RU2794996C2 - Antibodies specific to btn2 and their applications - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions concerns antibodies with specificity for BTN2 and their application. An anti-BTN2 antibody with specificity for human butyrophylin-2A1 (BTN2A1) and human butyrophylin-2A2 (BTN2A2) is proposed. The antibody has at least one of the following functions: inhibits the production of IFN-γ and TNF-α by activated T-cells γ V9/Vδ2; inhibits the cytolytic function of activated T-cellsγ V9/Vδ2; inhibits proliferation of activated T-cellsγ V9/Vδ2. Also the following are proposed: a nucleic acid molecule that encodes the heavy chain and light chain of an anti-BTN2 antibody, a host cell containing the said nucleic acid, a pharmaceutical composition containing an anti-BTN2 antibody.

EFFECT: inventions may be applicable in the treatment of autoimmune diseases.

9 cl, 5 dwg, 3 tbl

Description

The field of technology to which the invention belongs

The present invention relates to antibodies having specificity for BTN2 and their use.

State of the art

Leukocytes are cells of the immune system involved in the body's defense against pathogens. Along with the usual class I MHC-bound CTL CD8 ⁺ cells and NK cells, other non-standard T cells, in particular γδ T cells, have the same sensitivity and cytolytic potency as NK and T cells. The main subgroup of circulating γδ T cells are Vγ9/Vδ2 T cells, comprising 1-10% of human peripheral T cells.

Vγ9/Vδ2 T cells are important effectors of immune defense. They directly lyse pathogen-infected or abnormal cells. In addition, they regulate immune responses by inducing dendritic cell (DC) maturation as well as isotype switching and immunoglobulin production. This important cellular platform of the immune system is highly regulated by surface receptors, chemokines and cytokines. Vγ9/Vδ2 T cells are activated by non-peptide phosphorylated metabolites of the isoprenoid pathway, the so-called phosphoagonists (PAg).

T cell priming is modulated by specialized cells and secretion of chemotactic cytokines. We now know that T cell activation is the result of two synergistic events. The first is the interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC) conjugated to the treated antigen on the surface of antigen presenting cells (APCs). The second event is a costimulatory antigen-independent signal involving B7 molecules. The absence of a costimulatory signal causes anergy, that is, inhibition of T cell proliferation, cytokine secretion, and cytotoxic activity. The study of these pathways may provide insight into the triggering of such pathological phenomena as autoimmune or lymphoproliferative diseases.

Butyrophilins are a family of transmembrane proteins including butyrophilin (BTN), BTN-like (BTNL), and intraepithelial T cell selection and maintenance (SKINT) proteins (Arnett and Viney, 2014). Their extracellular portions contain IgV and IgC2 domains that share homology with the corresponding domains of B7 co-stimulatory molecules (Arnett and Viney, 2014), therefore, butyrophylins are considered members of the extended B7 or Ig superfamily.

BTN1A1, the first butyrophilin identified, is required for the formation, secretion, and stabilization of milk fat globules (Ogg et al., 2004). It was then suggested that the B7 genes and MHC class I and II genes may share a common ancestral gene and may encode proteins that have similar functions such as T-cell activation (Rhodes et al., 2001).

Subsequently, there has been increasing evidence that butyrophyllines play various roles in the immune system. The best established functions are for mouse SKINT1 and human BTN3A1, which are not conserved members of the family. SKINT1 controls the differentiation of Vγ5 ⁺ Vδ1 ⁺ T cells in the mouse thymus (Boyden et al., 2008).

The human BTN2 subfamily includes BTN2A1, BTN2A2, and the BTN2A3 pseudogene. The BTN2A1 and BTN2A2 protein isoforms exhibit IgV and IgC extracellular domains, a transmembrane domain, and an intracellular B30.2 domain characteristic of BTN3A1 and BTN3A3, but not of BTN3A2. In mice, the BTN2A2 gene is represented by a single copy and is an orthologue of the human BTN2A2 gene. The recombinant human BTN2A1-Fc protein showed that a specific BTN2A1 glycoform binds to the DC-SIGN lectin molecule found on dendritic cells (DCs). The binding of BTN2A1 to DC-SIGN depends on glycosylation of the high-mannose protein when expressed in tumor cells (Malcherek et al., 2007). However, no clear function has yet been established for BTN2A1/A2 in humans, although some experiments have been performed in mice using recombinant Fc proteins.

Smith et al., 2010 showed that recombinant mouse BTN2A2-Fc and BTN1A1-Fc bind to activated T cells, indicating the presence of one or more receptors on these cells. The immobilized BTN2A2-Fc or BTN2A1-Fc proteins, but not the MOG-Fc protein, inhibited the proliferation of anti-CD3 antibody-activated CD4 and CD8 T cells. Mouse BTN1A1 and BTN2A2 also inhibited T cell metabolism and secretion of IL-2 and IFN-γ.

Amman et al., 2013 found that binding of mouse BTN2A2-Fc to primary mouse CD3 ⁺ T cells stimulated with anti-CD3 antibody and anti-CD28 antibody reduced the number of proliferating cells and cell cycle entry. Binding of BTN2A2-Fc to stimulated anti-CD3 T cells inhibited CD3ε, Zap70 and subsequent Erk1/2 activation. Mouse BTN2A2-Fc also induces Foxp3 expression and T _reg differentiation in vitro.

Sarter et al., 2016 showed that Btn2a2 ^−/− mice show increased responses of effector CD4 ⁺ and CD8 ⁺ T cells, impaired induction of CD4 ⁺ T cells, increased antitumor responses, and exacerbated experimental autoimmune encephalomyelitis.

Currently, the treatment of autoimmune diseases and the prevention of transplant rejection in graft-versus-host disease (GVHD) depend on immunosuppressive drugs, which have serious side effects or are not always effective. Therefore, new immunosuppressants are required.

Currently, the treatment of autoimmune diseases and the prevention of transplant rejection in graft-versus-host disease (GvHD) simply depend on immunosuppressants. However, such immunosuppressants may not always be effective and/or have serious side effects.

Therefore, there is a need to identify new immunosuppressants and/or methods for inhibiting the immune response in patients in need of it.

The essence of the invention

The present invention relates to antibodies having specificity for BTN2 and their uses.

Specifically, provided herein is an antibody that binds to BTN2 (e.g., human BTN2A1 and BTN2A2 polypeptides) and exhibits at least one of the following properties:

• they inhibit the production of IFN-γ and/or TNF-α by activated Vγ9/Vδ2 and/or

• they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells and/or

• they inhibit the proliferation of activated Vγ9/Vδ2 T cells.

In certain embodiments, the anti-BTN2 antibodies of the present invention have specificity for both human butyrophylin 2A1 (BTN2A1) and human butyrophylin 2A2 (BTN2A2).

In certain embodiments, the anti-BTN2 antibodies of the present invention compete for binding to BTN2A2 with at least one of the following reference mouse antibodies:

i. mAb 4.15 derived from a hybridoma deposited with CNCM under deposit number CNCM I-5231;

ii. mAb 5.28 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5232;

iii. mAb 7.28 derived from a hybridoma deposited with CNCM under deposit number CNCM I-5233;

iv. mAb 7.48 derived from a hybridoma deposited with CNCM under deposit number CNCM I-5234;

v. mAb 8.15 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5235; or

vi. mAb 8.16 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5236.

In certain embodiments, the anti-BTN2 antibodies of the present invention comprise either:

i. a heavy chain and a light chain comprising 6 CDRs of mAb 4.15, mAb 4.15 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5231;

ii. a heavy chain and a light chain comprising 6 CDRs of mAb 5.28, with mAb 5.28 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5232;

iii. a heavy chain and a light chain comprising 6 CDRs of mAb 7.28, with mAb 7.28 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5233;

iv. a heavy chain and a light chain comprising 6 CDRs of mAb 7.48, mAb 7.48 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5234;

v. a heavy chain and a light chain comprising 6 CDRs of mAb 8.15, with mAb 8.15 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5235; or

vi. a heavy chain and a light chain comprising 6 CDRs of mAb 8.16, with mAb 8.16 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5236.

In other specific embodiments, the anti-BTN2 antibodies of the present invention comprise either:

(i) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 4.15 of SEQ ID NOs: 3-8, respectively;

(ii) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 5.28 of SEQ ID NOs: 11-16, respectively;

(iii) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 7.28 of SEQ ID NOs: 19-24, respectively;

(iv) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 from mAb 7.48 of SEQ ID NOs: 27-32, respectively;

(v) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 8.15 of SEQ ID NOs: 35-40, respectively; or

(vi) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 8.16 at SEQ ID NOs: 43-48, respectively.

In other specific embodiments, which may be combined with the previous embodiments, the anti-BTN2 antibodies of the invention comprise either:

(i) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 9, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 10;

(ii) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 17, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 18;

(iii) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 25, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 26;

(iv) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 33, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 34;

(v) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 41, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 42; or

(vi) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 49, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 50.

In certain embodiments, the anti-BTN2 antibodies of the present invention do not cross-react with human CD277, in particular they do not cross-react with any of human BTN3A1, BTN3A2 and BTN3A3.

In certain embodiments, the anti-BTN2 antibodies of the present invention inhibit the cytolytic function of activated Vγ9/Vδ2 T cells in the presence of the anti-CD277 mAb20.1 agonist antibody.

In certain embodiments, the anti-BTN2 antibodies of the present invention inhibit the cytolytic function of activated Vγ9/Vδ2 T cells, e.g. agents that induce the production of phosphoagonists (PAg).

In certain embodiments, these anti-BTN2 antibodies are human, chimeric, or humanized antibodies.

Another aspect of the present invention relates to nucleic acid molecules encoding the heavy chain and/or light chain of any of the antibodies against BTN2, as described above.

The invention also relates to host cells containing such nucleic acids, in particular for use in the preparation of any of the anti-BTN2 antibodies as described above.

Another aspect of the invention relates to antibodies against BTN2, as described above, for use in therapy, for example, in the treatment of autoimmune and inflammatory diseases and transplant rejection.

Another aspect of the invention relates to a method for treating autoimmune and inflammatory diseases and transplant rejection in subjects in need thereof, which comprises administering to the subject a therapeutically effective amount of an anti-BTN2 antibody as defined above.

Typically, these autoimmune and inflammatory diseases are selected from the group consisting of: rheumatoid arthritis (RA), insulin dependent diabetes mellitus (type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome , pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic leukopenia, idiopathic thrombocytosis penic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, protein-anaphylactic uveitis, primary biliary cirrhosis, primary myxedema, Reiter's syndrome, rigidity syndrome, thyrotoxicosis, ulcerative colitis and Wegener's granulomatosis.

The invention also relates to pharmaceutical compositions containing anti-BTN2 antibodies as defined above.

The invention also provides a method for inhibiting an immune response in a subject, comprising administering to the subject an effective amount of an anti-BTN2 antibody as set forth herein.

Disclosure of the essence of the invention

Definitions

In the present invention, the term "BTN2" has the meaning generally accepted in the art and refers to human BTN2 polypeptides, including BTN2A1 of SEQ ID NO: 1 or BTN2A2 of SEQ ID NO: 2.

SEQ ID NO: 1. BTN2A isoform-1 precursor (Homo sapiens)

MESAAALHFSRPASLLLLLSLCALVSAQFIVVGPTDPILATVGENTTLRCHLSPEKNAE

DMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQEN

GTYRCYFQEGRSYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRD

PYGGVAPALKEVSMPDADGLFMVTTAVIIRDKSVRNMSCSINNTLLGQKKESVIFIPESF

MPSVSPCAVALPIIVVILMIPIAVCIYWINKLQKEKKILSGEKEFERETREIALKELEKE

RVQKEEELQVKEKLQEELRWRRTFLHAVDVVLDPDTAHPDLFLSEDRRSVRRCPFRHLGE

SVPDNPERFDSQPCVLGRESFASGKHYWEVEVENVIEWTVGVCRDSVERKGEVLLIPQNG

FWTLEMHKGQYRAVSSPDRILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAF

SVPVRPFFRLGCEDSPIFICPALTGANGVTVPEEGLTLHRVGTHQSL

SEQ ID NO: 2. BTN2A isoform-2 precursor (Homo sapiens)

MEPAAALHFSLPASLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPE

KNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVT

AQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLT

VWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIFI

PESFMPSASPWMVALAVILTASPWMVSMTVILAVFIIFMAVSICCIKKLQREKKILSGEK

KVEQEEKEIAQQLQEELRWRRTFLHAADVVLDPDTAHPELFLSEDRRSVRRGPYRQRVPD

NPERFDSQPCVLGWESFASGKHYWEVEVENVMVWTVGVCRHSVERKGEVLLIPQNGFWTL

EMFGNQYRALSSPERILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAFTVPV

RPFFRLGSDDSPIFICPALTGASGVMVPEEGLKLHRVGTHQSL

In the present invention, the terms "antibody" and "immunoglobulin" have the same meanings and will be used equally in the present invention.

In the present invention, the term "antibody" refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, that is, molecules containing an antigen-binding site that immunospecifically binds an antigen. In this case, the term antibody covers not only whole molecules of antibodies, but also fragments of antibodies, as well as variants (including derivatives) of antibodies and fragments of antibodies.

In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main classes of heavy chains (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each strand contains individual sequence domains. The light chain includes two domains: a variable domain (V _L ) and a constant domain (C _L ). The heavy chain includes four domains: a variable domain (V _H ) and three constant domains (CH ₁ , _CH 2 and C _H 3, collectively referred to as _CH ). The variable regions of the light (V _L ) and heavy (V _H ) chains determine the recognition and specificity of antigen binding. The light ( _CL ) and heavy ( _CH ) constant region domains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding.

The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the binding site of the antibody and the antigenic determinant. The binding sites of antibodies consist of residues derived primarily from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may be involved in the binding site of an antibody or affect the overall structure of the domain and thereby the binding site. Complementarity determining regions or CDRs are those amino acid sequences that together determine the binding affinity and specificity of the natural Fv region of the native immunoglobulin binding site. Each of the immunoglobulin light and heavy chains contains three CDRs, which are referred to as L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, an antigen-binding site typically contains six CDRs, including CDRs from each of the heavy and light chain V regions. Frame regions (FRs) refer to the amino acid sequences inserted between the CDRs. The respective light and heavy chain variable regions typically contain 4 framework regions and 3 CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Residues in the variable domains of antibodies are usually numbered according to the system developed by Kabat et al. This system is outlined in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter “Kabat et al.”). This numbering system is used in the present description. The Kabat residue designations do not always directly correspond to the linear numbering of amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or more amino acids than strict Kabat numbering, corresponding to a truncation of a structural component or an insertion into the framework or complementarity determining region (CDR) of the basic structure of the variable domain. The correct Kabat numbering for a given antibody can be established by aligning homologous residues in the antibody sequence with the “standard” Kabat numbered sequence. The heavy chain variable CDR regions are residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The light chain variable CDR regions are at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

In certain embodiments, the antibodies provided herein are antibody fragments, in particular, any proteins containing antigen-binding domains of antibodies, as described here. Antibody fragments include, without limitation, Fv, Fab, F(ab') ₂ , Fab', dsFv, scFv, sc(Fv) ₂ and diabodies.

In the present invention, the term “specificity” refers to the ability of an antibody to bind detectably to an epitope presented on a BTN2 type antigen. In some embodiments, this refers to antibodies or proteins that bind to human BTN2A2 expressed in a cell line, for example, in the HEK293F cell line, as described in the examples, preferably with an EC ₅₀ value of less than 50 μg/ml, more preferably less than 10 μg /ml and even more preferably less than 1 µg/ml as defined in the examples and in FIGS. 1. In some embodiments, this refers to antibodies or proteins that bind to human BTN2A1 expressed in a cell line, for example, in the HEK293F cell line, in which all BTN3 and BTN2 isoforms have been knocked out, as described in the examples, preferably with an EC value ₅₀ less than 1 µg/‌ml, for example, less than 0.1 µg/ml; and/or refers to antibodies or proteins that bind to human BTN2A2 expressed in a cell line, e.g. HEK293F cell line, in which all BTN3 and BTN2 isoforms have been knocked out as described in the examples, preferably with an EC ₅₀ value of less than 50 μg/ml, for example, less than 1 μg/ml or less than 0.02 μg/ml. In other embodiments, they bind to a recombinant antigen polypeptide with a K _D value of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less.

“Isolated antibody” in the present invention means an antibody that is substantially free of other antibodies with a different antigenic specificity (e.g., an isolated antibody that specifically binds to BTN2 contains substantially no antibodies that specifically bind to antigens other than BTN2) . However, an isolated antibody that specifically binds to BTN2 may cross-react with other antigens such as related BTN2 molecules from other species. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemicals.

The terms "monoclonal antibody" or "monoclonal antibody composition" in the present invention refer to preparations of antibody molecules of the same molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

The expressions "antibody that recognizes an antigen" and "antibody having specificity for an antigen" are used here interchangeably with the term "antibody that specifically binds to an antigen".

The term “K _assoc ” or “K _a ” in the present invention refers to the rate of association in a particular antibody-antigen interaction, while the term “K _dis ” or “K _d ” in the present invention serves to indicate the rate of dissociation in a particular antibody-antigen interaction. antigen.

The term "K _D " in the present invention refers to the dissociation constant, which is obtained from the ratio of K _d and K _a (ie K _d /K _a ) and expressed as a molar concentration (M). Antibody K _D values can be determined by methods well known in the art. Preferred methods for determining mAb K _D values are given in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY (1992, 1993); and Muller, Meth. Enzymol. 92: 589-601 (1983); which are incorporated herein by reference in their entirety. One method for determining the K _D of antibodies is to use surface plasmon resonance or a biosensor system such as the ^Biacore® system.

Specificity can also be expressed, e.g., as an affinity/appearance ratio of about 10:1, 20:1, 50:1, 100:1, 10,000:1 or more when binding to a specific antigen compared to non-specific binding to other extraneous molecules (in this case, the specific antigen is the BTN2 polypeptide). The term "affinity" in the present invention means the binding strength of an antibody to an epitope.

In one aspect, the present invention relates to antibodies having specificity for BTN2, characterized in that they have at least one of the following properties:

i. they inhibit the production of IFN-γ and TNF-α by activated Vγ9/Vδ2 T cells and/or

ii. they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells and/or

iii. they inhibit the proliferation of activated Vγ9/Vδ2 T cells.

Anti-BTN2 antibodies of the present invention having such advantageous properties can be detected by screening against anti-BTN2 antibodies by cellular methods as described in the examples, in particular by CD107 degranulation on Daudi cells.

In the present invention, "inhibition of IFN-γ or TNF-α production" means that there is a significant reduction in the production of at least IFN-γ or TNF-α by activated Vγ9/Vδ2 T cells compared to control activated Vγ9/ T cells. Vδ2 (with IgG1 or IgG2a as control) and these Vγ9/Vδ2 T cells are activated either by co-culture with the target cell line (Daudi cells) or by phosphoagonists (PAg). In general, inhibition of IFN-γ or TNF-α production by activated Vγ9/Vδ2 T cells can be measured cellularly by intracellular labeling with anti-IFNγ or TNFα antibodies and flow cytometry. This method is described in more detail in the examples below.

In the present invention, "inhibition of the cytolytic function of activated Vγ9/Vδ2 T cells" means that there is a significant decrease in the cytolytic function of activated human Vγ9/Vδ2 T cells compared to control activated human Vγ9/Vδ2 T cells (with IgG1 or IgG2a as a control) and these human Vγ9/Vδ2 T cells are activated either by co-culturing with the target cell line (Daudi cells) or by phosphoagonists (PAg). In general, inhibition of the cytolytic function of activated Vγ9/Vδ2 T cells, for example in the presence of the mAb20.1 agonist antibody, can be measured by inhibition of induction of γδ T cell degranulation in a standard cell line and CD107 as a degranulation marker to detect positive degranulated T -cells γδ. This method is described in more detail in the examples below.

In the present invention, "inhibition of the proliferation of activated Vγ9/Vδ2 T cells" means that there is a significant reduction in the proliferation of activated Vγ9/Vδ2 T cells compared to the proliferation of Vγ9/Vδ2 T cells when activated with IgG1 or IgG2a as a control, moreover, these Vγ9/Vδ2 T cells are activated either by co-cultivation with the target cell line (Daudi cells) or by phosphoagonists (pAg). Typically, the proliferation of activated Vγ9/Vδ2 T cells can be measured cellularly by CFSE or Cell Trace Violet staining and flow cytometry.

In some embodiments, the antibodies of the present invention inhibit the cytolytic function of activated Vγ9/Vδ2 T cells to a level that is substantially equal to or superior to at least one of the reference antibodies: mAb 4.15, mAb 5.28, mAb 7.28, mAb 7.48, mAb 8.15, and mAb 8.16 as described below. In other specific embodiments, anti-BTN2 antibodies inhibit the cytolytic function of activated Vγ9/Vδ2 T cells to a level that is at least equal to or greater than mAb 103.2, this mAb 103.2 being disclosed in WO 2012/080351.

In some embodiments, the antibodies of the present invention inhibit the production of at least IFN-γ or TNF-α by activated Vγ9/Vδ2 T cells to a level that is substantially equal to or superior to at least one of the reference antibodies: mAb 4.15, mAb 5.28, mAb 7.28, mAb 7.48, mAb 8.15 and mAb 8.16 as described below. In other specific embodiments, anti-BTN2 antibodies inhibit the production of at least IFN-γ or TNF-α by activated Vγ9/Vδ2 T cells to a level that is at least equal to or greater than mAb 103.2, this mAb 103.2 being disclosed in WO 2012/080351.

In certain embodiments, the anti-BTN2 antibodies of the present invention are further characterized in that they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells even in the presence of the anti-CD277 mAb20.1 agonist antibody.

Anti-CD277 mAb20.1 was disclosed in WO 2012/080351, which antibody enhances the cytolytic function of activated Vγ9/Vδ2 T cells. In general, inhibition of the cytolytic function of activated Vγ9/Vδ2 T cells in the presence of mAb20.1 can be measured by inhibition of induction of γδ T cell degranulation on a standard cell line, e.g. Daudi cell line as a standard cell line and CD107 as a marker. degranulation to detect positive degranulated γδ T cells using mAb 20.1, for example at a concentration of 10 μg/ml. This method is described in more detail in the examples below.

Reference antibodies mAbs 1-6

The antibodies of the invention comprise reference mouse mAb1-mAb6 monoclonal antibodies which are produced by hybridomas deposited with the Collection Nationale de Cultures des Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) under the terms of the Budapest Treaty of September 14, 2017 under the corresponding deposit numbers, as described below in Table. 1.

Table 1. mAb1-mAb6

Antibody clone name Deposit number mAb1 4.15 CNCM I-5231 mAb2 5.28 CNCM I-5232 mAb3 7.28 CNCM I-5233 mAb4 7.48 CNCM I-5234 mAb5 8.15 CNCM I-5235 mAb6 8.16 CNCM I-5236

The invention also relates to any antibodies containing the respective V _H and V _L regions of any of the above reference antibodies.

The present invention also concerns hybridomas available from CNCM under deposit numbers CNCM I-5231, CNCM I-5232, CNCM I-5233, CNCM I-5234, CNCM I-5235 or CNCM I-5236.

Other antibodies of the invention include antibodies containing amino acids that have been mutated by deletion, insertion, or substitution of amino acids, but have at least 60%, 70%, 80%, 90%, 95%, or 100% CDR regions identical to the CDR regions of any from the above reference antibodies.

In some embodiments, the antibodies of the invention are mutant variants of any of the mAb1-mAb6 containing 6 CDR regions that are 100% identical to the corresponding 6 CDR regions of one of the reference mAb1-mAb6, and this mutant antibody variant includes mutant amino acid sequences, in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated by deletion, insertion or substitution of amino acids in the FR1, FR2, FR3 and FR4 regions compared to the corresponding framework regions of the corresponding reference antibodies.

In certain embodiments, the anti-BTN2 antibodies of the invention, preferably humanized anti-BTN2 antibodies, comprise either:

i. a heavy chain and a light chain comprising 6 CDRs of mAb 4.15, mAb 4.15 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5231;

ii. a heavy chain and a light chain comprising 6 CDRs of mAb 5.28, with mAb 5.28 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5232;

iii. a heavy chain and a light chain comprising 6 CDRs of mAb 7.28, with mAb 7.28 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5233;

iv. a heavy chain and a light chain comprising 6 CDRs of mAb 7.48, mAb 7.48 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5234;

v. a heavy chain and a light chain comprising 6 CDRs of mAb 8.15, with mAb 8.15 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5235; or

vi. a heavy chain and a light chain comprising 6 CDRs of mAb 8.16, with mAb 8.16 derived from a hybridoma deposited with CNCM under CNCM deposit number I-5236.

Thus, the antibodies of the invention also include murine anti-BTN2 antibodies isolated and structurally characterized by their heavy and light chain variable region amino acid sequences, as described in Table 1 below. 2.

Table 2. Amino acid sequences of heavy and light chain variable regions of mouse reference antibodies of the invention

Antibody Amino acid sequence V _H Amino acid sequence V _L mAb1 SEQ ID NO:9 SEQ ID NO:10 mAb2 SEQ ID NO:17 SEQ ID NO:18 mAb3 SEQ ID NO:25 SEQ ID NO:26 mAb4 SEQ ID NO:33 SEQ ID NO:34 mAb5 SEQ ID NO:41 SEQ ID NO:42 mAb6 SEQ ID NO:49 SEQ ID NO:50

The respective amino acid and nucleotide coding sequences for IgG4, IgG1 isotype constant regions and their mutant versions are well known in the art.

Exemplary amino acid sequences of CDR1 V _H (also referred to as HCDR1), CDR2 V _H (also referred to as HCDR2), CDR3 V _H (also referred to as HCDR1), CDR1 V L (also referred to as LCDR1), CDR2 _{V L (} also referred to as LCDR2), CDR3 V _L (also called HCDR3) of some antibodies according to the invention are given in table. 3.

In table. 3 CDR regions of some antibodies of the present invention are given according to the Chothia system (Chothia C, Lesk AM. 1987, J Mol Biol 196, 901-917).

For readability, the CDRs are hereinafter referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, respectively.

Table 3. CDR regions of reference mouse antibodies as defined by Chothia

parent antibody HCDR1 HCDR2 HDDR3 LCDR1 LCDR2 LCDR3 mAb1 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:7 SEQ ID NO:8 mAb2 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 SEQ ID NO:16 mAb3 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 mAb4 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:32 mAb5 SEQ ID NO:35 SEQ ID NO:36 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40 mAb6 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:48

In certain embodiments, isolated anti-BTN2 antibodies of the invention comprise either:

(a) a heavy chain variable region comprising HCDR1 of SEQ ID NO:3, HCDR2 of SEQ ID NO:4, HCDR3 of SEQ ID NO:5, and a light chain variable region comprising LCDR1 of SEQ ID NO:6, LCDR2 of SEQ ID NO:7 and LCDR3 of SEQ ID NO:8;

(b) a heavy chain variable region comprising HCDR1 of SEQ ID NO:11, HCDR2 of SEQ ID NO:12, HCDR3 of SEQ ID NO:13, and a light chain variable region comprising LCDR1 of SEQ ID NO:14, LCDR2 of SEQ ID NO:15 and LCDR3 of SEQ ID NO:16;

(c) a heavy chain variable region comprising HCDR1 of SEQ ID NO:19, HCDR2 of SEQ ID NO:20, HCDR3 of SEQ ID NO:21, and a light chain variable region comprising LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:23 and LCDR3 of SEQ ID NO:24;

(d) a heavy chain variable region comprising HCDR1 of SEQ ID NO:27, HCDR2 of SEQ ID NO:28, HCDR3 of SEQ ID NO:29, and a light chain variable region comprising LCDR1 of SEQ ID NO:30, LCDR2 of SEQ ID NO:31 and LCDR3 of SEQ ID NO:32;

(e) a heavy chain variable region comprising HCDR1 of SEQ ID NO:35, HCDR2 of SEQ ID NO:36, HCDR3 of SEQ ID NO:37, and a light chain variable region comprising LCDR1 of SEQ ID NO:38, LCDR2 of SEQ ID NO:39 and LCDR3 of SEQ ID NO:40; or

(f) a heavy chain variable region comprising HCDR1 of SEQ ID NO:43, HCDR2 of SEQ ID NO:44, HCDR3 of SEQ ID NO:45, and a light chain variable region comprising LCDR1 of SEQ ID NO:46, LCDR2 of SEQ ID NO:47 and LCDR3 of SEQ ID NO:48;

moreover, these antibodies against BTN2 have specificity for BTN2.

In other specific embodiments, isolated anti-BTN2 antibodies of the invention comprise either:

(a) a heavy chain variable region containing V _H according to SEQ ID NO:9 and a light chain variable region containing V _L according to SEQ ID NO:10;

(b) a heavy chain variable region containing V _H according to SEQ ID NO:17 and a light chain variable region containing V _L according to SEQ ID NO:18;

(c) a heavy chain variable region containing V _H according to SEQ ID NO:25 and a light chain variable region containing V _L according to SEQ ID NO:26;

(d) a heavy chain variable region containing V _H according to SEQ ID NO:33 and a light chain variable region containing V _L according to SEQ ID NO:34;

(e) a heavy chain variable region containing V _H according to SEQ ID NO:41 and a light chain variable region containing V _L according to SEQ ID NO:42; or

(f) a heavy chain variable region containing V _H according to SEQ ID NO:49 and a light chain variable region containing V _L according to SEQ ID NO:50;

moreover, these antibodies against BTN2 have specificity for BTN2.

Functional variants of antibodies

In a further embodiment, functional variants of the antibodies of the invention have full length heavy and light chain amino acid sequences; or the amino acid sequences of the heavy and light chain variable regions; or the amino acid sequences of all 6 CDR regions that are homologous or preferably identical to the corresponding amino acid sequences of the antibodies mAb1-mAb6 described above, in particular, in table. 1, 2 and 3, and such functional variants of the antibodies retain the desired functional properties of the original antibodies mAb1-mAb6.

Functional variants of V _L , V _H or CDR in the context of the monoclonal antibodies of the present invention allow the antibodies to retain at least a significant proportion (at least 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity/ avidity and/or specificity/selectivity of the parent antibody (i.e., any of the antibodies mAb1-mAb6), and in some cases, such monoclonal antibodies of the present invention may be associated with greater affinity, selectivity and/or specificity than the parent Ab.

The desired functional properties of the parent antibodies mAb1-mAb6 can be selected from the group consisting of:

i. they have specificity for BTN2, in particular they bind to human BTN2 expressed in a cell line, for example in HEK293F cell line expressing human BTN2A2 as described in the examples, preferably with an EC ₅₀ value of less than 50 μg/ml, more preferably less than 10 μg/ml, and even more preferably less than 1 μg/ml, as defined in the examples and in FIGS. 1;

ii. they bind to human BTN2A1 expressed in a cell line, e.g. HEK293F cell line, in which all BTN3 and BTN2 isoforms have been knocked out as described in the examples, preferably with an EC ₅₀ value of less than 1 μg/ml, e.g. less than 0 .1 µg/ml;

iii. they bind to human BTN2A2 expressed in a cell line, e.g. HEK293F, in which all BTN3 and BTN2 isoforms have been knocked out as described in the examples, preferably with an EC ₅₀ value of less than 50 μg/ml, e.g. less than 1 mcg/ml or less than 0.02 mcg/ml;

iv. they inhibit the production of IFN-γ or TNF-α by activated Vγ9/Vδ2 T cells;

v. they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells; and/or

vi. they inhibit the proliferation of activated Vγ9/Vδ2 T cells.

For example, the invention relates to functional variants of antibodies mAb1-mAb6 containing sequences of the variable region of the heavy chain (V _H ) and variable region of the light chain (V _L ), in which the CDR sequences, i. 6 CDR regions: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are at least 60%, 70%, 90%, 95%, or 100% identical to the corresponding CDR sequences of at least one of the mAb1-mAb6 antibodies, with antibody functional variants binding specifically to BTN2, and antibodies exhibit at least one of the following functional properties:

i. they inhibit the production of IFN-γ or TNF-α by activated Vγ9/Vδ2 T cells;

ii. they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells; and/or

iii. they inhibit the proliferation of activated Vγ9/Vδ2 T cells.

It also concerns functional variants of mAb1-mAb6 antibodies containing a heavy chain variable region and a light chain variable region that are at least 80%, 90% or at least 95% or 100% identical to the respective heavy and light chain variable regions of whichever - either of the antibodies mAb1-mAb6, presented, in particular, in table. 2; moreover, the functional variants of the antibodies specifically bind to BTN2 and exhibit at least one of the following functional properties:

i. they inhibit the production of IFN-γ or TNF-α by activated Vγ9/Vδ2 T cells;

ii. they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells; and/or

iii. they inhibit the proliferation of activated Vγ9/Vδ2 T cells.

In various embodiments, the antibodies may exhibit one or two of the desired functional properties listed above. The antibodies may be, for example, human antibodies, humanized antibodies, or chimeric antibodies. Preferably the antibodies or proteins are humanized human antibodies, more preferably humanized silent antibodies.

As used herein, the term "silent" antibody means an antibody that exhibits little or no ADCC activity as measured by in vitro ADCC activity by target cell lysis.

In one embodiment, the term "no or little ADCC activity" means that the silent antibody exhibits an ADCC activity that is less than 50%, e.g., less than 10% of the ADCC activity seen with the corresponding wild-type (non-silent) antibody, e.g. , with wild-type human IgG1 antibody. Preferably, the absence of detectable ADCC activity is detected by in vitro ADCC activity assay with a silent antibody compared to a Fab fragment of a control antibody.

Silent effector functions can be obtained by mutation in the constant Fc portion of antibodies and have been described in the art: Strohl 2009 (LALA &N297A); Baudino 2008, D265A (Baudino et al., J. Immunol. 181 (2008): 6664-69; Strohl, CO Biotechnology 20 (2009): 685-91). Examples of silent IgG1 antibodies include ADCC-reducing mutations at positions 234, 235, and/or 331 in the IgG1 Fc amino acid sequence (EU numbering). Another silent IgG1 antibody contains the N297A mutation, which produces aglycosylated or non-glycosylated antibodies.

The CDR sequences of variants may differ from the CDR sequences of the parent antibody (as shown, for example, in Table 3) mainly by conservative substitutions; for example, at least 10 substitutions, such as at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the variant are conservative amino acid residue substitutions. In the context of the present invention, conservative substitutions can be defined as substitutions within classes of amino acids expressed as follows:

aliphatic residues I, L, V and M

cycloalkenyl-linked residues F, H, W and Y

hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y

negatively charged residues D and E

polar residues C, D, E, H, K, N, Q, R, S, and T

positively charged residues H, K and R

small remnants of A, C, D, G, N, P, S, T, and V

very small remnants of A, G and S

residues involved in the formation of bends A, C, D, E, G, H, K, N, Q, R, S, P and T

flexible residues Q, T, K, S, G, P, D, E, and R.

More conservative groupings of substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Also, the CDRs of the variants are substantially conserved in terms of hydropathic/‌hydrophilic properties and residue weight/size compared to the CDRs of any of the mAbs 1-6. The importance of the hydropathic index of amino acids in conferring interactive biological function on proteins is generally understood in the art. It is assumed that the relative hydropathic nature of the amino acid contributes to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid has been assigned a hydropathic index based on their hydrophobicity and charge characteristics, namely: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also be measured using the similarity score, which is determined using the BLAST program (eg, BLAST 2.2.8, available from NCBI, using the standard settings BLOSUM62, Open Gap = 11 and Extended Gap = 1). Suitable variants typically exhibit at least 70% identity with the parent peptide. According to the present invention, that the first amino acid sequence is at least 70% identical to the second amino acid sequence means that the first sequence is identical to the second amino acid sequence by 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. According to the present invention, that the first amino acid sequence is at least 50% identical to the second amino acid sequence means that the first sequence is identical to the second amino acid sequence by 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

In some embodiments, the antibodies of the present invention are chimeric antibodies, typically mouse/human chimeric antibodies. The term "chimeric antibody" means a monoclonal antibody that contains a V _H domain and a V _L domain from an antibody derived from an animal, and a _CH domain and a _CL domain from a human antibody. Any animal can be used as an animal, such as mice, rats, hamsters, rabbits, etc. In particular, the mouse/human chimeric antibody may contain the V _H and V _L domains of any of the mAb1-mAb6 reference antibodies.

In some embodiments, the antibodies of the present invention are humanized antibodies. In certain embodiments, the antibodies of the present invention are humanized antibodies that contain 6 CDRs of any of the mAb1-mAb6 reference antibodies, for example, as shown in Table 1. 3. In the present invention, the term “humanized antibody” means a monoclonal antibody whose framework regions (FRs) have been modified by incorporating FRs from a donor immunoglobulin of a different species (e.g., human) compared to those of the parent immunoglobulin (e.g., mouse CDRs). ).

In some embodiments, the antibodies of the present invention are selected from the group consisting of Fab, F(ab') ₂ , Fab' and scFv. In the present invention, the term "Fab" refers to antibody fragments with a molecular weight of about 50,000 and having antigen-binding activity, in which about half of the N-terminal side of the H chain and the entire L chain of fragments obtained by treating IgG with papain protease are connected together with a disulfide bond. The term “F(ab′) ₂ ” denotes antibody fragments with a molecular weight of about 100,000 and having antigen-binding activity, which are slightly larger than the Fab disulfide-linked to the hinge region, from among the fragments obtained by treating IgG protease with pepsin. The term "Fab'" means fragments of antibodies with a molecular weight of about 50,000 and having antigen-binding activity, which are obtained by breaking the disulfide bond in the hinge region F(ab') ₂ . A single chain Fv polypeptide (“scFv”) is a covalently linked V _H ::V _L heterodimer that is typically expressed from a fusion gene comprising the genes encoding V _H and V _L joined by a peptide-encoding linker. The human scFv fragment of the invention contains CDRs that are maintained in the proper conformation, preferably using gene recombination techniques.

Functional variants of antibodies with mutant amino acid sequences can be generated by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the encoding nucleic acid molecules, followed by testing the encoded altered antibody for retention of function (i.e., the functions outlined above) using the methods described here functional analyses.

Antibodies that cross-block any of mAb1-mAb6 and/or bind to the same epitope as mAb1-mAb6

Additional antibodies with the same advantageous properties as the mAb1-mAb6 reference antibodies given here can be identified based on their ability to cross-compete (e.g., competitive binding inhibition) in a statistically significant manner with any of the mAb1-mAb6 reference antibodies as described above, in a standard BTN2 binding assay.

Antibodies of interest can first be screened for their binding affinity for BTN2, for example, from a library of recombinant human antibodies, for example, by phage display technology, or from transgenic mice expressing antibodies with human variable regions immunized with BTN2 antigens.

The ability of the test antibody to cross-compete or inhibit the binding of the antibodies of the present invention to human BTN2 indicates that the test antibody can compete with these antibodies for binding to human BTN2; such an antibody, according to a non-limiting theory, may bind to the same or related (e.g. structurally similar or spatially close) epitope on human BTN2 (e.g. BTN2A2 and/or BTN2A1) as the antibody with which it competes .

To screen for anti-BTN2 antibodies for their ability to bind to the same epitope as one of the mAb1-mAb6 reference antibodies, staining is performed, for example, on HEK293 cells transfected with human BTN2A2, or HEK293 cells that are knocked out for all BTN2 or BTN3 isoforms and express Human BTN2A2 or human BTN2A1 (as described in the examples), at a saturation concentration (10 μg/ml) of one of the mAb1-mAb6 reference antibodies for 30 minutes at 4°C. After 2 washes, various doses of test anti-BTN2 mAbs (30 min at 4° C.) are tested for their ability to compete with any of the mAb1-mAb6 reference antibodies. Those mAbs that compete for the same binding site as the reference antibody will not be able to recognize BTN2 in the presence of such reference antibodies. This data can be expressed as the mean fluorescence intensity.

Selected antibodies can be subjected to further testing for the useful properties of mAb1-mAb6, in particular in relation to the ability to inhibit activated Vγ9/Vδ2 T cells.

Accordingly, in one embodiment of the invention, isolated antibodies are provided that cross-block or cross-block with at least one of the mAb1-mAb6 antibodies for binding to BTN2, which antibodies:

i. have specificity for BTN2, in particular they bind to human BTN2 expressed in a cell line, for example in HEK293F cell line expressing human BTN2A2, as described in the examples, preferably with an EC ₅₀ value of less than 50 μg/ml, more preferably less than 10 µg/ml and even more preferably less than 1 µg/ml as defined in the examples and in FIG. 1;

ii. bind to human BTN2A1 expressed in a cell line, e.g. HEK293F cell line, in which all BTN3 and BTN2 isoforms have been knocked out as described in the examples, preferably with an EC ₅₀ value of less than 1 μg/ml, e.g. less than 0, 1 µg/ml;

iii. bind to human BTN2A2 expressed in a cell line, e.g. HEK293F cell line, in which all BTN3 and BTN2 isoforms have been knocked out as described in the examples, preferably with an EC ₅₀ value of less than 50 μg/ml, e.g. less than 1 μg /ml or less than 0.02 µg/ml;

iv. they inhibit the production of IFN-γ and/or TNF-α by activated Vγ9/Vδ2 T cells;

v. they inhibit the cytolytic function of activated Vγ9/Vδ2 T cells; and/or

vi. they inhibit the proliferation of activated Vγ9/Vδ2 T cells.

In another embodiment of the invention, antibodies are provided that bind to the same epitope as at least one of the anti-BTN2 mAb1-mAb6 antibodies as described herein.

In one specific embodiment, cross-blocking antibodies that bind to the same epitope on human BTN2 as any of the mAb1-mAb6 are chimeric, humanized, or human recombinant antibodies.

Production of transfectomas producing monoclonal antibodies

Antibodies of the present invention are obtained by any methods known in this field, such as, without limitation, any chemical, biological, genetic or enzymatic methods, either alone or in combination. Generally, with knowledge of the amino acid sequence of the desired sequence, one skilled in the art can readily prepare such antibodies by standard methods for producing polypeptides. For example, they can be synthesized by a well known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as those available from Applied Biosystems, Foster City, Calif.) and according to the manufacturer's instructions. On the other hand, the antibodies of the present invention can be synthesized by recombinant DNA methods well known in the art. For example, antibodies can be obtained as DNA expression products after incorporating the DNA sequences encoding the antibodies into expression vectors and introducing such vectors into suitable eukaryotic or prokaryotic cells that will express the desired antibodies from which they can subsequently be isolated by well known methods.

Accordingly, a further subject of the invention are nucleic acid molecules encoding the antibodies of the invention. In particular, the nucleic acid molecules encode the heavy chains or light chains of the antibodies of the present invention. In particular, the nucleic acid molecules contain regions encoding V _H or V _L that are at least 70%, 80%, 90%, 95% or 100% identical to the corresponding nucleic acids encoding the heavy chain variable region (V _H region) or the light chain variable region (V _L ) of any of the mAb1-mAb6 control antibodies.

Typically, such a nucleic acid is a DNA or RNA molecule that can be incorporated into any suitable vector such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector. In the present invention, the terms "vector", "cloning vector", and "expression vector" refer to a carrier by which a DNA or RNA sequence (eg, a foreign gene) can be introduced into host cells in order to transform them and cause expression ( e.g., transcription and translation) of the introduced sequence. So, the next subject of the invention is a vector containing the nucleic acid according to the invention. Such vectors may contain regulatory elements such as a promoter, enhancer, terminator, and the like, to cause or direct expression of the antibody when administered to a subject. Examples of promoters and enhancers used in animal cell expression vectors include the SV40 early promoter and enhancer, the LTR promoter and enhancer of the Moloney murine leukemia virus, the immunoglobulin H chain promoter and enhancer, and the like. Any animal cell expression vector can be used, as long as the gene encoding the C region of human antibodies can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1 beta d2-4, and the like. Other examples of plasmids include replicating plasmids containing an origin of replication or insertion plasmids such as, for example, pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenovirus, retroviral, herpesvirus and AAV vectors. Such recombinant viruses can be obtained by known methods such as transfection of packaging cells or short term transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and others. 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

A further subject of the present invention are host cells that have been transfected, infected or transformed with a nucleic acid and/or a vector as described above. In the present invention, the term "transformation" means the introduction of a "foreign" (i.e. foreign or extracellular) gene, DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to obtain the desired substance, usually a protein or an enzyme encoded by the introduced gene or sequence. Host cells that receive and express the introduced DNA or RNA are "transformed".

The nucleic acids of the invention can be used to produce antibodies of the invention in a suitable expression system. The term "expression system" means host cells and a compatible vector under suitable conditions, eg for expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cells. Common expression systems include E. coli cells and plasmid vectors, insect cells and baculovirus vectors, and mammalian cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, and the like). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., derived from lymphoblasts). , fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells with a defective dihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) (Urlaub G et al., 1980), rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as “YB2/0 cells”), and others.

The present invention also relates to a method for obtaining recombinant host cells expressing the antibodies of the invention, which includes the steps of: (i) introducing a recombinant nucleic acid or vector in vitro or ex vivo, as described above, into competent host cells, (ii) culturing the obtained recombinant host cells in vitro or ex vivo; and (iii) optionally selecting cells expressing and/or secreting these antibodies. Such recombinant host cells can be used to produce antibodies of the present invention.

The antibodies of the present invention are typically separated from the culture medium by standard immunoglobulin purification techniques such as, for example, protein A Sepharose, hydroxyapatite, gel electrophoresis, dialysis, or affinity chromatography.

In some embodiments, chimeric human antibodies of the present invention can be obtained by obtaining nucleotide sequences encoding the V _L and V _H domains as described previously, constructing an expression vector for chimeric human antibodies by inserting them into an expression vector for animal cells containing genes encoding C _H human antibodies and C _L human antibodies, and expressing the coding sequence by introducing the expression vector into animal cells. As the C _H domain of human chimeric antibodies, this can be any region that belongs to human immunoglobulin, but those of the IgG class are suitable, and any of the subclasses of the IgG class such as IgG1, IgG2, IgG3 and IgG4 can also be used. And as C _L chimeric human antibodies, it can be any region that belongs to Ig, and regions from the kappa class or the lambda class can be used. Methods for making chimeric antibodies include standard recombinant DNA and gene transfection methods well known in the art (see Morrison SL et al. (1984) and US 5,202,238 and US 5,204,244).

Humanized human antibodies of the present invention can be obtained by obtaining nucleotide sequences encoding CDR domains as described previously, constructing an expression vector for humanized antibodies by inserting them into an expression vector containing genes encoding (i) heavy chain constant region and variable framework regions. heavy chain regions identical to those of human antibodies; and (ii) a light chain constant region and variable light chain framework regions identical to those of human antibodies, and expressing genes by introducing an expression vector into a suitable cell line. Expression vector for humanized antibodies of the type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are in separate vectors, or of the type in which both genes are in the same vector (tandem type) . With respect to ease of construction of an expression vector for humanized antibodies, ease of introduction into cell lines, and balance between expression levels of antibody H and L chains in these cells, a tandem-type humanized antibody expression vector is preferred. Examples of expression vectors for humanized tandem-type antibodies include pKANTEX93 (WO 97/10354), pEE18, and others.

Techniques for humanizing antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (eg, see Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized by various methods known in the art, including, for example, CDR grafting (EP 239,400; PCT publication WO 91/09967; US. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneer, or ligation (EP 592,106; EP 519,596 Padlan EA (1991) Studnicka GM et al (1994) Roguska MA et al (1994) and strand shuffling (U.S. Pat. No. 5,565,332) The general recombinant DNA technology for making such antibodies is also known ( see European Patent Application EP 125023 and International Patent Application WO 96/02576).

The Fab fragments of the present invention can be obtained by treating antibodies specifically reactive with AMH with papain protease. The Fab can also be generated by inserting the DNA encoding the Fab of the antibody into a vector for a prokaryotic expression system or for a eukaryotic expression system, and introducing the vector into prokaryotes or eukaryotes (as required) to express the Fab.

F(ab') ₂ fragments of the present invention can be obtained by treating antibodies specifically reactive with AMH with pepsin protease. Also F(ab') ₂ can be obtained by linking Fab', described below, through a thioether bond or a disulfide bond.

Fab'-fragments of the present invention can be obtained by processing F(ab') ₂ specifically reactive with AMH, the reducing agent dithiothreitol. Fab′ can also be obtained by inserting DNA encoding the Fab′ fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into prokaryotes or eukaryotes (as required) for its expression.

The scFv of the present invention can be obtained by obtaining a cDNA encoding the V _H and V _L domains as previously described, constructing a DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or eukaryotic expression vector, and then introducing the expression vector into prokaryotes or eukaryotes (as required) for scFv expression.

To obtain humanized scFv fragments, a well-known CDR grafting technique can be used that involves selecting complementarity determining regions (CDRs) from a donor scFv fragment and grafting them into a human scFv fragment scaffold with a known three-dimensional structure (e.g., see WO 98/45322; WO 87/02671; US 5,859,205; US 5,585,089; US 4,816,567; EP 0173494).

The engineered antibodies of the present invention also include those in which modifications have been made to the framework residues within V _H and/or V _L , eg, to improve the properties of the antibodies. Typically, such framework modifications are carried out to reduce the immunogenicity of antibodies. For example, one approach is to “backmutate” one or more framework residues to the corresponding gamete sequence. In particular, an antibody that has undergone a somatic mutation may contain framework residues that differ from the gamete sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequences of the antibody with those gametic sequences from which the antibody is derived. To return the framework sequences to the gamete configuration, somatic mutations can be “back-mutated” into the gamete sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "back-mutated" antibodies are also within the scope of the invention. Another type of framework modifications involves the mutation of one or more residues within framework regions, or even within one or more regions of the CDR to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also called “deimmunization” and is described in more detail in US Patent Publication No. 2003/0153043 from Carr et al.

Engineering Fc

The antibodies of the invention may have one or more of the functional or structural features of the aspects described above, or any combination of selected functional and structural features.

The antibodies of the invention may be of any isotype. The choice of isotype is usually determined by the desired ADCC silencing-type effector functions. Typical isotypes are IgG1, IgG2, IgG3 and IgG4. Any of the human light chain constant regions can be used: kappa or lambda. If desired, the class of antibodies of the present invention can be switched by known methods. Typically, class switching techniques can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Thus, the effector functions of the antibodies of the present invention can be altered by isotype switching, eg, into IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies for various therapeutic applications. In some embodiments, the antibodies of the invention are full length antibodies. In some embodiments, the full length antibodies are IgG1 antibodies. In some embodiments, the full length antibodies are IgG4 antibodies. In some embodiments, the BTN2-specific IgG4 antibodies are stabilized IgG4 antibodies. Examples of suitable stabilized IgG4 antibodies are those in which arginine is at position 409 of the human heavy chain constant region of IgG4, which is listed in the EU index in the same way as in Kabat et al. above is replaced by lysine, threonine, methionine or leucine, preferably lysine (as described in WO 2006/033386) and/or the hinge region contains the Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO 2008/145142.

In some embodiments, the antibodies of the present invention do not contain an Fc portion that induces antibody-dependent cellular cytotoxicity (ADCC). The terms "Fc-domain", "Fc-part" and "Fc-region" refer to the C-terminal fragment of the heavy chain of antibodies, eg, from about amino acid (a.k.) 230 to a.k. 450 human gamma heavy chain or to the corresponding sequence in other types of heavy chains of antibodies (eg, α, δ, ε and μ for human antibodies) or their natural allotypes. Unless otherwise indicated, Kabat's conventional amino acid numbering for immunoglobulins is used throughout this specification (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda, MD). In some embodiments, the antibodies of the present invention do not contain an Fc domain capable of substantially binding to the FcγRIIIA (CD16) polypeptide. In some embodiments, the antibodies of the present invention lack an Fc domain (eg, lack a _CH 2 and/or _CH 3 domain) or contain an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the antibodies of the present invention consist of or include Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, diabodies, single chain antibody fragments, or multispecific antibodies containing several different antibody fragments. In some embodiments, the antibodies of the present invention are not associated with toxic molecules. In some embodiments, one or more of the amino acid residues can be replaced with other amino acid residues in order to alter the binding of the antibody to C2q and/or reduce or eliminate complement-dependent cytotoxicity (CDC). This approach is described in more detail in US Patent No. 6,194,551.

Another modification of antibodies contemplated by the present invention is pegylation. Antibodies can be pegylated, for example, to increase the biological (eg, serum) half-life of the antibodies. To pegylate antibodies, typically the antibody or antibody fragment is reacted with a polyethylene glycol (PEG) such as a reactive ester or an aldehyde derivative of PEG under conditions where one or more PEG moieties are attached to the antibody or antibody fragment. PEGylation can be carried out by an acylation reaction or by an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). In the present invention, the term "polyethylene glycol" embraces any form of PEG that is used to derivatize other proteins, such as mono-(C ₁ -C ₁₀ )-alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibodies to be pegylated are aglycosylated antibodies. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the present invention. For example, see EP 0154316 on Nishimura et al. and EP 0401384 to Ishikawa et al.

Another modification of antibodies provided by the invention is conjugation or fusion of at least the antigen-binding region of the antibody of the present invention with a serum protein such as human serum albumin or its fragment to increase the half-life of the resulting molecule.

In some embodiments of the invention, multispecific antibodies are also provided. Exemplary formats for multispecific antibody molecules of the invention include, without limitation, (i) two antibodies linked together by chemical heteroconjugation, one with specificity for BTN2 and the other with specificity for a second antigen; (ii) one antibody containing two different antigen-binding sites; (iii) a single chain antibody containing two different antigen binding sites, eg two scFvs linked in tandem via an additional peptide linker; (iv) a dual variable domain antibody (DVD-Ig) in which each light chain and heavy chain contains two variable domains in tandem through a short peptide ligament (Wu et al., Generation and characterization of a dual variable domain immunoglobulin (DVD- Ig™) molecule In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically linked bispecific (Fab') ₂ fragment; (vi) Tandab, which is the fusion of two single chain diabodies to form a tetravalent bispecific antibody that contains two binding sites for each of the target antigens; (vii) a flexitelo, which is a combination of scFv with a diabody to form a polyvalent molecule; (viii) a so-called "dock and lock" molecule based on the "dimerization and docking domain" in protein kinase A, which when applied to Fabs results in a trivalent bispecific binding protein consisting of two identical Fab fragments linked to another Fab fragment; (ix) a so-called Scorpion molecule containing, for example, two scFvs fused to both ends of a human Fab arm; and (x) diabody. Another typical format for bispecific antibodies are IgG-like molecules with complementary _CH 3 domains to enhance heterodimerization. Such molecules can be prepared by known technologies, such as technologies known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), buttonholes (Genentech), CrossMAb (Roche) and electrostatically fitted molecules (Amgen), LUZ -Y (Genentech), strand-exchange engineered domain antibodies (SEED) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies. In some embodiments, the bispecific antibodies are made or made by controlled Fab arm exchange, typically by DuoBody technology. WO 2008/119353 and WO 2011/131746 (both from Genmab A/S) describe methods for producing bispecific antibodies in vitro by controlled Fab arm exchange. In one exemplary method, described in WO 2008/119353, a bispecific antibody is formed by exchanging a "Fab arm" or "half molecule" (rearrangement of the heavy chain together with the light chain) between two monospecific antibodies containing IgG4-like C _H 3 regions, during incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms, which may have different sequences. In another exemplary method described in WO 2011/131746, the bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is an antibody of the present invention: a) obtaining a first antibody containing Fc- an immunoglobulin region, the Fc region comprising a first C _H 3 site; b) obtaining a second antibody containing the Fc-region of the immunoglobulin, and the Fc-region contains a second section of _CH 3; wherein the sequences of the first and second _CH 3 sites are different, so that the heterodimeric interaction between the first and second _CH 3 sites will be stronger than each of the homodimeric interactions between the first and second _CH 3 sites; c) incubating the first antibody together with the second antibody under reducing conditions; and d) obtaining an appropriate bispecific antibody, wherein the first antibody is an antibody of the present invention and the second antibody has a different binding specificity, or vice versa. Reducing conditions can be achieved, for example, by adding a reducing agent, eg from among 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. Step d) may further include returning the conditions to non-reducing or less reducing conditions, for example by removing the reducing agent, eg by desalting. Preferably, the sequences of the first and second _CH 3 regions are different, including only a few rather conservative asymmetric mutations, so that the heterodimeric interaction between the first and second _CH 3 regions will be stronger than either of the homodimeric interactions between the first and second _CH 3 regions. More details about these interactions and how they can be carried out, set out in WO 2011/‌131746, which is incorporated here by reference in its entirety. The following are exemplary combinations of such asymmetric mutations, optionally one or both of the Fc regions being of the IgG1 isotype.

Uses and Methods of the Invention

The antibodies or proteins of the present invention have in vitro and in vivo diagnostic and therapeutic applications. For example, these molecules can be administered to cells in culture, eg, in vitro or in vivo, or to subjects, eg, in vivo, to treat, prevent, or diagnose various diseases.

The methods are particularly suitable for treating, preventing or diagnosing BTN2 related diseases and/or autoimmune, inflammatory diseases and transplant rejection.

The invention also relates to methods for the manufacture of drugs for use in the treatment of inflammatory diseases, autoimmune diseases and rejection of organ or tissue transplants, and drugs contain antibodies against BTN2 of the present invention, as described in the previous sections.

As used herein, "BTN2-associated diseases" include diseases associated with or characterized by abnormal levels of BTN2A1 or BTN2A2 and/or diseases that can be treated by modulating BTN2A1 and/or BTN2A2 induced signaling activity in human blood cells, e.g., by inhibiting the production IFNγ or TNFα in activated Vγ9/Vδ2 T cells and/or cytolytic function of activated Vγ9/Vδ2 T cells. These include inflammatory diseases, autoimmune diseases, and rejection of organ or tissue transplants.

Examples of autoimmune diseases that can be treated include, without limitation, rheumatoid arthritis (RA), insulin dependent diabetes mellitus (type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic leukopenia, idiopathic thrombocytopenic purpura, male demon fetus, mixed disease connective tissue, myasthenia gravis, pernicious anemia, proteinaceous anaphylactic uveitis, primary biliary cirrhosis, primary myxedema, Reiter's syndrome, rigidity syndrome, thyrotoxicosis, ulcerative colitis and Wegener's granulomatosis.

The antibodies of the invention can be administered as the sole active ingredient or in combination, e.g. as an adjuvant or in combination with other drugs, e.g. immunosuppressive or immunomodulatory agents, or with other anti-inflammatory agents, e.g. for the treatment or prevention of the above diseases .

The subject of the present invention is a method for inhibiting an immune response in a subject, in particular inhibiting the cytolytic properties of Vγ9/Vδ2 T cells in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of an antibody of the present invention.

As used herein, the term "treatment" or "treat" refers to both prophylactic and curative or disease-modifying treatment, including the treatment of subjects at risk for or suspected of having a disease, as well as subjects suffering from or diagnosed with a particular disease or medical condition. , and includes the suppression of clinical relapses. Treatment may be administered to subjects with a medical condition or those who may eventually contract the disease in order to prevent, cure, slow the onset, lessen the severity or lessen one or more of the symptoms of the disease or relapse of the disease, or to prolong the life of the subject. more than expected in the absence of such treatment. By “therapeutic regimen” is meant a treatment regimen for a disease, eg a dosing regimen during therapy. The therapeutic mode may include an introductory mode and a maintenance mode. The term "introductory regimen" or "introduction period" means such a therapeutic regimen (or part of a therapeutic regimen) that is used for the initial treatment of the disease. The overall goal of the introductory regimen is to provide a high level of drug in the subject during the initial period of the treatment regimen. An introductory regimen may employ (partially or completely) a “loading regimen,” which may include administering a larger dose of the drug than the clinician would use on a maintenance regimen, administering the drug more frequently than the clinician would administer the drug on a maintenance regimen, or both. The expression “maintenance regimen” or “maintenance period” means such a therapeutic regimen (or part of a therapeutic regimen) that is used to maintain a subject during treatment of a disease, for example, to maintain a subject in remission for a long time (months or years). The maintenance regimen may include continuous therapy (eg, administration of the drug at regular intervals, eg, weekly, monthly, yearly, etc.) or intermittent therapy (eg, intermittent treatment, intermittent treatment, relapse treatment, or treatment upon reaching certain pre-set criteria (eg manifestations of the disease, etc.).

In the present invention, the term “therapeutically effective amount” means that amount which, at dosages and for the required period of time, is effective to achieve the desired therapeutic result. The therapeutically effective amount of the antibodies of the present invention may vary depending on such factors as the disease state, age, sex and weight of the individual, as well as the ability of the antibodies of the present invention to elicit the desired response in the individual. Also, a therapeutically effective amount is that amount such that any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. Effective dosages and dosage regimens for the antibodies of the present invention depend on the disease being treated and can be determined by those skilled in the art. A physician of ordinary skill in the art will readily be able to determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician may start doses of the antibodies of the present invention used in a pharmaceutical composition at a lower level than necessary to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention should be that amount of the compound that produces the lowest dose effective to produce a therapeutic effect according to the given dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize the course of a disease. Typically, the suitability of a compound for the treatment of, for example, autoimmune diseases is assessed in an animal model system predictive of efficacy in the treatment of autoimmune diseases. On the other hand, this property of the composition can be assessed by examining the ability of the compound to inhibit the induction of an immune response in in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound may reduce immune or inflammatory responses or otherwise relieve symptoms in a subject. One of ordinary skill in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen. A typical, without limitation, range for a therapeutically effective amount of antibodies of the present invention is 0.1-100 mg/kg, such as 0.1-50 mg/kg, such as 0.1-20 mg/kg, such as then 0.1-10 mg/kg, for example, about 0.5, about 0.3, about 1, about 3 mg/kg, about 5 mg/kg, or about 8 mg/kg. A typical, without limitation, range for a therapeutically effective amount of antibodies of the present invention is 0.02-100 mg/kg, such as 0.02-30 mg/kg, such as 0.05-10 mg/kg, or 0.1- 3 mg/kg, for example, 0.5-2 mg/kg. Administration, for example, may be intravenous, intramuscular, intraperitoneal, or subcutaneous, for example, may be administered proximal to the target site. Dosing regimens in the above methods of treatment and use are selected to obtain the optimal desired response (eg, therapeutic response). For example, one may administer as a single bolus, one may administer several divided doses over time, or one may proportionally decrease or increase the dose depending on the requirements of the therapeutic situation. In some embodiments, the effectiveness of the treatment is monitored during therapy, eg, at predetermined time points. In some embodiments, efficacy may be monitored by imaging of the lesion or other diagnostic methods described below, e.g., performing one or more PET-CT scans, for example, using a labeled antibody of the present invention, a fragment or mini-antibody derived from an antibody according to the present invention. If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more divided doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the human monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period of time, such as over 24 hours, to minimize any unwanted side effects. An effective dose of the antibodies of the present invention may also be administered on a weekly, bi-weekly, or three-week dosing schedule. The dosing period may be limited to eg 8 weeks, 12 weeks or until clinical progress is established. As non-limiting examples, treatment of the present invention may be given as a daily dose of antibodies of the present invention in an amount of 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1 ,1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg/kg per day on at least one of days 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after the start of treatment, or in any combination, using single or divided doses every 24, 12, 8, 6, 4 or 2 hours or in any combination.

Typically, the antibodies of the present invention are administered to subjects in the form of a pharmaceutical composition that contains a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, human serum albumin type serum proteins, phosphate buffers, glycine, sorbic acid, potassium sorbate, partial mixtures of saturated glycerides. vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium phosphate, monopotassium phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, wax, polyethylene block polymers - polyoxypropylene, polyethylene glycol and lanolin. For use in administration to patients, the compositions are formulated into dosage forms. Compositions of the present invention may be administered orally, parenterally, via an inhaler, topically, intrarectally, nasally, buccally, intravaginally, or via an implanted reservoir. The present invention employs subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion methods. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as the solvent or suspending medium. Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In the preparation of injectables, fatty acids such as oleic acid and its glyceride derivatives, as well as natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions, are useful. These oily solutions or suspensions may also contain a long chain alcohol diluent or a carboxymethyl cellulose type dispersant or similar dispersants that are commonly used in pharmaceutically acceptable dosage form formulations, including emulsions and suspensions. Other common surfactants such as Tween, Span, and other emulsifiers or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms can also be used to prepare dosage forms. Compositions of the present invention can be administered orally in any orally acceptable dosage form, including, without limitation, capsules, tablets, aqueous suspensions or solutions. In the case of oral tablets, common carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also commonly added. Useful diluents for oral administration in capsule form include, for example, lactose. When aqueous suspensions are required for oral administration, the active ingredient is mixed with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of the present invention may be administered as rectal suppositories. They can be prepared by mixing the agent with a suitable non-irritating excipient which will be solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of the present invention can also be administered topically, especially when the target for treatment includes areas or organs that are easily accessible for topical application, including diseases of the eyes, skin, or lower intestinal tract. Suitable topical forms are easily prepared for each of these areas or organs. For topical application, the compositions may be formulated as a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, without limitation, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, wax emulsifier, and water. Alternatively, the compositions may be formulated as a suitable lotion or cream containing the active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, without limitation, mineral oil, sorbitan monostearate, polysorbate 60, waxy cetyl esters, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application for the lower intestinal tract can be in the form of rectal suppositories (see above) or in the form of a suitable enema. You can also use patches. Compositions of the present invention may also be administered by nasal spray or inhalation. Such compositions are prepared according to techniques well known in the art of dosage forms and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons and/or other standard solubilizing or dispersing agents.

For example, the antibody in the pharmaceutical composition of the present invention can be supplied at a concentration of 10 mg/kg in disposable vials of 100 mg (10 ml) or 500 mg (50 ml). The product is formulated for intravenous administration in sterile water for injection with 9.0 mg/ml sodium chloride, 7.35 mg/kg sodium citrate dihydrate and 0.7 mg/kg polysorbate 80. It is adjusted to pH 6.5. A typical suitable dosage range for an antibody in a pharmaceutical composition of the present invention may be from 1 mg/m ² to 500 mg/m ² . However, it should be understood that these schedules are exemplary, and the optimal schedule and regimen can be adapted based on the affinity and tolerability of the particular antibody in the pharmaceutical composition, which must be determined in clinical trials. Pharmaceutical compositions of the invention for injection (eg IM, IV) can be formulated to contain sterile buffered water (eg 1 ml for IM) and 1 ng to 100 mg, eg 50 ng to 30 mg, more preferably 5 mg to 25 mg of an anti-BTN2 antibody of the invention.

The invention will now be illustrated with the following figures and examples. However, these examples and figures should in no way be interpreted as limiting the scope of the present invention.

Brief description of the figures

Fig. 1. BTN2 mAb binding on HEK293F cells. HEK293F cells were transfected with the BTN2A2-Flag plasmid. 1 day after transfection, BTN2A2 transfected HEK293F cells (A) and non-transfected HEK293F cells (B) were stained with purified mouse anti-human BTN2 mAb for 20 min at 4°C. An anti-Flag monoclonal antibody served as a positive transfection control. After 2 washes, the cells were incubated with secondary goat anti-mouse IgG c PE for 20 min at 4°C and with a cell viability marker. Cells were registered on LSRFortessa (BD) and analyzed using FlowJo (TreeStar).

Fig. 2. BTN2 is expressed in cancer cell lines. Cancer cells derived from solid tumors (A) or hematopoietic neoplasms (B) were stained with purified anti-BTN2 mAb (8.16) (10 μg/ml) for 20 min at 4°C. After 2 washes, the cells were incubated with secondary goat anti-mouse IgG c PE for 20 min at 4°C and with a cell viability marker. Cells were registered on LSRFortessa (BD) and analyzed using FlowJo (TreeStar). Cells originated from lines: prostate cancer (PC3, DU145, LNCaP), melanoma (Gerlach), pancreatic cancer (L-IPC, Panc-1, Mia-PACA-2), colorectal cancer (Caco-2), Hela (cancer cervix), kidney cancer (A498), lung cancer (A549), breast cancer (MDA-MB-134, MDA-MB-231, SKBR3), Burkitt's lymphoma (Daudi, Raji), non-Hodgkin's lymphoma (RL), chronic myelogenous leukemia (K562), acute myeloid leukemia (U937).

Fig. 3. Anti-BTN2 mAbs inhibit the cytolytic function of Vγ9Vδ2 T cells. γδ T cells were expanded from PBMCs from 6 healthy donors (see Materials and Methods). Purified γδ T cells were stimulated overnight with IL-2 (200 U/ml). γδ T cells were then co-cultured at 37°C with target Daudi cells (at an effector:target ratio (E:T) of 1:1) with anti-CD107a antibodies and anti-CD107b and GolgiStop antibodies, in the presence of anti-BTN2 mAbs (4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33) (10 µg/ml) or without. After 4 hours, cells were harvested, fixed and permeabilized, and then stained with intracellular mAbs (IFN-γ, TNF-α) and analyzed by flow cytometry. The figure shows (A) degranulation of γδ T cells and production of inflammatory cytokines (B) TNF-α, (C) IFN-γ. The lower dotted line represents the basal cytolytic function of γδ T cells against Daudi cells.

Fig. 4. Anti-BTN2 mAbs inhibit the agonistic effect of the anti-CD277 mAb (20.1) on the cytolytic function of Vγ9Vδ2 T cells. Purified γδ T cells were stimulated overnight with IL-2 (200 U/ml). γδ T cells were then co-cultured at 37°C with target Daudi cells (effector:target ratio (E:T) = 1:1) with anti-CD107a and anti-CD107b and GolgiStop, in the presence of anti-BTN2 mAbs (4.15 , 5.28, 7.28, 7.48, 8.15, 8.16, 8.33) or not with or without anti-CD277 mAb 20.1. After 4 hours, cells were harvested and analyzed by flow cytometry. The figure shows the percentage of positive for CD107 (marker of degranulation) cells among γδ T-cells. The lower dotted line represents basal degranulation of γδ T cells against Daudi cells. The upper dotted line represents the mean level of degranulation in the presence of anti-CD277 antibody 20.1.

Fig. 5. Anti-BTN2 mAbs do not cross-react with HEK293F cells transfected with CD277 isoforms (BTN3A1, BTN3A2, or BTN3A3). HEK293F cells transfected with BTN3A1, BTN3A2, BTN3A3 and untransfected HEK293F cells were stained with purified mouse anti-human BTN2 mAb or mouse anti-human CD277 mAb 20.1 or isotype control IgG1 for 20 min at 4°C. An anti-Flag antibody served as a positive transfection control. After 2 washes, the cells were incubated with secondary goat anti-mouse IgG with PE for 20 min at 4°C and with a cell viability marker. Cells were registered on LSRFortessa (BD) and analyzed using FlowJo (TreeStar).

EXAMPLES

Materials and methods

Cell cultures

Peripheral blood mononuclear cells (PBMC) were obtained from healthy voluntary donors (HV) provided by the local blood bank (EFS-Marseille-France) and isolated using a density gradient (Eurobio).

Burkitt's Daudi lymphoma cells were obtained from the American Type Culture Collection (ATCC) and cultured (0.5×10 ⁶ /ml) in RPMI 1640 medium with 10% FCS.

Propagation of T-cells γδ

γδ effector T cells were generated as previously described. On day 0, HV PBMCs were stimulated with zoledronate (Sigma, 1 μM) and rhIL-2 (Proleukin, 200 IU/ml). From day 5, rhIL-2 was renewed every two days and the cells were stored at 1.15×10 ⁶ /ml for 15 days. On the last day, the purity of γδ T cells was determined by flow cytometry. Only cell cultures that reached more than 80% of γδ T cells were selected for use in functional tests. Purified γδ T cells were thawed before use.

Production of monoclonal antibodies (mAbs)

Mouse antibodies against human BTN2 (clones 4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33 with IgG1 isotype) and mouse anti-human CD277 (also known as BTN3A; clone 20.1 with IgG1 isotype) were isolated from cell culture supernatants.

flow cytometry

PBMCs, purified γδ T cells or cancer line cells were incubated with the indicated mAbs and analyzed for LSRFortessa (Becton Dickinson) using the DIVA program (BD Bioscience). Antibodies used for the γδ T cell degranulation assay used the following: anti-CD107a-FITC (BD Biosciences), anti-CD107b-FITC (BD Biosciences), anti-CD3-PeVio700 (Miltenyi), anti-Tgd-PE (Miltenyi), live /dead in near IR (ThermoFisher). The following antibodies were used to screen for BTN2 expression in cancer cells: purified against BTN2 (clone 8.16, 10 μg/mL), FcR Block reagent (Miltenyi), goat against PE mouse (Jackson Immunoresearch), live/dead in near IR (ThermoFisher ).

Functional assays on γδ T cells

Purified HV γδ T cells were cultured overnight with IL-2 (200 U/ml). Then, γδ T-cells were cultured at 37°C together with target Daudi cells (at an effector:target ratio (E:T) = 1:1) and cytotoxic tests were performed in 4-hour samples in the presence of GolgiStop and soluble CD107(a&b) -FITC, with or without anti-BTN2 mAbs (4.15, 5.28, 7.28, 7.48, 8.15, 8.16, 8.33) and/or with anti-CD277 mAb 20.1 (10 µg/mL), with or without phosphoagonist (Pag) activation. After 4 hours, cells were harvested, fixed and permeabilized and then stained with intracellular mAbs (IFN-γ, TNF-α). Finally, cells were resuspended in PBS with 2% paraformaldehyde and immediately analyzed for BD LSRFortessa (BD Biosciences, San Jose, CA). The level of cytolytic function of γδ T cells was measured based on the percentage of cells positive for CD107a and CD107b (degranulation) and/or production of inflammatory cytokines (IFN-γ, TNF-α).

Anti-BTN2 mAb binding assay on BTN2A1 and BTN2A2 expressing cells

HEK-293F cells carrying CRISPR-Cas9-mediated deletions of all BTN3 and BTN2 isoforms (293F BTN3/BTN2 KO) were generated (data not shown), cultured in DMEM (Life Technologies) with 10% fetal bovine serum (FBS, Gibco) and 1 mM sodium pyruvate (ThermoFisher Scientific) and transfected independently with pcDNA3-Zeo-BTN2A1-CFP or pcDNA3-Zeo-BTN2A2-CFP encoding CFP(Nter) fusion proteins BTN2A1 and BTN2A2 using Lipofectamine 2000 reagent (ThermoFisher) according to manufacturer's instructions.

flow cytometry

Cells were harvested 24 hours after transfection (5×10 ⁴ per sample) and stained in duplicate at the indicated concentrations (2-fold dilutions starting at 20 μg/mL and up to 64 pg/mL) of all 7 purified anti-human BTN2 antibodies in 50 µl of staining buffer (DPBS1X (ThermoFisher Scientific), 1% FBS, 1 mM EDTA (ThermoFisher Scientific)) for 30 min at 4°C. The same concentrations of mouse anti-IgG1 antibody (Miltenyi) were used as isotype control for staining. Cells were then washed twice with 200 μl of staining buffer and incubated with PE-conjugated goat anti-mouse Ig at a 1:200 dilution (Jackson ImmunoResearch) in staining buffer for 30 min at 4°C in the dark. Finally, cells were washed twice in staining buffer and then fixed with BD Cytofix reagent (BD Bioscience) according to the manufacturer's instructions. The mean fluorescence intensity (MFI) per channel for PE in the CFP positive population was determined for each sample on Cytoflex LX (Beckman Coulter) and analyzed using FlowJo v10.4.2 software (FlowJo, LLC 2006-2018).

Statistics

EC ₅₀ of purified anti-human BTN2 mAbs were determined on transfected BTN2A1 and BTN2A2 293F BTN3/BTN2 KO cells based on log(dose)-response curves after non-linear regression on a variable slope model. These analyzes were performed using GraphPad Prism 7.04 (GraphPad).

T cell proliferation γδ

γδ T-cells were isolated from PBMCs from healthy donors using an anti-TCR γδ microbead kit (Miltenyi Biotec). The purity of γδ T cells as assessed by flow cytometry was over 80%. γδ T cells were labeled with CellTrace Violet for 20 min at 37°C. Then 5×10 ⁵ CellTrace labeled cells were cultured in 96-well round bottom plates in the presence of 200 IU/ml IL-2 with or without PAg and with or without anti-BTN2 antibodies (10 μg/ml). After 5 days of culture, CellTrace dilution was determined by flow cytometry on a BD LSRFortessa (BD Biosciences, San Jose, CA).

Statistics

Results were expressed as median ± SEM. Statistical analysis was performed using Spearman correlation, Wilcoxon test and Mann-Whitney t-test. p<0.05 values were considered significant. Analyzes were performed using the GraphPad Prism software.

RESULTS

Identification of reference antibodies mAb1-mAb7

Reference antibodies mAb1-mAb7 were prepared as follows.

Mice were immunized with BTN2A1-Fc antigen. Mouse splenocytes were harvested and fused with myeloma to form hybridomas. Hybridomas producing antibodies with the highest affinity for BTN2 were screened and isolated, resulting in hybridomas deposited as CNCM I-5231, CNCM I-5232, CNCM I-5233, CNCM I-5234, CNCM I-5235, CNCM I-5236 and CNCM I-5237 capable of producing mAb1-mAb7, respectively.

A hybridoma producing mAb7 (mAb 8.33), which serves as a reference control for the antibodies of the present invention, was deposited on September 14, 2017 at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France ) in accordance with the terms of the Budapest Treaty.

The deposited hybridoma for mAb 8.33 has CNCM deposit number CNCM I-5237.

Binding of mAbs 1-6 to HEK293F cells

On the graphs from Fig. 1 shows affinity titration curves for mouse anti-human BTN2 mAbs on human BTN2A2 transfected HEK293F cells (FIG. 1A) or untransfected HEK293F cells (FIG. 1B).

The table below lists the EC ₅₀ values for each antibody. All antibodies tested were able to recognize and bind to BTN2A2 on HEK cells, with the exception of mAb 8.33 (mAb7), which did not bind to BTN2A2 according to flow cytometry.

Antibody clone name EC ₅₀ (mcg/ml) mAb1 mAb 4.15 0.07 mAb2 mAb 5.28 0.07 mAb3 mAb 7.28 31.3 mAb4 mAb 7.48 0.08 mAb5 mAb 8.15 0.10 mAb6 mAb 8.16 0.11 mAb7 mAb 8.33 -

BTN2 polypeptide is expressed in cancer cell lines

Cancer cell lines were incubated with mAb6 (mouse anti-BTN2 mAb 8.16) and then with secondary goat anti-mouse IgG with PE. As can be seen from FIG. 2, broad expression of the BTN2 protein was observed in a panel of cancer cell lines derived from solid tumors or hematopoietic neoplasms, including the Daudi cell line (Burkitt's lymphoma), a standard cell line used in the Vγ9Vδ2 T cell degranulation assay.

mAbs 1-6 inhibit cytolytic function of Vγ9Vδ2 T cells

Vγ9Vδ2 T cells isolated from PBMCs from healthy donors were expanded. Vγ9Vδ2 T cells were co-cultured with target Daudi cells. As can be seen from FIG. 3, the addition of anti-BTN2 mAb 1-6 leads to inhibition of the cytolytic function of Vγ9Vδ2 T cells against target Daudi cells as measured by CD107 degranulation and production of inflammatory cytokines (TNF-α, IFN-γ).

The control antibody mAb7 (mAb 8.33), which does not bind to BTN2A2 as determined by flow cytometry, does not affect Vγ9Vδ2 T cell degranulation (cytolytic function) or inflammatory cytokine production.

Anti-CD277 agonist antibody 20.1 serves as a reference for an antibody that promotes Vγ9Vδ2 T cell degranulation, and anti-CD277 antagonist antibody 103.2 serves as a reference for an antibody that inhibits Vγ9Vδ2 T cell degranulation.

mAbs 1-6 inhibit the agonistic effect of anti-CD277 mAb (20.1) on the cytolytic function of Vγ9Vδ2 T cells

We also tested the effect of a combination of anti-BTN2 mAbs 1-6 in the presence of an anti-CD277 agonist mAb 20.1 (as described in WO 2012/080351), which enhances Vγ9Vδ2 T cell degranulation.

As can be seen from FIG. 4, mAbs 1, 2, and 4 (anti-BTN2 mAbs 4.15, 5.28, 7.48, respectively) unexpectedly inhibit the agonistic effect of anti-CD277 antibody 20.1 on the cytolytic function of Vγ9Vδ2 T cells against target Daudi cells.

mAbs 5 and 6 (anti-BTN2 mAbs 8.15, 8.16, respectively) partially inhibited the agonistic effect of anti-CD277 antibody 20.1 on the cytolytic function of Vγ9Vδ2 T cells against target Daudi cells.

mAb3 (anti-BTN2 mAb 7.28) and mAb7 (anti-BTN2 mAb 8.33) did not significantly inhibit the agonistic effect of anti-CD277 antibody 20.1 on the cytolytic function of Vγ9Vδ2 T cells against target Daudi cells.

The combination of a 103.2 antagonist and a 20.1 anti-CD277 agonist serves as a control: antagonist antibody 103.2 inhibits the agonistic effect of antibody 20.1 on the cytolytic function of Vγ9Vδ2 T cells against Daudi target cells, as previously described.

Characterization results for mAbs 1-6 (and control mAb7) are summarized in the table below.

Clone EC ₅₀ (μg/ml) on HEK-BTN2 cells Effect on γδ/CD107 T cells Effect on γδ/CD107 T-cells in pres. 20.1 4.15 0.07 inhibition inhibition 5.28 0.07 inhibition inhibition 7.28 31.30 inhibition No 7.48 0.08 inhibition inhibition 8.15 0.10 inhibition frequent inhibition 8.16 0.11 inhibition frequent inhibition 8.33 - No No

mAbs 1-6 do not cross-react with HEK293F cells transfected with CD277 isoforms (BTN3A1, BTN3A2, or BTN3A3)

The binding of anti-BTN2 mAbs to HEK293F cells transfected with any CD277 isoform (BTN3A1, BTN3A2, or BTN3A3) was similar to that observed on untransfected HEK293F cells (see FIG. 5). It can be concluded that anti-BTN2 mAbs do not cross-react with any CD277 isoform.

mAbs 1-6 have binding specificity for human BTN2A1 and BTN2A2 isoforms

mAbs 1-6 binding on HEK293F BTN3/BTN2 KO cells

The EC ₅₀ values of each BTN2A1 and BTN2A2 antibody are shown in the table below. All tested anti-BTN2 antibodies bound to both isoforms, with the exception of mAb 8.33 (mAb7), which showed no staining on flow cytometry.

Antibody clone name BTN2A1:
EC ₅₀ (mcg/ml) BTN2A2:
EC ₅₀ (mcg/ml) mAb1 mAb 4.15 0.06 0.01 mAb2 mAb 5.28 0.04 0.01 mAb3 mAb 7.28 0.02 11.8 mAb4 mAb 7.48 0.02 0.01 mAb5 mAb 8.15 0.08 0.5 mAb6 mAb 8.16 0.08 2.4 mAb7 mAb 8.33 - -

pivot table

Degranulation Tγδ
(inhibition in %) Antibody clone name BTN2A1:
EC ₅₀ (mcg/ml) BTN2A2:
EC ₅₀ (mcg/ml) on target cells (Daudi) on Daudi cells + mAb to BTN3 (20.1) mAb1 mAb 4.15 0.06 0.01 >90% >90% mAb2 mAb 5.28 0.04 0.01 mAb3 mAb 7.28 0.02 11.8 ≤10% mAb4 mAb 7.48 0.02 0.01 >90% mAb5 mAb 8.15 0.08 0.5 ≤50% mAb6 mAb 8.16 0.08 2.4 mAb7 mAb 8.33 - - No No

Nucleotide and amino acid sequences for the implementation of the claimed invention

SEQID NO: Short description Subsequence 1 a.k. BTN2A1 sequence MESAAALHFS RPASLLLLLL SLCALVSAQF IVVGPTDPIL ATVGENTTLR CHLSPEKNAE DMEVRWFRSQ FSPAVFVYKG GRERTEEQME EYRGRTTFVS KDISRGSVAL VIHNITAQEN GTYRCYFQEG RSYDEAILHL VVAGLGSKPL
ISMRGHEDGG IRLECISRGW YPKPLTVWRD PYGGVAPALK EVSMPDADGL FMVTTAVIIR DKSVRNMSCS INNTLLGQKK ESVIFIPESF MPSVSPCAVA LPIIVVILMI PIAVCIYWIN KLQKEKKILS GEKEFERETR EIALKELEKE
RVQKEEELQV KEKLQEELRW RRTFLHAVDV VLDPDTAHPD LFLSEDRRSV RRCPFRHLGE SVPDNPERFD SQPCVLGRES FASGKHYWEV EVENVIEWTV GVCRDSVERK GEVLLIPQNG FWTLEMHKGQ YRAVSSPDRI LPLKESLCRV
GVFLDYEAGD VSFYNMRDRS HIYTCPRSAF SVPPVRPFFRL GCEDSPIFIC PALTGANGVT VPEEGLTLHR VGTHQSL 2 a.k. BTN2A2 sequence MEPAAALHFS LPASLLLLLL LLLLSLCALV SAQFTVVGPA NPILAMVGEN TTLRCHLSPE KNAEDMEVRW FRSQFSPAVF VYKGGRERTE EQMEEYRGRI TFVSKDINRG SVALVIHNVT AQENGIYRCY FQEGRSYDEA ILRLVVAGLG
SKPLIEIKAQ EDGSIWLECI SGGWYPEPLT VWRDPYGEVV PALKEVSIAD ADGLFMVTTA VIIRDKYVRN VSCSVNNTLL GQEKETVIFI PESFMPSASP WMVALAVILT ASPWMVSMTV ILAVFIIFMA VSICCIKKLQ REKKILSGEK KVEQEEKEIA QQLQEELRWR RTFLHAADVV LDPDTAHP EL FLSEDRRSVR RGPYRQRVPD NPERFDSQPC VLGWESFASG KHYWEVEVEN VMVWTVGVCR HSVERKGEVL LIPQNGFWTL EMFGNQYRAL SSPERILPLK ESLCRVGVFL
DYEAGDVSFY NMRDRSHIYT CPRSAFTVPV RPFFRLGSDD SPIFICPALT GASGVMVPEE GLKLHRVGTH QSL 3 a.k. HCDR1
mAb 4.15 SYDIN 4 a.k. HCDR2
mAb 4.15 WIFPGDDSIIQNEKFKG 5 a.k. HDDR3
mAb 4.15 LGPLRGFTY 6 a.k. LCDR1
mAb 4.15 RASESVDRYGSSSFMH 7 a.k. LCDR2
mAb 4.15 RASNLES 8 a.k. LCDR3
mAb 4.15 QQSNEDPWT 9 a.k. _VH mAb 4.15 MGWSWVFLFLLSVTAGVHSQVQLQQSGAELVKPGASVKLSCKASGYIFTSYDINWVRQRPEQGLEWIGWIFPGDDSIIQNEKFKGKATLTTDKSSSTVYMQLSRLTSEDSAVYFCARLGPLRGFTYWGQGTLVTVSA 10 a.k. V _L mAb 4.15 METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDRYGSSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPWTFGGGTKLEIK eleven a.k. HCDR1
mAb 5.28 DYSMN 12 a.k. HCDR2
mAb 5.28 RINTETGEPTYADDFKG 13 a.k. HDDR3
mAb 5.28 DYAKR 14 a.k. LCDR1
mAb 5.28 KASQDVITAVA 15 a.k. LCDR2
mAb 5.28 STSYRYT 16 a.k. LCDR3
mAb 5.28 LQHYTTPWT 17 a.k. _VH mAb 5.28 MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWVGRINTETGEPTYADDFKGRFAFSLETSASTAYLQIKNLKNEDTATYFCVRDYAKRWGQGTSVTVSS 18 a.k. V _L mAb 5.28 MGIKMESQIQVFVFVSLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVITAVAWYQQKPGQSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTISSVQAEDLAVYYCLQHYTTPWTFGGGTKLEIK 19 a.k. HCDR1
mAb 7.28 SYWIE 20 a.k. HCDR2
mAb 7.28 EILPGSGSTKYNEKFRG 21 a.k. HDDR3
mAb 7.28 LKGYYGGGAMDY 22 a.k. LCDR1
mAb 7.28 RASKSISKYLA 23 a.k. LCDR2
mAb 7.28 SGSTLQS 24 a.k. LCDR3
mAb 7.28 QQHNEYPWT 25 a.k. V _H mAb 7.28 MEWTWVFLFLLSVTAGVHSQVHLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGREWIGEILPGSGSTKYNEKFRGKATFAADTSSNTAYVQLSSLTSEDSAVYYCARLKGYYGGGAMDYWGQGTSVTVSS 26 a.k. V _L mAb 7.28 MRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNELLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK 27 a.k. HCDR1
mAb 7.48 DFYMY 28 a.k. HCDR2
mAb 7.48 TISDGGSHTYYPDSVKG 29 a.k. HDDR3
mAb 7.48 DTTIITPY thirty a.k. LCDR1
mAb 7.48 RSSTGAVTTSNYAN 31 a.k. LCDR2
mAb 7.48 GTNNRAP 32 a.k. LCDR3
mAb 7.48 GLWYSNHWV 33 a.k. _VH mAb 7.48 MNFGLSLIFLVLVLKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFTFSDFYMYWVRRTPEKRLEWVATISDGGSHTYYPDSVKGRFTISRDNAKNNLYLQMRSLKSEDTAMYYCGRDTTIITPYWGQGTLVTVSA 34 a.k. V _L mAb 7.48 MAWISLILSLLALSSGAISQSVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCGLWYSNHWVFGGGTKLTVL 35 a.k. HCDR1
mAb 8.15 GYWMT 36 a.k. HCDR2
mAb 8.15 EINPDSSTINYTPSLRD 37 a.k. HDDR3
mAb 8.15 GSYYPSY 38 a.k. LCDR1
mAb 8.15 RASKVSSSGYSYMN 39 a.k. LCDR2
mAb 8.15 LASNLES 40 a.k. LCDR3
mAb 8.15 QHSRELPHT 41 a.k. V _H mAb 8.15 MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASGFDFSGYWMTWVRQAPGKGLEWIGEINPDSSTINYTPSLRDKFIISRDNAKNTLYLQMSKVRSEDTALYFCARGSYYPSYWGQGTLVTVSA 42 a.k. V _L mAb 8.15 METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSYMNWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEDEDAATYYCQHSRELPHTFGGGTKLEIK 43 a.k. HCDR1
mAb 8.16 GYWMT 44 a.k. HCDR2
mAb 8.16 EINPDSSTINYTPSLRD 45 a.k. HDDR3
mAb 8.16 GSYYPSY 46 a.k. LCDR1
mAb 8.16 RASKVSSSGYSYMN 47 a.k. LCDR2
mAb 8.16 LASNLES 48 a.k. LCDR3
mAb 8.16 QHSRELPHT 49 a.k. V _H mAb 8.16 MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASGFDFSGYWMTWVRQAPGKGLEWIGEINPDSSTINYTPSLRDKFIISRDNAKNTLYLQMSKVRSEDTALYFCARGSYYPSYWGQGTLVTVSA 50 a.k. V _L mAb 8.16 METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSYMNWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEDEDAATYYCQHSRELPHTFGGGTKLEIK 51 a.k. HCDR1
mAb 8.33 SGYYWN 52 a.k. HCDR2
mAb 8.33 YISYDGNNNYNPSLKN 53 a.k. HDDR3
mAb 8.33 PLYDGYYWYFDV 54 a.k. LCDR1
mAb 8.33 ITSTDIDDDMN 55 a.k. LCDR2
mAb 8.33 EANTLRP 56 a.k. LCDR3
mAb 8.33 LQSDNLPYT 57 a.k. V _H mAb 8.33 MKVLSLLYLLTAIPGILSDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGNNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCASPLYDGYYWYFDVWGAGTTVTVSS 58 a.k. V _L mAb 8.33 MTMFSLALLLSLLLLCVSDSRAETTVTQSPASLSLAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEANTLRPGVPSRFSSSGRGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK 59 nt. HCDR1
mAb 4.15 AGCTATGATATAAAC 60 nt. HCDR2
mAb 4.15 TGGATTTTTCCTGGAGATGATAGTATTATTCAGAATGAGAAGTTCAAGGGC 61 nt. HDDR3
mAb 4.15 TTGGGCCCATTACGAGGGTTTACTTAC 62 nt. LCDR1
mAb 4.15 AGAGCCAGTGAAAGTGTTGATCGTTATGGCAGTAGTTTTATGCAC 63 nt. LCDR2
mAb 4.15 CGTGCATCCAACCTAGAATCT 64 nt. LCDR3
mAb 4.15 CAGCAAAGTAATGAGGATCCGTGGACG 65 nt. _VH mAb 4.15 ATGGGATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAACTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACATCTTCACAAGCTATGATATAAACTGGTGAGGCAGAGGCCTGAACAGGACTTGAGTGGATTGGATGGATTTTTCCTGGAGATG ATAGTATTATTCAGAATGAGAAGTTCAAGGGCAAGGCCACACTGACTACAGACAAATCCTCCAGCACAGTCTACATGCAGCTCAGCAGGCTGACATCTGAGACTCTGCTGTCTATTTCTGTGCAAGATTGGGCCCATTACGAGGGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGGGTTTACTTACTGGGGCCAAGGGACTCTGGTCACTG TCTCTGCA 66 nt. V _L mAb 4.15 ATGGAGACAGAACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTGACATTGTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATCGTTATGGCAGTAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAA CCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 67 nt. HCDR1
mAb 5.28 GACTATTCAATGAAC 68 nt. HCDR2
mAb 5.28 AGGATCAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAGGGA 69 nt. HDDR3
mAb 5.28 GACTACGCTAAGCGG 70 nt. LCDR1
mAb 5.28 AAGGCCAGTCAGGATGTGATTACTGCTGTAGCC 71 nt. LCDR2
mAb 5.28 TCGACATCCTACCGGTACACT 72 nt. LCDR3
mAb 5.28 CTGCAACATTATACTACTCCGTGGACG 73 nt. _VH mAb 5.28 ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCAAAGTATCCAAGCACAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGAACTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGGTGGGCAGGATCAACACTGAG ACTGGTGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAAAAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGTTAGACTACGCTAAGCGGTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 74 nt. V _L mAb 5.28 ATGGGCATCAAAATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTCTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGATTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGACATCCTA CCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGTCAGGCTGAAGACCTGGCAGTTTATTACTGTCTGCAACATTATACTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 75 nt. HCDR1
mAb 7.28 AGCTACTGGATAGAG 76 nt. HCDR2
mAb 7.28 GAGATTTTACCTGGAAGTGGAAGTACTAAGTACAATGAGAAGTTTAGGGGC 77 nt. HDDR3
mAb 7.28 TTGAAGGGTTACTACGGAGGAGGTGCTATGGACTAC 78 nt. LCDR1
mAb 7.28 AGGGCAAGTAAGAGCATTAGCAAATATTTAGCC 79 nt. LCDR2
mAb 7.28 TCTGGATCCACTTTGCAATCT 80 nt. LCDR3
mAb 7.28 CAACAGCATAATGAATACCCGTGGACG 81 nt. V _H mAb 7.28 ATGGAATGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCACCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAAATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCGTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGA AGTACTAAGTACAATGAGAAGTTTAGGGGCAAGGCCACATTCGCTGCAGATACATCCTCCAACACAGCCTACGTGCAACTCAGCAGCCTGACATCTGAGACTCTGCCGTCTATTACTGTGCAAGATTGAAGGGTTACTACGGAGGAGGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTTACCGTCTCTTCA 82 nt. V _L mAb 7.28 ATGAGGTTCCAGGTTCAGGTTCTGGGGCTCCTTCTGCTCTGGATATCAGGTGCCCAGTGTGTGATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATGAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGA ATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATAATGAATACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 83 nt. HCDR1
mAb 7.48 GACTTTTACATGTAT 84 nt. HCDR2
mAb 7.48 ACCATTAGTGATGGTGGTAGTCACACCTACTATCCAGACAGTGTGAAGGGG 85 nt. HDDR3
mAb 7.48 GATACTACGATAATTACTCCTTAC 86 nt. LCDR1
mAb 7.48 CGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAAC 87 nt. LCDR2
mAb 7.48 GGTACCAAACAACCGAGCTCCA 88 nt. LCDR3
mAb 7.48 GGTCTTTGGTACAGCAACCATTGGGTG 89 nt. _VH mAb 7.48 ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTTTTACATGTATTGGGTTCGCCGGACTCCGGAAAAGGCTGGAGTGGGTCGCAACCATTAGTGATG GTGGTAGTCACACCTACTATCCAGACAGTGTGAAGGGGCGATTACCATCTCCAGAGACAATGCCAAGAACAACCTCTACCTACAAATGAGAAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGGAAGAGATACTACGATAATTACTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 90 nt. V _L mAb 7.48 ATGGCCTGGATTTCACTTATACTCTCTCTCCTGGCTCTCAGCTCAGGGGCCATTTCCCAGTCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCTCCAG GTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGGTCTTTGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 91 nt. HCDR1
mAb 8.15 GGATACTGGATGACT 92 nt. HCDR2
mAb 8.15 GAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAGGGAT 93 nt. HDDR3
mAb 8.15 GGGAGCTACTATCCCTCTTAC 94 nt. LCDR1
mAb 8.15 AGGGCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGAAC 95 nt. LCDR2
mAb 8.15 CTTGCATCCAACCTAGAATCT 96 nt. LCDR3
mAb 8.15 CAGCACAGTAGGGAGCTTCCGCACACG 97 nt. V _H mAb 8.15 ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAAGTGAAGCTTCTCGAGTCTGGAGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTGTGGATACTGGATGACTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGC AGTACGATAAACTATACGCCATCTCTAAGGGATAAATTCATCATCTCCAGAGACAACGCCAAGAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTTCTGTGCAAGAGGGAGCTACTATCCCTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 98 nt. V _L mAb 8.15 ATGGAGACAGAACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGGACATTGTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGCCAGCAAAAGTGTCAGTTCATCTGGGCTATAGTTATATGAACTGGTACCAGCAGAAACCAGGACAGCCACCAAAACTCCTCATCTATCTTGCATCC AACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGATGAGGATGCTGCAACCTATTACTGTCAGCAACAGTAGGGAGCTTCCGCACACGTTCGGAGGGGGACCAAGCTGGAAATAAAA 99 nt. HCDR1
mAb 8.16 GGATACTGGATGACT 100 nt. HCDR2
mAb 8.16 GAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAGGGAT 101 nt. HDDR3
mAb 8.16 GGGAGCTACTATCCCTCTTAC 102 nt. LCDR1
mAb 8.16 AGGGCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGAAC 103 nt. LCDR2
mAb 8.16 CTTGCATCCAACCTAGAATCT 104 nt. LCDR3
mAb 8.16 CAGCACAGTAGGGAGCTTCCGCACACG 105 nt. V _H mAb 8.16 ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGTCCAGTGTGAAGTGAAGCTTCTCGAGTCTGGAGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTGTGGATACTGGATGACTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGC AGTACGATAAACTATACGCCATCTCTAAGGGATAAATTCATCATCTCCAGAGACAACGCCAAGAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTTCTGTGCAAGAGGGAGCTACTATCCCTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 106 nt. V _L mAb 8.16 ATGGAGACAGAACACACTCCTGTTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGGACATTGTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGCCAGCAAAAGTGTCAGTTCATCTGGGCTATAGTTATATGAACTGGTACCAGCAGAAACCAGGACAGCCACCAAAACTCCTCATCTATCTTGCATCC AACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGATGAGGATGCTGCAACCTATTACTGTCAGCAACAGTAGGGAGCTTCCGCACACGTTCGGAGGGGGACCAAGCTGGAAATAAAA 107 nt. HCDR1
mAb 8.33 AGTGGTTATTACTGGAAC 108 nt. HCDR2
mAb 8.33 TACATAAGCTACGACGGTAACAATAACTACAACCCATCTCTCAAAAAAT 109 nt. HDDR3
mAb 8.33 CCCTCTATGATGGTTATTACTGGTACTTCGATGTC 110 nt. LCDR1
mAb 8.33 ATAACCAGCACTGATATTGATGATGATGATATGAAC 111 nt. LCDR2
mAb 8.33 GAAGCCAATACTCTTCGTCCT 112 nt. LCDR3
mAb 8.33 TTGCAAAGTGATAACTTGCCGTACACG 113 nt. V _H mAb 8.33 ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCAGGAAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAACAATA ACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACGTCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAAGCTACATATTACTGTGCAAGTCCTCTCTATGATGGTTATTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA 114 nt. V _L mAb 8.33 ATGACCATGTTCTCACTAGCTCTTCTCTCAGTCTTCTTCTCCTCTGTGTCTCTGATTCTAGGGCAGAAACAACTGTGACCCAGTCTCAGCATCCCTGTCCCTGGCTATAGGAGAAAAAGTCACCATCAGATGCATAACCAGCACTGATATTGATGATGATATGAACTGGTACCAGCAGAAGCCAGGGGAACCTCCTAAGCTCCTTATTTCAGAAGCCAATACTCTTCGTC CTGGAGTCCCATCCCGATTCTCCAGCAGTGGCCGTGGTACAGATTTTGTTTTTACAATTGAAAACATGCTCTCAGAAGATGTTGCAGATTACTACTGTTTGCAAAGTGATAACTTGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

Links

In the present application, various references describe the prior art to which this invention pertains. The contents of these references are hereby incorporated by reference into the present specification.

Claims (25)

1. Anti-BTN2 antibody with specificity for human butyrophilin-2A1 (BTN2A1) and human butyrophilin-2A2 (BTN2A2), characterized in that it contains either: (i) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 4.15 sequences SEQ ID NOs: 3-8, respectively; (ii) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 5.28 sequences SEQ ID NOs: 11-16, respectively; (iii) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 7.28 sequences SEQ ID NOs: 19-24, respectively; (iv) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 7.48 sequences SEQ ID NOs: 27-32, respectively; (v) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 8.15 sequences SEQ ID NOs: 35-40, respectively; or (vi) H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 from mAb 8.16 sequences SEQ ID NOs: 43-48 respectively, and is also characterized in that it has at least one of the following functions: vii) it inhibits the production of IFN-γ and TNF-α by activated Vγ9/Vδ2 T cells; viii) it inhibits the cytolytic function of activated Vγ9/Vδ2 T cells; ix) it inhibits the proliferation of activated Vγ9/Vδ2 T cells. 2. Anti-BTN2 antibody according to claim 1, which contains either: (i) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 9, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 10; (ii) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 17, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 18; (iii) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 25, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 26; (iv) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 33, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 34; (v) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 41, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 42; or (vi) a heavy chain in which the V _H region is at least 95% identical to SEQ ID NO: 49, and a light chain in which the V _L region is at least 95% identical to SEQ ID NO: 50. 3. An anti-BTN2 antibody according to any one of the preceding claims, characterized in that it does not cross-react with human CD277. 4. An anti-BTN2 antibody according to any one of the preceding claims which inhibits the cytolytic function of Vγ9/Vδ2 T cells in the presence of the anti-CD277 mAb20.1 agonist antibody. 5. An anti-BTN2 antibody according to any one of the preceding claims, which is a human, chimeric or humanized antibody. 6. A nucleic acid molecule that encodes a heavy chain and a light chain of an anti-BTN2 antibody according to any one of paragraphs. 1-5. 7. A host cell comprising a nucleic acid according to claim 6 for use in the treatment of a disease selected from the group consisting of: rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (type 1 diabetes), multiple sclerosis (MS), Crohn's disease , systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain's syndrome -Barre, disease Hashimoto's, idiopathic leukopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, proteinaceous anaphylactic uveitis, primary biliary cirrhosis, primary myxedema, Reiter's syndrome, rigidity syndrome, thyrotoxicosis, ulcerative colitis, and Wegener's granulomatosis. 8. An anti-BTN2 antibody according to any one of paragraphs. 1-5 for use in the treatment of a disease selected from the group consisting of: rheumatoid arthritis (RA), insulin dependent diabetes mellitus (type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, syndrome Sjogren, pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic leukopenia, idiopathic thrombocytopenic purpura, male infertility , mixed connective tissue disease, myasthenia gravis, pernicious anemia, proteinaceous anaphylactic uveitis, primary biliary cirrhosis, primary myxedema, Reiter's syndrome, rigidity syndrome, thyrotoxicosis, ulcerative colitis and Wegener's granulomatosis. 9. Pharmaceutical composition containing an antibody against BTN2 according to any one of paragraphs. 1-5 and at least one pharmaceutically acceptable carrier, for use in the treatment of a disease selected from the group consisting of: rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (type 1 diabetes), multiple sclerosis (MS), Crohn's disease, systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, pemphigus vulgaris, pemphigoid, Addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome , Hashimoto's disease, idiopathic leukopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, proteinaceous anaphylactic uveitis, primary biliary cirrhosis, primary myxedema, Reiter's syndrome, rigidity syndrome, thyrotoxicosis, ulcerative colitis and Wegener's granulomatosis.

Images