds to the CDR2 encoded by Vδ2 on the opposite side of the TCR and the CDR3 loop encoded by the γ-chain. This represents a different model of Ag sensing distinct from the canonical MHC-Ag complex recognition by αβ T cells.

[0413] In one previous study using short hairpin RNA (shRNA) knockdown, no obvious role of BTN2A1 in pAg presentation was found (S. Vavassori et al. (2013)). However, since the knockdown efficiency was only 81% and BTN2A1 protein was not measured, it is possible that the remaining BTN2A1 had the retained function. To date, BTN2A1 has been scarcely characterized, and there is only one initial study that identified the glycosylation-dependent receptor CD209 (G. Malcherek et al. (2007)). Since the inventors found that N-linked glycans are not required for the binding of BTN2A1 to the γδ TCR (Figure 24), it is unlikely that CD209 plays a role in this interaction. Little is known about the expression pattern of BTN2A1, but RNA analysis predicts broad expression on the surface of immune cells. The inventors confirmed that BTN2A1 is expressed on the surface of circulating T cells, B cells, NK cells, and monocytes, as well as on the surface of Vγ9Vδ2 + T cells (Figure 24). This may explain how pγδ T cells present pAg to themselves (C.T. Morita et al. (1995)).

[0414] Recent studies have shown that human BTNL3 and BTNL8 associate simultaneously to be stimulatory for human Vγ4 + γδ T cells, and it has been revealed that BTNL3 interacts with a region called the HV4 loop encoded by the germline of the γ-chain variable domain (R. Di Marco Barros et al. (2016); D. Melandri et al. (2018)). Similarly, mouse BTNL1 and BTNL6 are linked and are intestinal Vγ7 +It is important for the function of γδ T cells and appears to bind to a similar region of γδ TCR (R. Di Marco Barros et al. (2016); D. Melandri et al. (2018)). In contrast, the BTN2A1-Vγ9 binding interface appears to rely more on the outer surface of the ABED β-sheet of the Vγ9 TCR than on the HV4 loop. This indicates that the BTN2A1 binding footprint on Vγ9 may be far from the CDR loops and closer to the Cγ domain. Since butyrophilin molecules tend to dimerize (e.g., BTN3A1 can form stable Vkoji-shaped homodimers and also form heterodimers with BTN3A2 (S. Gu et al. (2017)), and can form BTNL3-BTNL8 heterodimers (D. Melandri et al. (2018))), the association between BTN2A1 and BTN3 (e.g., BTN3A1) may show a direct interaction, but its molecular basis is still unclear.

[0415] Compared with other Ag-presenting molecules (MHC molecules and MHC-like molecules), the recognition of the heteromeric butyrophilin complex is a fundamentally different class of immune recognition. It is still unknown how pAg changes this complex to induce antigenicity, but it may involve remodeling of the butyrophilin dimer or multimer, and / or conformational changes to BTN2A1 and BTN3. Other related molecules such as ABCA1 (B. Castella et al. (2017)) may be directly required.

[0416] These findings indicate that BTN2A1 is a direct target for agonistic and / or antagonistic interventions in γδ T cell-mediated immunotherapy for infectious diseases, cancer, and autoimmunity.

[0417] Tumor killing / inhibition assay

[0418] In the following experiments, γδ T cells were enriched by MACS using anti-γδ TCR conjugated with PE-Cy7, followed by magnetic bead amplification mediated by anti-phycoerythrin (Miltenyi Biotec). After enrichment, CD3 + Vδ2 + γδ T cells were further purified by sorting using Aria III (BD). Enriched γδ T cells were stimulated in vitro for 48 hours with plate-bound anti-CD3ε (OKT3, 10 μg / ml, Bio-X-Cell), soluble anti-CD28 (CD28.2, 1 μg / ml, BD Pharmingen), phytohemagglutinin (0.5 μg / ml, Sigma), IL-15 (50 ng / ml), and recombinant human IL-2 (100 U / ml, PeproTech), and then maintained with IL-2 and IL-15 for 14 - 21 days. Cells were cultured in complete medium consisting of a 50:50 (v / v) mixture of RPMI-1640 and AIM-V (Invitrogen) supplemented with 10% (v / v) FCS (JRH Biosciences), penicillin (100 U / ml), streptomycin (100 μg / ml), Glutamax (2 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), and HEPES buffer (15 mM), pH 7.2 - 7.5 (all from Invitrogen Life Technologies) plus 50 μM 2-mercaptoethanol (Sigma-Aldrich).

[0419] LM-MEL-62 melanoma cells and LM-MEL-75 melanoma cells were seeded at 1 × 10 per well in RPMI1640 medium supplemented with 10% FBS in 96-well flat-bottom plates 4Cells were seeded at [quantity] and left overnight for adhesion. T25 gamma-delta T cells were added to TCR RPMI with 100 U / ml of IL-2 at an effector:target ratio of 2:1 and stimulated with 5 μM of zoledronate or 0.5 ng / ml of HMBPP, or left unstimulated. The agonistic antibodies 253, 259, or the isotype control BM4 were added to each well at 10 μg / ml. All conditions were repeated in triplicate. Cells were incubated at 37 °C and on day 3, Vd2+ cells were acquired by flow cytometry. Live cells were gated and activation was determined by analysis of CD25 expression. The viability of melanoma cells was determined by MTS assay. The MTS reagent was added to RPMI medium at a ratio of 1:5, 100 μl per well. Cells were incubated at 37 °C for 30 minutes and the plates were read at 490 nm using a Spectrostar Nano plate reader.

[0420] Measurement of gamma-delta T cell activation in the presence of agonistic antibodies

[0421] In some experiments, γδ T cells were enriched by MACS using anti-γδ TCR conjugated with PE-Cy7 followed by anti-phycoerythrin-mediated magnetic bead purification (Miltenyi Biotec). After enrichment, CD3+ Vδ2+ γδ T cells were further purified by sorting using Aria III (BD). The enriched γδ T cells were stimulated in vitro for 48 hours with plate-bound anti-CD3ε (OKT3, 10 μg / ml, Bio-X-Cell), soluble anti-CD28 (CD28.2, 1 μg / ml, BD Pharmingen), phytohemagglutinin (0.5 μg / ml, Sigma), IL-15 (50 ng / ml), and recombinant human IL-2 (100 U / ml, PeproTech), and then maintained for 14 - 21 days with IL-2 and IL-15. Cells were cultured in complete medium consisting of a 50:50 (v / v) mixture of RPMI-1640 and AIM-V (Invitrogen) supplemented with 10% (v / v) FCS (JRH Biosciences), penicillin (100 U / ml), streptomycin (100 μg / ml), Glutamax (2 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), and HEPES buffer (15 mM), pH 7.2 - 7.5 (all from Invitrogen Life Technologies) plus 50 μM 2-mercaptoethanol (Sigma-Aldrich).

[0422] Previously expanded in vitro CD3+ Vδ2+ γδ T cells (5×10 5 cells) were cultured for 24 hours with HMBPP (0.5 ng / ml) ± 10 μg / ml neutralizing anti-BTN2A1 mAb or isotype control. The expression of CD25 was revealed by flow cytometry and the concentration of IFN-γ was determined by cytometric bead assay (BD Bioscience) according to the manufacturer's instructions.

[0423] Identification of BTN2A1 agonistic antibodies

[0424] The inventors screened a panel of antibodies specific for BTN2A1 as described above and identified those that can act as agonists of BTN2A1. The inventors evaluated the ability of anti-BTN2A1 antibodies to activate γδ T cells. The inventors evaluated the upregulation of CD25 on the surface of previously expanded γδ T cells after culturing the cells overnight with 10 μg / ml of anti-BTN2A1 antibody or isotype control antibody (BM4). As shown in Figure 26A, all of antibodies 244, 253, and 259 were able to increase the percentage of γδ T cells expressing CD25.

[0425] The inventors further measured the levels of interferon γ secreted by γδ T cells after culturing overnight in the presence of 10 μg / ml of anti-BTN2A1 antibody or isotype control antibody (BM4). Secretion of interferon γ is another indicator of TCR-dependent activation. As shown in Figure 26B, all of antibodies 244, 253, and 259 were able to increase the levels of secreted interferon γ.

[0426] The inventors further examined, in co-culture experiments, the ability of anti-BTN2A1 antibodies to activate γδ T cells to kill cancer cells and / or to inhibit the growth of cancer cells. The cultures were performed using γδ T cells and melanoma cells (LM-MEL-75 or LM-MEL-62) at a ratio of 2:1. The cells were cultured for 3 days with antibody 253 or 259 or BM4 (isotype control) or zoledronate (positive control) or HMBPP (positive control). As shown in Figure 27, cells cultured in the presence of antibody 253 or 259 induced lysis of at least one of the melanoma cell lines at a level similar to that of the positive control. Figure 27B shows the activation level of δ T cells evaluated by γCD25 upregulation. In particular, Figure 27B shows that the level of expression is upregulated in γδ T cells cultured in the presence of antibody 253 or 259.

[0427] Activation of γδ T cells and induction of cell death in the absence of phosphoantigens

[0428] Materials and methods

[0429] Luminex (PBMC)

[0430] Blood from one healthy donor (Red Cross Australia) was ficoll-treated, the PBMC layer was recovered, washed, and 5×10 5 cells were treated in duplicate with 10 μg / ml of the indicated antibody in 500 μl of TCRPMI supplemented with 100 u / ml of IL-2 and incubated at 37°C and 5% CO2 for 16 hours. After the supernatant was recovered, it was submitted to Crux Biolabs (Scoresby, VIC, Australia) for Luminex Human 20-plex Inflammation panel analysis (EPX200-12185-901). In this analysis, the following analytes were measured: GM-CSF; ICAM-1, IFN-α; IFN-g, IL-1α, IL-1b, IL-10, IL-12p70, IL-13, IL-17A, IL-4, IL-8, IP-10, MCP-1, IL-6, MIP-1a, MIP-1b, sE-selectin, sP-selectin, TNF-α. All samples were run with appropriate controls and standards. Treatment with antibody 259 was performed in only one well.

[0431] Luminex (Vγ9Vδ2)

[0432] Briefly, Vγ9Vδ2 cells were isolated from PBMCs derived from one healthy donor (Red Cross Australia) and one cancer patient using the TCRγδ+ T Cell Isolation Kit (Miltenyi). Cells were stimulated in TCRPMI supplemented with 100 u / ml of IL-2, CD3 (10 μg / ml), and CD28 (1 μg / ml) for 48 hours. Cells were washed and then grown in TCRPMI supplemented with 100 u / ml of IL-2 at 37°C and 5% CO2 for 14 days before being frozen. The patient provided informed consent and the study was approved under HREC 14 / 425. Vγ9Vδ2 cells were thawed and left overnight at 37°C and 5% CO2 in TCRPMI supplemented with 50 u / ml of IL-2. The next day, 2×10 5 cells were treated with the indicated antibodies (in duplicate) at 10 μg / ml, or zoledronic acid (4 μM), or HMBPP (0.5 ng / ml) in 200 ul of TCRPMI supplemented with 100 u / ml of IL-2 and incubated at 37°C and 5% CO2 for 16 hours. After collecting the supernatant, it was submitted to Crux Biolabs (Scoresby, VIC, Australia) for Luminex Human 20-plex Inflammation panel analysis (EPX200-12185-901). All samples were run with appropriate controls and standards.

[0433] In vitro killing assay

[0434] Vγ9Vδ2 cells were isolated from PBMCs derived from one healthy donor (Red Cross Australia) or one cancer patient using the TCRγδ+ T Cell Isolation Kit (Miltenyi). The patient submitted an informed consent, and the study was approved under HREC 14 / 425. The cells were stimulated in TCRPMI supplemented with 100 u / ml of IL-2, CD3 (10 μg / ml), and CD28 (1 μg / ml) for 48 hours. The cells were washed and then grown in TCRPMI supplemented with 100 u / ml of IL-2 at 37°C and 5% CO2 for 14 days before being frozen. Vγ9Vδ2 cells were thawed and left overnight at 37°C and 5% CO2 in TCRPMI supplemented with 50 u / ml of IL-2.

[0435] In all assays, LM-MEL-62 melanoma cells were seeded at 10,000 cells / well in 100 μl of RF10 medium in 96-well flat-bottom plates and left overnight at 37°C and 5% CO2 to adhere.

[0436] E:T titration

[0437] The next day, Vγ9Vδ2 cells were washed and counted, and then added to the melanoma cells in TCRPMI supplemented with 100 u / ml of IL-2 and either 4 μM of zoledronate or 10 ug / ml of antibody 259 at an E:T ratio of 2:1, 1:1, 1:2, 1:4, 1:8, or 1:16.

[0438] Antibody titration

[0439] Vγ9Vδ2 cells were washed, counted, and seeded at 10,000 cells / well in TCRPMI supplemented with 100 u / ml of IL-2. Anti-BTN2A1 antibodies 253, 259, or BM4 isotype control were added in duplicate to the wells at dilutions of 10, 1, 0.1, and 0.01 ug / ml. The cells were incubated at 37 °C and 5% CO2. On day 3, the Vγ9Vδ2 cells were washed and 100 μl of MTS reagent was added to the wells for 1 hour according to the manufacturer's protocol (Promega, USA). The plates were then read at 490 nm using a Spectrostar Nano microplate reader (BMG Labtech), and the survival rate corrected for background absorbance was determined.

[0440] Flow cytometry

[0441] Vγ9Vδ2 cells were stained as described above to look for CD3, Vδ2, CD25, and viability discrimination dye, and analyzed with a Canto flow cytometer (BD). Cells were gated for lymphocytes, single cells, live cells, CD3+Vδ2+, and activation was revealed based on CD25 expression.

[0442] Discussion

[0443] Vγ9Vδ2 cells react by upregulating activation markers (including CD25 and CD69) and expressing cytokines when phosphoantigens are presented. For this, the expression of BTN2A1 and BTN3A1 on the surface of antigen-presenting cells is required. Anti-BTN2A1 antibodies 259 and 253 can mimic phosphoantigen-mediated activation to varying degrees in the absence of these intermediates of the mevalonate / non-mevalonate pathway (Figure 28A).

[0444] Functionally, this activation results in a dose-dependent increase in the ability of pre-expanded Vγ9Vδ2 T cells to recognize and kill melanoma tumor cells (referred to herein as LM-MEL-62). Vγ9Vδ2 cells derived from two different donors (melanoma patients) were pre-expanded and incubated with melanoma cells (at a 1:1 ratio) and treated with different amounts of antibody 253 or antibody 259. Similar to the activation data in Figure 28A, treatment with antibody 259 resulted in lower survival of LM-MEL-62 cells compared to antibody 253. Higher concentrations of antibody 259 enhanced tumor cell killing mediated by Vγ9Vδ2 cells, and maximum killing in both donors was achieved between 1 and 10 μg / ml (Figure 28B).

[0445] Tumor cell killing was dependent not only on the dose of the antibody but also on the ratio of effector (Vγ9Vδ2) cells to target (LM-MEL-62) cells (Figure 28C). Compared to treatment with zoledronic acid, antibody 259-mediated cell killing showed a correlation with E:T (more effector cells led to more killing), but killing occurred to a lesser extent in both donors. Interestingly, Vγ9Vδ2 cells derived from one cancer patient (patient 1) appeared to have less tumor cell killing ability than those from healthy donors, independent of the stimulation used. This suggests a (reversible) functional change of Vγ9Vδ2 cells in the cancer setting, which may extend beyond the tumor microenvironment (cells were isolated from the circulation).

[0446] These differences were, in part, due to the cytokine / chemokine profiles reflected in Vγ9Vδ2 cells grown in vitro and activated with zoledronate, HMBPP, or the different antibodies shown (Figs. 29A and B, Tables 3 and 4). In Vγ9Vδ2 cells derived from donors and patients, treatment with zoledronate and HMBPP increased the expression / secretion of GMCSF, ICAM-1, IFNγ, IL-13, MIP-1a, MIP-1b, sE-selectin, sP-selectin, and TNFα. All of these, except ICAM1, were more highly expressed when a stronger stimulus (HMBPP) was used, and the expression of ICAM-1 was upregulated to a similar extent by zoledronate and HMBPP. Secretion of IL-17A and IL-4 could only be detected in the context of HMBPP activation. Since IL-17 has immunosuppressive functions, this demonstrates that activation needs to be finely tuned to block signals in order to achieve the most desirable outcomes. Treatment with the two agonistic anti-BTN2A1 antibodies 253 and 259 showed a pattern similar to that of treatment with zoledronate and HMBPP, but 253 was a weaker stimulus.

[0447] [[Table 3]]

[0448] [[Table 4]]

[0449] In Vγ9Vδ2 cells derived from healthy donors, treatment with antibody 253 led to only a slight increase in the amount of GMCSF secreted, but this was not the case for the other cytokines / chemokines measured. In Vγ9Vδ2 cells derived from cancer patients, no increase was detected for any of the analytes, although the basal levels of many analytes were often higher than those in cells derived from healthy donors where no detectable expression was seen when treated with the isotype antibody only (BM4).

[0450] With antibody 259, a substantial increase in numerous analytes was observed in both donors, including GMCSF, IFNγ, IL-13, IL-17 (very low), MIP1α and MIP1β, sE- and sP-selectin, and TNFα. ICAM1 increased only in cells derived from healthy donors upon treatment with antibody 259, and in cancer-derived cells, de novo expression of IL-4 exceeded 400 pg / ml. In healthy donors, IL-4 was not detected when treated with antibody 259.

[0451] The inventors further used a BTN2A1 antagonist antibody to explore which cytokines with baseline expression in pre-expanded Vγ9Vδ2 cells could be blocked by inhibition of BTN2A1 gamma-delta TCR binding / activation. In healthy donor cells, treatment with 34C1 downregulated GMCSF as well as MIP1α and b compared to the isotype (BM4) treated control. In cancer patient-derived cells with much higher baseline cytokine expression, a decrease in levels could be detected for GMCSF, ICAM1 (to undetectable levels), IFNγ, IL-13, MIP1α, and MIP1β, as well as sE-selectin, suggesting that BTN2A1 blockade could be a potentially effective therapeutic strategy for reducing these factors.

[0452] To examine how our agonistic anti-BTN2A1 antibody 259 affects cytokine / chemokine expression in the context of PBMC, the inventors treated freshly isolated PBMC from one healthy donor with antibody 259 and antibody 229 (antagonistic anti-BTN2A1 antibody), as well as an isotype control. For the inhibitory signal for cytokine expression at baseline settings with respect to the BTN2A1 blocking signal and activation signal (259), the inventors explored the consequences of the inhibitory signal from other immune cell subsets expressing BTN2A1 and / or 3A1, or secondary effects of Vγ9Vδ2 cell activation.

[0453] Consistent with the initial data from the isolated Vγ9Vδ2 cell cultures, antibody 259 increased the expression of IFNγ and sE-selectin in the context of total PBMCs, and both the BTN2A1 inhibitory antibody and the BTN3A1 inhibitory antibody blocked the baseline expression (Figure 30). Other signals present in the highly enriched Vγ9Vδ2 cell cultures were not detected in the context of total PBMCs. Most notably, an increase in the level of TNFα was not detected when contacted with antibody 259 (Table 5). This may be due to the low number of Vγ9Vδ2 cells in PBMCs and the dilution of signals exceeding the detection threshold.

[0454]

Table 5

[0455] Interestingly, the inventors observed that additional cytokines / chemokines were upregulated by 259, which were not detected in monocultures. Among them was IL-8 (CXCL8), another chemotactic substance for immune cells (mainly neutrophils and T cells) (Henkels et al 2011), and IL-8 may play a significant role by attracting T cells in autoimmune conditions such as psoriasis (Zheng et al 1998). In addition, the inventors also saw an increase in CCL2 (MCP-1), a potent chemotactic substance for dendritic cells, and IL-6 as a prototype and key interleukin related to the inflammatory process when treated with 259 (Erlandsson et al, 2017; Hashizume et al., 2015). All of these cytokines / chemokines were downregulated by the blockade of the BTN2A1 / 3A1 signaling axis using 229. This confirms that they are dependent on signaling through this complex.

[0456] Several cytokines and chemokines were decreased by the antagonist antibody below the level of the isotype control, but their expression was not enhanced by treatment with antibody 259. Among them were ICAM-1, MIP1α and MIP1β, and sE-selectin.

[0457] Activation of Vδ2-γδ T cells

[0458] Method

[0459] Mouse 3T3 fibroblasts were transfected with the full-length human CD1c or CD1d heavy chain using the pMSCV-IRES-GFP plasmid and Fugene transfection reagent, or with a control construct (BTNL3). When the 3T3 cells expressed CD1c or CD1d on the surface after about 2 days, they were cultured for 24 hours with a human T cell line expressing a human γδ TCR specific for CD1c (Vγ9Vδ1+) or CD1d (Vγ9Vδ1+ or Vγ5Vδ1+), and then the level of activation regarding the T cell line was determined by flow cytometry using CD69. The T cell line was cultured on immobilized anti-CD3 / anti-CD28 as a positive control or with untransfected 3T3 cells as a negative control.

[0460] Discussion

[0461] To examine the ability of BTN2A1 to increase the reactivity of Vδ2 - γδ T cells to the cognate ligands of Vδ2 - γδ T cells, the inventors performed in vitro assays using two γδ T cell lines (both Vγ9Vδ1 γδTCR+) that react with CD1c and CD1d, respectively (Figure 31). The inventors also included a control Vγ5Vδ1+ γδ T cell line (clone 9C2; A.P. Uldrich et al. (2013)) that also reacts with CD1d but should not bind to BTN2A1 because it lacks Vγ9. The inventors transfected mouse 3T3 APCs with either human CD1c or CD1d, in addition to either BTN2A1 or an irrelevant control construct (human BTNL3), cultured them with the γδ T cell lines, and measured activation (CD69) after 24 hours. The data show that BTN2A1 is able to (a) induce some activation of these Vγ9+ γδ T cell lines even without an additional TCR ligand and (b) increase the activation of both the CD1c - specific γδTCR and the CD1d - specific γδTCR. This appeared to be specific for the Vγ9+ TCR. Because 9C2 (Vγ5+) reacted specifically with CD1d, which was not enhanced by BTN2A1 expression.

[0462] These findings indicate that in addition to being important for the activation of Vγ9Vδ2+ γδ T cells, BTN2A1 can directly induce the activation of Vδ2 - γδ T cells and can also increase the response of these cells to their cognate Ag.

[0463] References

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Claims

1. 1. A method of inhibiting activation of γδ T cells expressing a Vγ9+ TCR in a subject, comprising administering to the subject a BTN2A1 antagonist, wherein the BTN2A1 antagonist: i) inhibiting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) inhibiting BTN2A1 binding to Vγ9; iii) inhibiting the BTN2A1 / BTN3A1 complex from binding to Vγ9+ TCR; and / or iv) A method of decreasing the activity and / or survival of cells expressing BTN2A1.

2. The method of claim 1, which inhibits activation of Vy9V52+ γδ T cells.

3. The method of claim 1 or 2, which inhibits activation of Vγ9Vδ2- γδ T cells.

4. The method of any one of claims 1 to 3, wherein the BTN2A1 / BTN3A1 complex comprises one or more additional molecules.

5. The method of claim 4, wherein the BTN2A1 / BTN3A1 complex comprises BTN3A2 and / or BTN3A3.

6. 6. The method of any one of claims 1 to 5, wherein one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of said γδ T cells is inhibited.

7. The method of any one of claims 1 to 6, wherein the BTN2A1 antagonist inhibits phosphoantigen-mediated activation of the γδ T cells.

8. The method of any one of claims 1 to 7, wherein the BTN2A1 antagonist inhibits the association of BTN2A1 with BTN3A1.

9. The method of claim 8, wherein the BTN2A1 antagonist inhibits the direct association of BTN2A1 and BTN3A1.

10. 10. The method of any one of claims 1 to 9, wherein the BTN2A1 antagonist inhibits binding of BTN2A1 to the germline coding region of Vγ9 and / or the distal delta chain.

11. 11. The method of any one of claims 2 to 10, wherein the BTN2A1 antagonist inhibits binding of the BTN2A1 / BTN3A1 complex to a germline coding region of V52 (such as the CDR2 and / or CDR3 loops of the TCR gamma chain).

12. The method of any one of claims 1 to 11, wherein the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch the BTN2A1 molecule from a stimulatory BTN2A1 to a non-stimulatory BTN2A1.

13. The method of any one of claims 7 to 10, wherein the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule and inhibits phosphoantigen activation.

14. 1. A method of suppressing or inhibiting a Vy9+ γδ T cell response in a subject, comprising administering to the subject a BTN2A1 antagonist, the BTN2A1 antagonist comprising: i) inhibiting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) inhibiting BTN2A1 binding to Vγ9+ TCR; iii) inhibiting the BTN2A1 / BTN3A1 complex from binding to Vγ9+ TCR; and / or iv) A method of decreasing the activity and / or survival of cells expressing BTN2A1.

15. The method of claim 14, which suppresses or inhibits Vy9V52+ γδ T cell responses.

16. The method of claim 14 or 15, which suppresses or inhibits Vγ9Vδ2- γδ T cell responses.

17. The method of any one of claims 14 to 16, wherein the BTN2A1 / BTN3A1 complex comprises one or more additional molecules.

18. The method of claim 17, wherein the BTN2A1 / BTN3A1 complex comprises BTN3A2 and / or BTN3A3.

19. 19. The method of any one of claims 14 to 18, wherein one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of said γδ T cells is inhibited.

20. The method of any one of claims 14 to 20, wherein the BTN2A1 antagonist inhibits activation of the γδ T cells.

21. 21. The method of claim 20, wherein the BTN2A1 antagonist inhibits phosphoantigen-mediated activation of the γδ T cell.

22. The method of any one of claims 14 to 21, wherein the BTN2A1 antagonist inhibits the association of BTN2A1 with BTN3A1.

23. The method of claim 22, wherein the BTN2A1 antagonist inhibits the direct association of BTN2A1 and BTN3A1.

24. 24. The method of any one of claims 14 to 23, wherein the BTN2A1 antagonist inhibits binding of BTN2A1 to the germline coding region of Vγ9 and / or the distal delta chain.

25. 25. The method of any one of claims 15 to 24, wherein the BTN2A1 antagonist inhibits binding of the BTN2A1 / BTN3A1 complex to a germline coding region of V52 (such as the CDR2 and / or CDR3 loops of the TCR gamma chain).

26. The method of any one of claims 14 to 25, wherein the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch the BTN2A1 molecule from a stimulatory BTN2A1 to a non-stimulatory BTN2A1.

27. The method of any one of claims 21-26, wherein the BTN2A1 antagonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule and inhibits phosphoantigen activation.

28. 1. A method of inhibiting activation of γδ T cells expressing a Vγ9+ TCR in vitro or ex vivo, comprising culturing the γδ T cells and a cell expressing BTN2A1 in the presence of a BTN2A1 antagonist, the BTN2A1 antagonist being: i) inhibiting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) inhibiting BTN2A1 from binding to Vγ9; and / or iii) A method for inhibiting the binding of the BTN2A1 / BTN3A1 complex to Vγ9+ TCR.

29. The method of claim 28, further defined by any one of the features defined in claims 2 to 13.

30. 26. The method of claim 24 or 25, further comprising administering the γδ T cells to a subject in need thereof.

31. A method for preventing, treating, delaying the progression of, preventing the recurrence of, or alleviating a symptom of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect, comprising administering a BTN2A1 antagonist to a subject in need thereof in an amount sufficient to prevent, treat, delay the progression of, prevent the recurrence of, or alleviate said symptom of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect in the subject.

32. A method for preventing, treating, slowing the progression of, preventing the recurrence of, or alleviating a symptom of cancer or an infectious disease, comprising administering a BTN2A1 antagonist to a subject in need thereof in an amount sufficient to prevent, treat, slow the progression of, prevent the recurrence of, or alleviate said symptom of cancer or an infectious disease in the subject.

33. 1. A method of activating γδ T cells expressing a Vγ9+ TCR in a subject, comprising administering to the subject a BTN2A1 agonist, wherein the BTN2A1 agonist: i) promoting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) induces ligation of Vγ9+ TCR on the surface of γδ T cells; and / or iii) A method for increasing the activity and / or survival of cells expressing BTN2A1.

34. 34. The method of claim 33, wherein Vy9V52+ γδ T cells are activated.

35. The method of claim 31 or 32, wherein Vγ9Vδ2- γδ T cells are activated.

36. The method of claim 33 or 34, wherein the BTN2A1 / BTN3A1 complex comprises one or more additional molecules.

37. The method of claim 36, wherein the BTN2A1 / BTN3A1 complex comprises BTN3A2 and / or BTN3A3.

38. 38. The method of any one of claims 33 to 37, wherein one or more of the cytolytic function, cytokine production of one or more cytokines, or proliferation of said γδ T cells is activated.

39. The method of any one of claims 33-38, wherein the BTN2A1 agonist activates the γδ T cells independently of phosphoantigen binding.

40. The method of any one of claims 33 to 39, wherein the BTN2A1 agonist promotes the association of BTN2A1 with BTN3A1.

41. The method of claim 38, wherein the BTN2A1 agonist promotes direct association of BTN2A1 and BTN3A1.

42. The method of any one of claims 33 to 41, wherein the BTN2A1 agonist is bispecific for BTN2A1 and BTN3A1.

43. 43. The method of claim 42, wherein the BTN2A1 agonist cross-reacts with BTN3A1.

44. The method of any one of claims 33-43, wherein the BTN2A1 agonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch the BTN2A1 molecule from a non-stimulatory BTN2A1 to a stimulatory BTN2A1.

45. 1. A method of inducing or enhancing a Vγ9+ γδ T cell response in a subject, comprising administering to the subject a BTN2A1 agonist, the BTN2A1 agonist comprising: i) promoting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) induces ligation of Vγ9+ TCR on the surface of γδ T cells; and / or iii) A method for increasing the activity and / or survival of cells expressing BTN2A1.

46. 46. ​​The method of claim 45, which induces a Vy9V52+ γδ T cell response.

47. The method of claim 45 or 46, which induces a Vγ9Vδ2- γδ T cell response.

48. The method of claim 45 or 46, wherein the BTN2A1 / BTN3A1 complex comprises one or more additional molecules.

49. The method of claim 48, wherein the BTN2A1 / BTN3A1 complex comprises BTN3A2 and / or BTN3A3.

50. 50. The method of any one of claims 46 to 49, wherein one or more of the following is induced: cytolytic function, cytokine production of one or more cytokines, or proliferation of said γδ T cells.

51. 51. The method of any one of claims 45-50, wherein the BTN2A1 agonist activates the γδ T cells independently of phosphoantigen binding.

52. The method of any one of claims 45 to 51, wherein the BTN2A1 agonist promotes the association of BTN2A1 with BTN3A1.

53. The method of claim 52, wherein the BTN2A1 agonist promotes direct association of BTN2A1 and BTN3A1.

54. The method of any one of claims 45 to 53, wherein the BTN2A1 agonist is bispecific for BTN2A1 and BTN3A1.

55. 55. The method of claim 54, wherein the BTN2A1 agonist cross-reacts with BTN3A1.

56. The method of any one of claims 45-55, wherein the BTN2A1 agonist alters one or more of the extracellular domains (IgV and / or IgC) of the BTN2A1 molecule to switch the BTN2A1 molecule from a non-stimulatory BTN2A1 to a stimulatory BTN2A1.

57. 1. A method of in vitro or ex vivo activation of γδ T cells expressing a Vγ9+ TCR, comprising culturing the γδ T cells and a cell expressing BTN2A1 in the presence of a BTN2A1 agonist, the BTN2A1 agonist being: i) promoting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) induces ligation of Vγ9+ TCR on the surface of γδ T cells; and / or iii) A method for increasing the activity and / or survival of cells expressing BTN2A1.

58. The method of claim 57, further defined by any one of the features defined in claims 33 to 44.

59. 59. The method of claim 57 or 58, further comprising administering the activated γδ T cells to a subject in need thereof.

60. A method for preventing, treating, slowing the progression of, preventing the recurrence of, or alleviating a symptom of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect, comprising administering a BTN2A1 agonist to a subject in need thereof in an amount sufficient to prevent, treat, slow the progression of, prevent the recurrence of, or alleviate said symptom of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect in the subject.

61. A method for preventing, treating, slowing the progression of, preventing the recurrence of, or alleviating a symptom of cancer or an infectious disease, comprising administering a BTN2A1 agonist to a subject in need thereof in an amount sufficient to prevent, treat, slow the progression of, prevent the recurrence of, or alleviate said symptom of cancer or an infectious disease in the subject.

62. Binds specifically to BTN2A1, i) the formation of a BTN2A1 / BTN3A1 complex on the cell surface; ii) BTN2A1 binds to Vγ9; and / or iii) BTN2A1 / BTN3A1 complex binds to Vγ9+ TCR BTN2A1 antagonist inhibits

63. 63. The BTN2A1 antagonist of claim 62, further defined by any one of the features defined in claims 2-12.

64. Binds specifically to BTN2A1, i) promoting the formation of the BTN2A1 / BTN3A1 complex on the cell surface; ii) induces ligation of Vγ9+ TCR on the surface of γδ T cells; and / or iii) increasing the activity and / or survival of cells expressing BTN2A1 BTN2A1 agonist.

65. 65. The BTN2A1 agonist of claim 64, further defined by any one of the features defined in claims 33-44.

66. The method of any one of claims 1 to 65, wherein the BTN2A1 antagonist or agonist is a protein comprising an antigen-binding domain.

67. The protein is (i) single chain Fv fragment (scFv); (ii) dimeric scFv; (iii) Fv fragment; (iv) single domain antibodies (sdAbs); (v) nanobodies; (vi) diabodies, triabodies, tetrabodies, or higher multimers; (vii) a Fab fragment; (viii) Fab′ fragment; (ix) F(ab') fragment; (x) F(ab') 2 Fragment; (xi) any one of (i)-(x) linked to an Fc region of an antibody; (xii) any one of (i)-(x) fused to an antibody or antigen-binding fragment thereof that binds to an immune effector cell; or (xiii) Antibodies 67. The method of claim 66,

68. 68. The method of claim 67, wherein the protein of the disclosure is an affinity matured antibody, a chimeric antibody, a CDR grafted antibody, or a humanized antibody, or an antigen-binding fragment thereof.

69. The method of any one of claims 1 to 32, or the BTN2A1 antagonist of claim 62 or 63, wherein the BTN2A1 antagonist is a soluble Vγ9+ TCR.

70. The method of claim 69, wherein the soluble Vγ9+ TCR is monomeric.

71. The method of claim 69, wherein the soluble Vγ9+ TCR is a multimer.

72. The BTN2A1 antagonist comprises a heavy chain variable region (V H ) and a light chain variable region (V L 64. The method of any one of claims 1 to 32, or the BTN2A1 antagonist of claim 62 or 63, which is an antibody comprising:

73. The BTN2A1 antagonist comprises a heavy chain variable region (V H ) and a light chain variable region (V L 64. The method of any one of claims 1 to 32, or the BTN2A1 antagonist of claim 62 or 63, which is an antibody comprising:

74. The BTN2A1 antagonist comprises a heavy chain variable region (V H ) and a light chain variable region (V L 64. The method of any one of claims 1 to 32, or the BTN2A1 antagonist of claim 62 or 63, which is an antibody comprising:

75. The BTN2A1 antagonist comprises a heavy chain variable region (V H ) and a light chain variable region (V L 64. The method of any one of claims 1 to 32, or the BTN2A1 antagonist of claim 62 or 63, which is an antibody comprising:

76. The BTN2A1 antagonist comprises a heavy chain variable region (V H ) and a light chain variable region (V L 64. The method of any one of claims 1 to 32, or the BTN2A1 antagonist of claim 62 or 63, which is an antibody comprising:

77. Binds specifically to BTN2A1: (i) activating γδ T cells and / or increasing the number of activated γδ T cells in a population of cells; and / or (i) increasing the proportion of γδ T cells expressing markers of γδ T cell activation; and / or (ii) increasing cytokine secretion by γδ T cells; and / or (iii) inducing γδ T cells to kill cancer cells and / or inhibit the proliferation of cancer cells and / or to kill infected cells and / or inhibit the proliferation of infected cells; and / or (iv) a BTN2A1 agonist that increases the amount of a marker of γδ T cell activation expressed on the cell surface of γδ T cells.

78. Binds specifically to BTN2A1: (i) increasing the proportion of γδ T cells expressing CD25 on their cell surface; and / or (ii) increasing the secretion of interferon-γ by γδ T cells; and / or (iii) inducing γδ T cells to kill and / or inhibit the proliferation of cancer cells; and / or (iv) a BTN2A1 agonist that increases the amount of CD25 expressed on the cell surface of γδ T cells.

79. (i) a light chain variable region (V) comprising the sequence set forth in SEQ ID NO: 140 or its complementarity determining region (CDR) L ) and a heavy chain variable region (V H ). (ii) a V sequence comprising the sequence set forth in SEQ ID NO: 148 or its CDRs; L and a V sequence comprising the sequence set forth in SEQ ID NO: 152 or its CDR. H ; (iii) a V sequence comprising the sequence set forth in SEQ ID NO: 156 or its CDR. L and a V sequence comprising the sequence set forth in SEQ ID NO: 160 or its CDRs. H 78. The BTN2A1 agonist of claim 77, which is an antibody comprising:

80. 80. A method of activating γδ T cells expressing a Vγ9+ TCR in a subject, comprising administering to said subject a BTN2A1 agonist of any one of claims 77-79.

81. 80. A method of inducing or enhancing a Vγ9+ γδ T cell response in a subject, comprising administering to said subject a BTN2A1 agonist of any one of claims 77-79.

82. 80. A method of in vitro or ex vivo activation of γδ T cells expressing a Vγ9+ TCR, comprising culturing said γδ T cells and cells expressing BTN2A1 in the presence of a BTN2A1 agonist of any one of claims 77-79, and optionally administering the activated γδ T cells to a subject in need thereof.

83. 80. A method of preventing, treating, slowing the progression of, preventing the recurrence of, or alleviating a symptom of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect, comprising administering to a subject in need thereof a BTN2A1 agonist of any one of claims 77-79 in an amount sufficient to prevent, treat, slow the progression of, prevent the recurrence of, or alleviate said symptom of an autoimmune disease, transplant rejection, graft-versus-host disease, or graft-versus-tumor effect in the subject.

84. 80. A method of preventing, treating, slowing the progression of, preventing the recurrence of, or alleviating a symptom of cancer or an infectious disease, comprising administering to a subject in need thereof a BTN2A1 agonist of any one of claims 77-79 in an amount sufficient to prevent, treat, slow the progression of, prevent the recurrence of, or alleviate said symptom of cancer or an infectious disease in the subject.

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