Antibodies with specificity for BTN2 and uses thereof

Abstract

The present invention relates to antibodies with specificity for BTN2A and their use, in particular for the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to anti-BTN2A1 activating antibodies that bind to BTN2A1 and shift the macrophage population towards anti-tumor M1 macrophages, directly activate NK cells, and have cytotoxic effects against cancer cells. Alternatively, or in combination, the antibodies may activate Vy9 / V52 T cells. Such antibodies are particularly useful for the treatment of cancer. [Background technology]

[0002] Macrophages exhibit a variable phenotype ranging from the classical activated M1 type to the alternative M2 type. Activated by bacterial derived products such as LPS (polysaccharide) as well as infection-associated signals such as IFNγ, M1 macrophages rapidly differentiate from monocytes after migration. M1 macrophages are highly inflammatory with high phagocytic and bactericidal potential. M1 macrophages secrete important proinflammatory cytokines such as TNFα, IL-1, IL-6, and IL-12 as well as reactive oxygen species. In contrast, M2 macrophages are present later in the healing process resulting in granulation tissue formation and antagonize the inflammatory response, thus allowing healing to begin. These anti-inflammatory cells recruit fibroblasts and release proangiogenic factors to recruit endothelial progenitor cells and activate them to differentiate into myofibroblasts, allowing new blood vessel formation. This occurs via the secretion of the key anti-inflammatory cytokines IL-4, IL-10, and IL-13, a process that is also associated with reduced production of ROS, nitric oxide (NO), and TNFα.

[0003] The tumor microenvironment (TME), especially in tumors recovering from cancer treatment, strongly polarizes macrophages toward an M2-like phenotype, which is not only highly proangiogenic but also immunosuppressive. Thus, in most solid cancers, increased infiltration by tumor-associated macrophages (TAMs) has long been associated with poor patient prognosis, highlighting their value as potential diagnostic and prognostic biomarkers in cancer.

[0004] Therefore, reprogramming and selective killing of M2 macrophages has been suggested as a promising therapeutic strategy (Zhu Y et al., Cancer Res, 2014).

[0005] Furthermore, as innate immune cells, natural killer (NK) cells play a pivotal role in immune surveillance against cancer. NK cells can eliminate a variety of abnormal or stressed cells without prior sensitization, and preferentially kill stem-like cells or cancer stem cells. Upon forming an immunological synapse with a target cell, NK cells release preformed cytolytic granules containing perforin and granzymes, whose function is to induce cell lysis.

[0006] Based on this assumption, several studies have successfully utilized adoptive transfer of NK cells against various tumors, especially hematological malignancies. However, cancers use various tactics to delay, alter or even halt antitumor immunity, resulting in failure of control over tumor growth. Antitumor responses of NK cells also face many limitations. In particular, the tumor microenvironment (TME) remains a major barrier to the efficacy of NK cells, especially adoptively transferred NK cells. For example, tumor-infiltrating immune cells such as dendritic cells (DCs), suppressive or tolerogenic macrophages, and regulatory T (Treg) cells, as well as cancer-associated fibroblasts embedded in the extracellular matrix, can intervene in the activation of NK cells via the secretion of immunosuppressive cytokines or by interfering with receptor expression. For example, in the TME, TGF-β (secreted by M2-polarized macrophages, among others) is recognized as the major inhibitory cytokine of NK cells, limiting the number and anti-metastatic function of NK cells.

[0007] It would therefore be extremely valuable from a therapeutic point of view to have tools that could stimulate antitumor activity by both i) inhibiting the immunosuppressive effects of the tumor environment and ii) directly inducing NK cell activation and NK cell-mediated cytotoxicity.

[0008] Butyrophilins constitute a family of transmembrane proteins that includes butyrophilin (BTN), BTN-like (BTNL) proteins, and SKINT (selection and upkeep of intraepithelial T cell) proteins (Arnett and Viney, Nat Rev Immunol, 2014). The extracellular portion of butyrophilins contains IgV-like and IgC-like domains that exhibit homology to corresponding domains of the B7 costimulatory molecule (Arnett and Viney, 2014), and therefore butyrophilins are considered to be members of the extended B7 or Ig superfamily.

[0009] The butyrophilin (BTN) family of genes consists of 13 genes forming eight distinct groups in humans (Abeler-Dorner et al., Trends Immunol, 2012; Afrache et al., Immunogenetics, 2012). The seven human BTN genes are divided into three subfamilies: BTN1, BTN2, and BTN3, which are clustered in the MHC class I region of chromosome 6 and form a phylogenetically related group. The BTN1 subfamily contains only the archetypal single copy BTN1A1 gene, whereas the BTN2 and BTN3 subfamilies each contain three genes, BTN2A1, BTN2A2, and BTN2A3 (which are pseudogenes), BTN3A1, BTN3A2, and BTN3A3, respectively. Several genetic polymorphisms have been described for BTN gene family members that are associated with different diseases, including hypertension, chronic renal failure, inclusion body myositis, type 1 and type 2 diabetes, or HCV infection (Chen et al., Int J Clin Exp Pathol, 2015; Horibe et al., Am J Hypertens, 2011 and 2014; Milman et al., Clin Respir, 2011; Murakata et al., Biomed Rep, 2014; Oguri et al., J Med Genet, 2013; Pacheco et al., Orphanet J Rare Dis, 2016). Single nucleotide polymorphism variants for BTNL2, BTN2A1, BTN3A2, and BTN3A3 have been described, as well as deleterious copy number variations involving BTNL3 and BTNL8 (Aigner et al., BMC Genet, 2013).

[0010] The first butyrophilin identified, BTN1A1, is required for milk fat globule formation, secretion, and stabilization (Ogg et al., Proc Natl Acad Sci, 2004). It has since been proposed that the B7 gene and the MHC class I and II genes share a common ancestor gene and may code for proteins involved in similar functions, such as T cell activation (Rhodes et al., Genomics, 2001; HarlyC et al., Blood, 2012). The BTN2A1 and BTN2A2 protein isoforms exhibit the extracellular domains of IgV and IgC, the transmembrane domain, and the characteristic intracellular B30.2 domain, and are similar to BTN3A1 and BTN3A3, but not to BTN3A2. In mice, BTN2A2 is a single copy gene and is the orthologue of the human BTN2A2 gene. Recombinant human BTN2A1-Fc protein revealed that specific glycoforms of BTN2A1 bind to DC-SIGN, a lectin molecule found on dendritic cells (DCs). Binding of BTN2A1 to DC-SIGN is dependent on high-mannose glycosylation of the protein when expressed by tumor cells (Malcherek et al., J Immunol, 2007).

[0011] Growing evidence subsequently suggested that butyrophilin plays diverse roles in the immune system. RNA-seq from 53 human tissue samples derived from the Genotype-Tissue Expression Project (GTEx Consortium, 2013) shows ubiquitous expression of BTN2A1 transcripts in normal tissues. Comparison of RNA-seq data from GTEx with data from the Cancer Genome Atlas (TCGA) database using Gene Expression Profiling Interactive Analysis (Tang, Z. et al., Nucleic Acids Res, 2017) indicates modulation of BTN2A1 transcript expression in several cancers, including cervical squamous cell carcinoma and adenocarcinoma, small cell lung carcinoma, ovarian cancer, pancreatic cancer, and endometrial cancer.

[0012] The above (WO 2019057933) reports an antibody that recognizes both isoforms of BTN2A, which inhibits the production of IFN-γ and / or TNF-α by activated Vγ9 / Vδ2 T cells, and / or inhibits the cytolytic function of activated Vγ9 / Vδ2 T cells, and / or inhibits the proliferation of activated Vγ9 / Vδ2 T cells.

[0013] Vγ9 / Vδ2 T cells are key effectors of immune defense. They directly lyse pathogen-infected or abnormal cells. In addition, Vγ9 / Vδ2 T cells regulate immune responses by inducing dendritic cell (DC) maturation as well as isotype switching and immunoglobulin production. This key cellular platform of the immune system is tightly regulated by surface receptors, chemokines, and cytokines. Vγ9 / Vδ2 T cells are activated by non-peptidic phosphorylated isoprenoid pathway metabolites, termed pAg (phosphoagonist).

[0014] The development of antibodies that target BTN2A1 and activate more than one immune cell compartment, such as immune cells, particularly macrophages, NK cells, and / or γδ T cells, particularly Vγ9 / Vδ2 T cells, may be particularly important in cancer therapy, among other things, and for the treatment of infectious diseases. Summary of the Invention [Means for solving the problem]

[0015] The present disclosure provides, for the first time, an antibody that binds to BTN2A (specifically, binds to the BTN2A1 isoform (e.g., a human BTN2A1 polypeptide)) that has the following characteristics: i. the property of inhibiting the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. The property of directly inducing NK cell activation; iv. Properties that enhance NK cell-mediated cytotoxicity The present invention provides an antibody that exhibits at least one of the following:

[0016] In particular, the antibodies of the present disclosure exhibit at least one of properties i) and ii) and at least one of properties iii) and iv), and more particularly, the antibodies of the present disclosure exhibit properties i)-iv).

[0017] Thus, such antibodies according to the present disclosure: Shifting macrophage phenotype and functionality towards pro-inflammatory M1 macrophages, thereby preferentially providing an anti-tumor microenvironment, resulting in the secretion of pro-inflammatory cytokines; and / or Directly induce the activation of NK cells, thereby further enhancing their cytolytic activity is possible.

[0018] Thus, in accordance with the present disclosure, such antibodies represent powerful tools that can be used in a variety of strategies for cancer treatment.

[0019] In particular embodiments, the anti-BTN2A antibodies of the disclosure have the following properties for binding to BTN2A: (i) mAb 101G5, a reference murine antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20; or mAb 107G3, a reference murine antibody comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2. conflicts with one of

[0020] In some embodiments, the anti-BTN2A antibody of the disclosure is Positions 65, 68, 69, 72, 78, 84, 85, 95, 97, and 100 of SEQ ID NO: 17, or Positions 212, 213, 218, 220, 224, and 229 of SEQ ID NO:17 An epitope comprising an amino acid residue located at Bind to.

[0021] In particular embodiments, the anti-BTN2A antibodies of the disclosure are CDR1 is a heavy chain variable region comprising SEQ ID NO:3, CDR2 is a heavy chain variable region comprising SEQ ID NO:4, CDR3 is a heavy chain variable region comprising SEQ ID NO:5, CDR1 is a light chain variable region comprising SEQ ID NO:6, CDR2 is a light chain variable region comprising SEQ ID NO:7, and CDR3 is a light chain variable region comprising SEQ ID NO:8, or CDR1 is a heavy chain variable region comprising SEQ ID NO:21, CDR2 is a heavy chain variable region comprising SEQ ID NO:22, CDR3 is a heavy chain variable region comprising SEQ ID NO:23, CDR1 is a light chain variable region comprising SEQ ID NO:24, CDR2 is a light chain variable region comprising SEQ ID NO:25, and CDR3 is a light chain variable region comprising SEQ ID NO:26. Includes.

[0022] In particular embodiments, the anti-BTN2A antibodies of the disclosure are a heavy chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:2; or A heavy chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20. Includes.

[0023] In particular embodiments, the antibodies of the present disclosure have the following characteristics: the ability of Vγ9Vδ2 T cells to secrete cytolytic molecules; the property of activating the cytolytic function of Vγ9Vδ2 T cells, and / or Activating properties of Vγ9Vδ2 T cells The present invention further exhibits at least one of the following:

[0024] Most particularly, anti-BTN2A antibodies according to such embodiments may typically compete for binding to BTN2A with mAb 107G3, a reference murine antibody comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0025] More particularly, such anti-BTN2A antibodies include CDR1 is a heavy chain variable region comprising SEQ ID NO:3, CDR2 is a heavy chain variable region comprising SEQ ID NO:4, CDR3 is a heavy chain variable region comprising SEQ ID NO:5, CDR1 is a light chain variable region comprising SEQ ID NO:6, CDR2 is a light chain variable region comprising SEQ ID NO:7, and CDR3 is a light chain variable region comprising SEQ ID NO:8, or a heavy chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:2; or A heavy chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2. may include.

[0026] In some embodiments of the present disclosure, the antibodies of the present disclosure have specificity for BTN2A1.

[0027] In particular embodiments, the anti-BTN2A antibodies of the disclosure are human, chimeric, or humanized antibodies.

[0028] The present disclosure also includes nucleic acid molecules encoding the heavy and / or light chains of the anti-BTN2A antibodies described above.

[0029] The present disclosure also relates to host cells containing such nucleic acid, particularly for use in the production of any one of the anti-BTN2A antibodies described above.

[0030] Another aspect of the disclosure relates to an anti-BTN2A antibody as described above for use in therapy, particularly in the treatment of cancer or infectious disease.

[0031] Typically, the cancers according to the present disclosure are cervical cancer, such as squamous cell carcinoma and cervical adenocarcinoma, ovarian cancer, skin cancer, including squamous cell carcinoma and melanoma, lung cancer, including small cell lung carcinoma, prostate cancer, colon cancer, pancreatic cancer, and endometrial cancer, more particularly squamous cell carcinoma, including hematological and solid cancers, including cervical adenocarcinoma, squamous cell carcinoma, ovarian cancer, small cell lung carcinoma, prostate cancer, colon cancer, pancreatic cancer, and endometrial cancer.

[0032] The present disclosure also relates to pharmaceutical compositions comprising the anti-BTN2A antibodies described above and at least one pharma- ceutically acceptable carrier.

[0033] The present disclosure further provides a method for activating an immune response in a subject, the method comprising administering to the subject an effective amount of an anti-BTN2A antibody disclosed herein.

[0034] [Detailed Description of the Invention] definition As used herein, the term "BTN2" has its general meaning in the art and refers to a human BTN2 polypeptide, including BTN2A1 of SEQ ID NO:17 or BTN2A2 of SEQ ID NO:18.

[0035] SEQ ID NO: 17: BTN2A isoform 1 precursor (Homo sapiens): MESAAAALHFSRPASLLLLLLSLCALVSAQFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGR SYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVIIRDKSVRNMSCSINNTLLGQKKESVIFIPESFMPSVSPCAVALPIIVVILMIPIA VCIYWINKLQKEKKILSGEKEFERETREIALKELEKERVQKEEELQVKEKLQEELRWRRTFLHAVDVVLDPDTAHPDLFLSEDRRSVRRCPFRHLGESVPDNPERFDSQPCVLGRESFASGKHYWEVEVENV IEWTVGVCRDSVERKGEVLLIPQNGFWTLEMHKGQYRAVSSPDRILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAFSVPVRPFFRLGCEDSPIFICPALTGANGVTVPEEGLTLHRVGTHQSL

[0036] SEQ ID NO: 18: BTN2A isoform 2 precursor (Homo sapiens): MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCY FQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIFIPESFMPSASPWMVALAVILTA SPWMVSMTVILAVFIIFMAVSICCIKKLQREKKILSGEKKVEQEEKEIAQQLQEELRWRRTFLHAADVVLDPDTAHPELFLSEDRRSVRRGPYRQRVPDNPERFDSQPCVLGWESFASGKHYWEVEVENVM VWTVGVCRHSVERKGEVLLIPQNGFWTLEMFGNQYRALSSPERILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAFTVPVRPFFRLGSDDSPIFICPALTGASGVMVPEEGLKLHRVGTHQSL

[0037] As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and will be used interchangeably in this disclosure.

[0038] As used herein, the term "antibody" refers to immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments, as well as variants (including derivatives) of antibodies and antibody fragments.

[0039] As used herein, the term "antibody" also includes bispecific or multispecific molecules. An antibody may be derivatized and linked to another functional molecule, such as another peptide or protein (e.g., another antibody, or a ligand for a receptor), to create a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, an antibody may be derivatized and linked to more than one other functional molecule to create a multispecific molecule that binds to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the present invention may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimic, resulting in a bispecific molecule. In addition, in embodiments where the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the first and second target epitopes. In one embodiment, the bispecific molecule disclosed herein comprises as a binding specificity at least one antibody or antibody fragment thereof, including, for example, a Fab, Fab', F(ab')2, Fv, unibody or single chain Fv. The antibody may also be any minimal fragment thereof, such as a light chain dimer or a heavy chain dimer, or an Fv construct or a single chain construct as described in Ladner et al., U.S. Pat. No. 4,946,778.

[0040] Other antibodies that can be used in the bispecific molecules disclosed herein are murine mAbs, chimeric mAbs, and humanized monoclonal antibodies (mAbs).

[0041] The bispecific molecules of the present disclosure can be prepared by conjugating the components of the binding specificity using methods known in the art. For example, each component is conjugated to each other. When the binding specificity is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2pyridyldithio)propionic acid (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). Other methods include those described in Brennan et al., 1985; Glennie et al., 1987; Paulus, 1985. Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. The bispecific molecules of the present disclosure may be single-chain molecules comprising one single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. Binding of the bispecific molecules to their specific targets may be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition and apoptosis), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by using a labeling reagent (e.g., an antibody) specific for the complex of interest.

[0042] 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 major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains significantly different sequence domains. Light chains contain two domains, the variable domain (VL) and the constant domain (CL). Heavy chains contain four domains, the variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The light (CL) and heavy (CH) chain constant region domains confer important biological properties, such as antibody chain assembly, secretion, transplacental transport, complement binding, and Fc receptor (FcR) binding.

[0043] An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of one light chain variable portion and one heavy chain variable portion. The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is mainly composed of residues from the hypervariable or complementarity determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or affect the overall domain structure, thereby affecting the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and binding specificity of the native Fv region of the native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically comprises six CDRs, including a set of CDRs from each of the heavy and light chain V regions. "Framework region" (FR) refers to the amino acid sequence inserted between the CDRs. Thus, the light and heavy chain variable regions typically comprise four framework regions and three CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0044] Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al., which is set forth in Kabat et al., 1987, "Sequences of Proteins of Immunological Interest", US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues in the sequence according to SEQ ID NO:. The actual linear amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering, corresponding to the shortening of, or insertion into, structural components of the basic variable domain structure, whether framework or complementarity determining regions (CDRs). The correct Kabat numbering for residues can be determined for a given antibody by alignment of the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0045] In particular embodiments, the antibodies provided herein are antibody fragments, more particularly any protein that contains the antigen-binding domain of an antibody disclosed herein, including but not limited to Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies.

[0046] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds BTN2A1 is substantially free of antibodies that specifically bind to antigens other than BTN2A). However, an isolated antibody that specifically binds BTN2 may have cross-reactivity with other antigens, such as related BTN2 molecules from other species. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0047] The affinity of an antibody refers to the strength with which the antibody binds to an epitope presented by an antigen, such as BTN2A1 in this disclosure, via its antigen-binding site (paratope). Affinity can be evaluated based on the evaluation of the Kd value.

[0048] As used herein, "K D The term "k off k on (i.e., k off / k on ) and expressed as a molar concentration (M). D The value relates to the concentration of the antibody (the amount of antibody needed for a particular experiment), so a low K D value (low concentration) and therefore high affinity of the antibody. D Values ​​can be determined using methods well established in the art. DPreferred methods for determining K values ​​can be found 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, 1983, the references of which are incorporated herein by reference in their entireties. DMethods for determining affinity are by using surface plasmon resonance or by using biosensor systems such as the Biacore® (for more information on affinity evaluation see also Rich RL, Day YS, Morton TA, Myszka DG, "High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using BIACORE®", Anal Biochem 2001) or the Octet® system. The Octet® platform is based on the Biolayer Interferometry (BLI) technology. The principle of the BLI technology is based on the optical interference pattern of white light reflected from two surfaces (an immobilized protein layer and an internal reference layer). Binding of the analyte in solution with the ligand immobilized on the biosensor chip surface leads to an increase in the optical thickness at the biosensor chip, which results in a shift in the interference pattern, measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in optical thickness of the biological layer, and when this shift is measured over time and its magnitude is plotted as a function of time, a classical association / dissociation curve is obtained. This interaction is measured in real time, allowing monitoring of binding specificity, association and dissociation rates, and concentration (see Abdiche et al., 2008, but also see details in Results). Affinity measurements are typically performed at 25° C.

[0049] As used herein, "k assoc " or "k a " or "k on " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas "k dis " or "k d " or "k off The term "rate of dissociation of a particular antibody-antigen interaction" is intended to refer to the off-rate of a particular antibody-antigen interaction.

[0050] As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0051] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen, such as the BTN2A isoforms in this disclosure (including BTN2A1 and BTN2A2). The term "specificity" typically refers to a K of 10 nM or less, 5 nM, 1 nM or less, 100 pM or less, or 10 pM or less for human BTN2A, particularly BTN2A1. D Typically, K D is 10 -3 Typically, the antibodies of the present disclosure are specific for BTN2A1, or both BTN2A1 and BTN2A2, and have a K D In some embodiments, specificity may also be assessed by expressing BTN2A1 in a cell line (e.g., a HEK-293T cell line) and staining the transfected cells with increasing concentrations (e.g., ranging from 5 ng / mL to 75 μg / mL) of a purified anti-BTN2A1 mAb disclosed herein, or a negative control, such as its control isotype. Nonlinear regression analysis of the mean fluorescence intensity data yields the EC 50 Typically, antibodies specific for BTN2A1 according to the present disclosure have an EC value for BTN2A1 of less than 50 μg / mL, in particular less than 40 μg / mL (see the Examples section), in particular between 0.1 μg / mL and 50 μg / mL, or between 0.5 μg / mL and 20 μg / mL. 50 Combine with.

[0052] As used herein, the terms "an antibody that recognizes an antigen" and "an antibody having specificity for an antigen" are used interchangeably with the term "an antibody that specifically binds to an antigen."

[0053] "Selectively binding" typically means that an antibody binds more strongly to a target, such as an epitope, for which it is specific, as compared to binding to another target. An antibody binds more strongly to a first target as compared to a second target if its affinity for the first target is greater than its affinity for the second target. Typically, an antibody binds more strongly to a first target as compared to a second target if it binds to the first target with an equilibrium dissociation constant (K D ) or EC 50 The smaller the equilibrium dissociation constant or EC 50 When the agent binds at a specific target, it binds more strongly to the first target compared to the second target. Most particularly, the agent does not bind appreciably to the second target at all. In some embodiments of the present application, the antibody is selective for BTN2A, and most particularly for BTN2A1. Selectivity is also further exhibited by an affinity ratio of, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1, or more, in binding to the specific antigen versus non-specific binding to other unrelated molecules (e.g., the specific antigen can be a BTN2A polypeptide, particularly BTN2A1, and the other "unrelated molecules" can be, for example, BTN3 isoforms).

[0054] The selectivity of the antibodies disclosed herein can be examined by applying cross-reactivity assays to other closely related proteins (e.g., BTN3 isoforms) compared to the intended target protein (BTN2A isoform). An antibody is typically considered selective if no such cross-reactivity is detected while at the same time, and at the same antibody dilution, it results in a strong signal of the intended target (see results detailed in the Examples corresponding to FIG. 3). An antibody that "cross-reacts with an antigen" is one that has a K of 10 nM or less, 1 nM or less, or 100 pM or less for this antigen. D An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen with a K of 100 nM or greater. D , or a K of 1 μM or more D , or a K of 10 μM or more D "Antibody" is intended to refer to an antibody that binds to an antigen. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding to these proteins in standard binding assays (typically see Table 3 or FIG. 3A).

[0055] In particular embodiments, the anti-BTN2A1 antibodies of the disclosure have the following structure: It cross-reacts with the cynomolgus monkey BTN2A1 orthologue (cynoBTN2A1; NCBI ref.XP_015304392.1), which has the sequence of SEQ ID NO: 35, as defined by

[0056] In some of these embodiments, the antibodies disclosed herein bind to the extracellular domain of cynoBTN2A1 and the corresponding EC domain found in huBTN2A1. 50 EC equal to (10% more or less than) 50 Combine with.

[0057] The term "identity" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. If a position in both compared sequences is occupied by the same base or the same amino acid residue, the respective molecules are identical at this position. The percentage of identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100. In general, the comparison is made when the two sequences are aligned to give the maximum identity. Identity can be calculated, for example, by alignment using the cumulative program of GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) or any of the sequence comparison algorithms such as BLAST, FASTA, or CLUSTALW.

[0058] A functional variant of a reference molecule according to the present disclosure exhibits functional properties that are substantially equivalent to or superior to the corresponding functional properties of the reference molecule (e.g., 107G3 mAb). By "substantially equivalent" herein, it is intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the corresponding functional properties of the reference molecule.

[0059] In one aspect, the disclosure relates to an antibody having specificity for BTN2A1 as defined above. Typically, such an antibody has a K of 10 nM or less for human BTN2A1 as defined above. D It is characterized in that it binds at

[0060] In some embodiments, the antibodies of the disclosure do not cross-react with BTN3 isoforms.

[0061] An antibody having specificity for BTN2A, but in particular BTN2A1 according to the present invention, also typically has the following properties: i. the property of inhibiting the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. The ability to induce direct activation of NK cells; iv. Properties that enhance NK cell-mediated cytotoxicity The compound is further characterized in that it has at least one of:

[0062] Preferably, an antibody of the disclosure exhibits at least one of properties i and ii, and at least one of properties iii and iv, and most likely exhibits properties i-iv.

[0063] Anti-BTN2A antibodies of the disclosure having such advantageous properties can be screened among anti-BTN2A antibodies, for example, using the assays detailed in the Examples section, which are outlined below, and their implementation.

[0064] In the macrophage polarization assay, M2 macrophages are generated from macrophages in the presence of anti-BTN2A antibodies, in particular the anti-BTN2A1 antibody defined above, or in the presence of a negative control, such as its control isotype, as conventionally done in the art. During M-CSF (macrophage colony-stimulating factor)-induced M2 polarization as a control for inhibition of M2 differentiation, GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFN-γ can be added to monocytes. Conversely, M1 macrophages polarized in the presence of GM-CSF can also typically be used as a phenotypic control. After polarization, the expression of M1- and M2-associated markers at the cell membrane can be evaluated by flow cytometry in the resulting macrophages.

[0065] M1 markers that may be conveniently detected according to the present disclosure include, but are not limited to, PDL1, CD86, CD40, CD80, and / or SOCS3. Typically, PDL1 and / or CD86 are detected. M2 markers that may be conveniently detected according to the present disclosure include, but are not limited to, CD14, CD163, CD206, and CD209, with CD14 and / or CD163 being typically detected.

[0066] Typically, inhibition of monocyte polarization into M2 macrophages is if a significant increase in at least one M1 marker is observed when M2 macrophages are generated in the presence of an anti-BTN2A antibody of the present disclosure compared to a control isotype thereof; and / or if a significant reduction in at least one M2 marker is observed when M2 macrophages are generated in the presence of an anti-BTN2A antibody compared to a negative control, such as a control isotype. induced by anti-BTN2A of the present disclosure.

[0067] Furthermore, typically by using a suitable commercially available kit (e.g., an ELISA-like kit), the cytokine secretion profile, which is a characteristic distinguishing feature between M1 and M2 macrophages, including, inter alia, M2-associated anti-inflammatory IL-10 cytokine and M1-associated pro-inflammatory TNFα cytokine, can be quantified in the culture supernatant. Additional cytokines, which can also be easily detected to characterize the M1 or M2 macrophage phenotype, include IL-1, IL-6, IL-12, and IL-23 for M1-associated cytokines, and TGFβ and IL-10 for M2-associated cytokines.

[0068] Thus, in some embodiments, inhibition of monocyte polarization into M2 macrophages is also and / or if a significant decrease in the secretion of at least one M2-associated cytokine is observed when M2 macrophages are cultured in the presence of an anti-BTN2A antibody of the present disclosure compared to a negative control, such as a control isotype thereof; If a significant increase in the secretion of at least one M1-associated cytokine is observed when M2 macrophages are cultured in the presence of an anti-BTN2A antibody of the present disclosure compared to a negative control, such as a control isotype thereof. This may be believed to result from the action of the anti-BTN2A antibodies of the present disclosure.

[0069] Typically, anti-BTN2A antibodies according to the present disclosure may dose-dependently inhibit the polarization of monocytes into M2 macrophages, as assessed by a reduction in the expression of M2-associated markers (such as CD14 and / or CD163) and / or a reduction in the secretion of M2-associated cytokines (such as IL-10). The median inhibitory concentration (IC) of such antibodies with respect to the secretion of said at least one M2-associated cytokine or the expression of said at least one M2-associated marker may be 0.01 to 0.05. 50 ) can be determined in a dose-response curve as detailed in the Examples section. In some detailed embodiments, the anti-BTN2A antibodies of the invention For expression of M2-associated markers (typically CD14 and / or CD163), IC in the range of 0.05 μg / mL, particularly 0.1 μg / mL to 100 μg / mL, particularly 50 μg / mL. 50 and / or For secretion of M2-associated cytokines (typically IL-10), an IC in the range of 0.01 μg / mL, particularly 0.05 μg / mL to 100 μg / mL, particularly 50 μg / mL, and most particularly 0.1 to 20 μg / mL. 50 It presents.

[0070] Additionally, or alternatively, anti-BTN2A antibodies according to the present disclosure may dose-dependently bias monocyte polarization towards M1 macrophages despite M2-promoting stimuli, as assessed by increased expression of M1-associated markers (such as CD86 and / or PDL1) and / or decreased secretion of M2-associated cytokines (such as IL-10). The 50% effective concentration (EC) of such antibodies with respect to secretion of said at least one M1-associated cytokine or expression of said at least one M1-associated marker may be determined. 50 ) can be determined in a dose-response curve, as detailed in the Examples section. In some detailed embodiments, the anti-BTN2A antibodies of the invention For expression of M1-associated markers (typically CD86 and / or PDL1), an EC value in the range of 0.01 μg / mL, particularly 0.1 μg / mL to 100 μg / mL, particularly 50 μg / mL, and most particularly 1 to 50 μg / mL. 50 and / or For secretion of M1-associated cytokines (typically TNFα), an EC value in the range of 0.01 μg / mL, particularly 0.05 μg / mL to 100 μg / mL, particularly 50 μg / mL, and most particularly 0.1 to 10 μg / mL. 50 It presents.

[0071] In the M2 macrophage reversion assay, typically, M2 macrophages generated from monocytes in the presence of M-CSF can be cultured with or without polysaccharide (LPS) in the presence of a negative control, such as an anti-BTN2A antibody (most particularly, the anti-BTN2A1 antibody, as defined supra), or a control isotype thereof. As a positive control for M2 reversion, GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFN-γ can be added to the M2 culture. M1 macrophages polarized in the presence of GM-CSF can typically be used as a phenotypic control. After the reversion experiment, macrophages reverted in the absence of LPS can be analyzed for the expression of M1- or M2-associated markers by flow cytometry, as described above. Cytokine secretion can also be quantified, as described above.

[0072] Typically, reversion of M2 macrophages to M1 macrophages occurs as follows: a significant increase in M1 markers is observed when M2 macrophages are cultured in the presence of said anti-BTN2A antibody compared to its control isotype; and / or If a significant decrease in M2 markers is observed when M2 macrophages are cultured in the presence of the anti-BTN2A antibody compared to a negative control, such as a control isotype. is induced by the anti-BTN2A antibodies of the present disclosure.

[0073] Natural killer (NK) cell activation may be assessed by culturing NK cells (typically from a healthy donor) with an anti-BTN2A antibody as defined herein, or a control isotype for negative control, with or without the addition of IL-2 and / or IL-15. After at least 48 hours, and in particular after at least 4 days, the NK cells may then be phenotyped ex vivo for their activation markers, such as CD69 and / or CD25. Typically, NK cell activation is considered to be induced by an anti-BTN2A antibody of the present disclosure when a significant increase in NK cell activation markers, such as CD69 and / or CD25, is observed in the presence of said anti-BTN2A (with or without further activation by IL-2 and / or IL-15) compared to a negative control, such as its isotype control. The results presented herein clearly support that the antibodies of the present invention induce direct activation of NK cells.

[0074] Enhancement of cytotoxicity by NK cells can be further evaluated in vitro by assessing degranulation of NK cells. In such functional assays, cancer cell lines, such as leukemia cell lines (myeloid leukemia) or carcinoma (e.g., colon cancer, breast adenocarcinoma, or lung adenocarcinoma) cell lines, are co-cultured in the presence of NK cells as described above (preactivated with anti-BTN2A antibody or its negative control as described above) and in the presence or absence of IL-2 and / or IL-15. Degranulation of NK cells can typically be assessed by the percentage of CD107 positive NK cells (in the presence or absence of IL-2 and / or IL-15) in flow cytometry.

[0075] Typically, NK cell cytotoxicity is considered to be induced by an anti-BTN2A antibody if NK cell degranulation (e.g., percentage of CD107 positive NK cells) is significantly increased following activation of NK cells with said anti-BTN2A antibody compared to a negative control, such as a control isotype. Typically, the anti-BTN2A antibodies of the present disclosure also induce NK cell degranulation (especially against various cancer cell lines, as described supra) in a dose-dependent manner. The 50% effective concentration (EC) of such antibodies for NK cell degranulation is 50 ) can be determined with dose-response curves, as detailed in the Examples section.

[0076] The reference antibodies, 101G5 and / or 107G3, may be used as positive controls in the functional assays described above.

[0077] In some embodiments, the anti-BTN2A antibodies of the disclosure inhibit monocyte polarization into M2 macrophages, induce reversion of M2 macrophages to anti-tumor M1 macrophages, trigger direct activation of NK cells, and / or enhance NK cell-mediated cytotoxicity to levels substantially equivalent to or superior to reference antibodies, mAb 101G5 or mAb 107G3, as described more fully below. As used herein, by "inhibit polarization of monocytes into M2 macrophages, induce reversion of M2 macrophages to anti-tumor M1 macrophages, trigger direct activation of NK cells, and / or enhance NK cell-mediated cytotoxicity to a level substantially equivalent to or superior to that of a reference antibody" is intended to mean that less than a 20%, more particularly less than a 15%, more particularly less than a 10% variation in the test functional activity of the test anti-BTN2A1 antibody compared to any one of the reference mAbs, 107G3 or 101G5, is observed, typically less than a 5% variation.

[0078] The present disclosure also provides 1) Antibodies having specificity for BTN2A, and in particular for BTN2A1, as defined supra, in particular having the following characteristics: Typically, the antibody binds to human BTN2A1 expressed in a cell line, for example a HEK293T cell line transfected with a plasmid encoding human BTN2A1, as described in the Examples, and more particularly has an EC of less than 50 μg / mL, more particularly less than 40 μg / mL. 50 or binds to BTN2A1 with a K of 10 nM or less. D and / or have a ratio of binding affinity to BTN2A1 relative to non-specific binding of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1, or more; Does not cross-react with BTN3 an antibody having at least one of the following: and 2) Additionally or alternatively, functional properties: the property of activating the production of cytolytic molecules, in particular IFN-γ, by Vγ9 / Vδ2 T cells, and / or the property of activating the cytolytic function of Vγ9 / Vδ2 T cells, and / or Activating properties of Vγ9 / Vδ2 T cells Also included are antibodies that exhibit one or more of the following:

[0079] It will be appreciated that proliferation, cytolytic function and production of cytolytic molecules (especially IFN-γ) by Vy9 / V52 T cells are typically achieved by activated Vy9 / V52 T cells, which is achieved by the antibodies disclosed herein.

[0080] Anti-BTN2A1 antibodies of the present disclosure having such advantageous properties may be screened among anti-BTN2A1 antibodies using the cellular assays described in the Examples, in particular by ELISA-based assessment of IFNγ secretion by Vy9 / V52 T cells, and / or by CD107 degranulation assays in various cancer cell lines, such as the Daudi, Jurkat, L-IPC, or MDA-MB-134 cell lines.

[0081] The cytotoxic molecule according to the present disclosure is typically present in an IFNγ cytokine or a TNFα cytokine.

[0082] As used herein, "activating the production of cytolytic molecules" (typically IFNγ / or TNFα production) means that a significant increase in at least IFNγ or TNFα production by activated Vy9 / V52 T cells is observed when compared to control activated Vy9 / V52 T cells (activated with IgG1 or hybridoma culture medium as controls), where said Vy9 / V52 T cells are activated by co-culture with a target cell line (Daudi, Jurkat, L-IPC or MDA-MB-134 cell line) or by pAg (phosphoantigen). Typically, activation of IFNγ or TNFα production by activated Vy9 / V52 T cells can be measured by ELISA-based amounts of IFNγ or TNFα secreted by Vy9 / V52 T cells in cell assays by intracellular labeling with antibodies against IFNγ or TNFα, or in their culture medium, assessed in flow cytometry. Such assays are described in more detail in the Examples below (see Materials and Methods section).

[0083] As used herein, "activating the cytolytic function of activated Vy9 / V52 T cells" means that a significant increase in the cytolytic function of activated human Vy9 / V52 T cells is observed when compared to control activated human Vy9 / V52 T cells (activated with IgG1 or hybridoma culture medium as control), where said human Vy9 / V52 T cells are activated by co-culture with a target cell line (Daudi, Jurkat, L-IPC or MDA-MB-134 cell line) or by pAg (phosphoantigen). Typically, activation of the cytolytic function of activated Vy9 / V52 T cells can be measured following measurement of activation of induction of Vy9 / V52 T cell degranulation against a standard cell line using CD107a in combination with CD107b as degranulation markers to detect positive degranulated Vy9 / V52 T cells. Treatment of Vy9 / V52 T cells with phorbol 12-myristate 13-acetate (PMA) with ionomycin can typically be used as a positive control for Vy9 / V52 T cell activation. Such an assay is described in more detail in the Examples below.

[0084] As used herein, "activating the proliferation of activated Vy9 / V52 T cells" means that a significant increase in the proliferation of activated Vy9 / V52 T cells is observed when compared to the proliferation of Vy9 / V52 T cells activated with IgG1 as a control, where said Vy9 / V52 T cells are activated by co-culture with a target cell line (Daudi, Jurkat, L-IPC or MDA-MB-134 cell line) or by pAg (phosphoantigen). Typically, the proliferation of activated Vy9 / V52 T cells can be measured in cell assays by CFSE or Cell Trace violet staining and flow cytometry on Vy9 / V52 T cells purified from peripheral blood, or by monitoring the expansion of the Vy9 / V52 T cell compartment in peripheral blood mononuclear cells with or without stimulation.

[0085] In some embodiments, the anti-BTN2A1 antibodies of the disclosure activate the cytolytic function of activated Vy9 / V52 T cells to a level substantially equivalent to or superior to that of mAb 107G3, a reference antibody described below. By "activating the cytolytic function of activated Vy9 / V52 T cells to a level substantially equivalent to that of a reference antibody" herein is intended to mean observing less than 15%, in particular less than 10%, and typically less than 5% variation in the cytolytic function of activated Vy9 / V52 T cells by the test anti-BTN2A1 antibody compared to the reference mAb 107G3.

[0086] In some embodiments, the anti-BTN2A1 antibodies of the present disclosure activate the production of cytolytic molecules (i.e., at least IFNγ or TNFα) by activated Vy9 / V52 T cells to levels substantially equivalent to or superior to the reference antibody, mAb 107G3, described below. By "activating at least IFNγ or TNFα production by activated Vy9 / V52 T cells to levels substantially equivalent to the reference antibody" herein is intended to mean observing less than 15%, particularly less than 10%, and typically less than 5% variation in the production of cytolytic molecules by activated Vy9 / V52 T cells by the test anti-BTN2A1 antibody compared to the reference mAb, 107G3.

[0087] In some embodiments, the anti-BTN2A1 antibodies of the disclosure activate the cytolytic function of activated Vy9 / V52 T cells to a level substantially equivalent to or superior to that of mAb 107G3, a reference antibody described below. By "activating the cytolytic function of activated Vy9 / V52 T cells to a level substantially equivalent to that of a reference antibody" herein is intended to mean observing less than 15%, in particular less than 10%, and typically less than 5% variation in the cytolytic function of activated Vy9 / V52 T cells by the test anti-BTN2A1 antibody compared to the reference mAb 107G3.

[0088] Reference antibodies, mAb 101G5, mAb 107G3, and their variants The antibodies disclosed herein include reference monoclonal antibodies, mAb 101G5, which comprises VH and VL regions defined in SEQ ID NOs: 19 and 20, respectively, and mAb 107G3, which comprises VH and VL regions defined in SEQ ID NOs: 1 and 2, respectively.

[0089] Other antibodies of the disclosure include antibodies having at least 90%, particularly at least 95, 96, 97, 98, 99, or 100 percent identity to the VH and VL regions set forth in SEQ ID NOs: 1 and 2, respectively, or the VH and VL regions set forth in SEQ ID NOs: 19 and 20, respectively.

[0090] In certain embodiments, an anti-BTN2A antibody according to the present disclosure, typically a humanized anti-BTN2A1 antibody, comprises CDR1, a heavy chain variable region comprising SEQ ID NO:3, CDR2, a heavy chain variable region comprising SEQ ID NO:4, CDR3, a heavy chain variable region comprising SEQ ID NO:5, CDR1, a light chain variable region comprising SEQ ID NO:6, CDR2, a light chain variable region comprising SEQ ID NO:7, and CDR3, a light chain variable region comprising SEQ ID NO:8. In particular embodiments of the antibodies disclosed herein, the six CDR regions are 100% identical to the six CDR regions of the reference mAb 107G3, defined in SEQ ID NOs:3-8.

[0091] In other detailed embodiments, an anti-BTN2A1 antibody according to the present disclosure, typically a humanized anti-BTN2A1 antibody, comprises CDR1, a heavy chain variable region comprising SEQ ID NO:21, CDR2, a heavy chain variable region comprising SEQ ID NO:22, CDR3, a heavy chain variable region comprising SEQ ID NO:23, CDR1, a light chain variable region comprising SEQ ID NO:24, CDR2, a light chain variable region comprising SEQ ID NO:25, and CDR3, a light chain variable region comprising SEQ ID NO:26.

[0092] In particular embodiments of the antibodies disclosed herein, the six CDR regions are 100% identical to the six CDR regions of the reference mAb, 101G5, as defined in SEQ ID NOs: 21-26. Other antibodies disclosed herein include antibodies that have been mutated by amino acid deletion, insertion, or substitution, but still have amino acids in the CDR regions that have at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100 percent identity with the CDR regions of the reference mAb, 101G5. Typically, according to the present disclosure, an antibody may have between one, two, three, or four amino acid variations (including deletions, insertions, or substitutions) in one or more CDRs compared to the CDR sequences of the reference antibody, mAb 107G3, as defined in SEQ ID NOs: 21-26.

[0093] In other detailed embodiments of the antibodies disclosed herein, the six CDR regions are 100% identical to the six CDR regions of the reference mAb 107G3, as defined in SEQ ID NOs: 3-8. Other antibodies disclosed herein include antibodies that have been mutated by amino acid deletion, insertion, or substitution, but still have amino acids in the CDR regions that have at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100 percent identity with the CDR regions of the reference mAb 107G3. Typically, according to the present disclosure, an antibody may have between one, two, three, or four amino acid variations (including deletions, insertions, or substitutions) in one or more CDRs compared to the CDR sequences of the reference antibody mAb 107G3, as defined in SEQ ID NOs: 3-8.

[0094] In some embodiments, an antibody of the disclosure is a mutant variant of mAb 101G5 or mAb 107G3 having six CDR regions that are 100% identical to the corresponding six CDR regions of reference mAb 101G5 or 107G3, respectively, where the mutant variant antibody comprises a variant amino acid sequence in which one, two, three, four, or five amino acids have been mutated by amino acid deletion, insertion, or substitution in the FR1, FR2, FR3, and FR4 regions when compared to the corresponding framework regions of the corresponding reference antibody.

[0095] Functional variant antibodies As shown by the experimental data presented in the Examples, the reference mAb, 107G3, binds to residues at positions 65, 68, 69, 72, 78, 84, 85, 95, 97, 100 of human BTN2A1. Thus, the present disclosure encompasses mAbs that bind to a conformational epitope comprising amino acid residues located at positions 60-100 of SEQ ID NO:17, most particularly comprising amino acid residues at positions 65, 68, 69, 72, 78, 84, 85, 95, 97, 100 of SEQ ID NO:17, and have one or more of the functional properties defined supra and further recalled below, in particular one or more of the functional properties of the reference mAb, 107G3.

[0096] As also shown by the experimental data presented in the Examples, the reference mAb, 101G5, binds to residues at positions 212, 213, 218, 220, 224, 229 of BTN2A1. Thus, the present disclosure encompasses mAbs that bind to a conformational epitope comprising amino acid residues located at positions 210-230 of SEQ ID NO: 17, most particularly comprising residues at positions 212, 213, 218, 220, 224, 229 of SEQ ID NO: 17, and have one or more of the functional properties defined supra and further recalled below, in particular having one or more of the functional properties of the reference mAb, 101G5.

[0097] In yet other embodiments, a functional variant antibody of the present disclosure has a full-length heavy chain amino acid sequence and a full-length light chain amino acid sequence, or a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence, or an amino acid sequence of all six CDR regions, that are homologous or, more particularly, identical to the corresponding amino acid sequence of any one of the above-described reference antibodies, mAb 101G5 or mAb 107G3, wherein such a functional variant antibody retains the desired functional properties of said reference antibody.

[0098] A functional variant of the reference mAb, 101G5 antibody, or the reference antibody, mAb 107G3, particularly a functional variant of a VL, VH, or CDR, as used in the context of the monoclonal antibodies of the present disclosure, is an antibody that has at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of the affinity (typically measured by surface plasmon resonance (SPR) or by using the Octet® platform, based on Biolayer Interferometry (BLI) technology, typically performed at 25° C.) relative to the parent antibody (e.g., mAb 101G5 or mAb 107G3). D It is possible for such monoclonal antibodies to still retain affinity, selectivity, and / or specificity (as assessed by mAb 101G5 or mAb 107G3), and in some cases, such monoclonal antibodies of the present disclosure may be associated with greater affinity, selectivity, and / or specificity than the parent Ab (e.g., mAb 101G5 or mAb 107G3).

[0099] The desired functional properties of the reference mAb, 101G5 or 107G3, or a variant of said reference antibody disclosed herein, are: i. Specificity for BTN2A1, in particular the characteristics of binding to human BTN2A1 expressed in a cell line, e.g., HEK-293T BTN2 KO cells transfected with a plasmid encoding human BTN2A1, as described in the Examples, more particularly an EC of less than 50 μg / mL, more particularly less than 40 μg / mL. 50or a K of 10 nM or less as measured by surface plasmon resonance (SPR) (typically at 25° C.) or by Luminex assay (exemplified in the Examples) or Octet® (Abdiche et al., 2008). D and / or having a ratio of binding affinity to BTN2A1 compared to non-specific binding of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1, or more; and / or ii. Inhibition of monocyte polarization into M2 macrophages, typically assessed as illustrated in the Examples section; and / or iii. Induction of reversion of M2 macrophages to anti-tumor M1 macrophages, typically as assessed as exemplified in the Examples section; and / or iv. Direct induction of NK cell activation, typically assessed as illustrated in the Examples section; and / or v. Enhancement of NK cell-mediated cytotoxicity, typically as assessed as illustrated in the Examples section. may be selected from the group consisting of:

[0100] In some more particular embodiments, the desired functional properties of the reference mAb 107G3, or a variant of said reference antibody disclosed herein, are: vi. Activation of the production of cytolytic molecules (e.g., IFNγ or TNFα) from Vy9 / V52 T cells, typically assessed as illustrated in the Examples; vii. activation of the cytolytic function of Vy9 / V52 T cells, typically assessed as illustrated in the Examples; and / or viii. Activation of Vγ9 / Vδ2 T cell proliferation, typically assessed as illustrated in the Examples. It may further be selected from the group consisting of:

[0101] Typically, the functional properties of a functional variant of the reference mAb 101G5 or 107G3 according to points (ii) to (v) above are substantially equivalent to or superior to the corresponding functional properties of the corresponding reference antibody mAb 101G5 or 107G3 described above. By "substantially equivalent" herein is intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding functional properties of the reference mAb 107G3.

[0102] Typically, the functional properties of the functional variant of the reference mAb 107G3 according to points (vi) to (viii) above are substantially equivalent to or superior to the corresponding functional properties of the reference antibody mAb 107G3 described above. By "substantially equivalent" herein is intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding functional properties of the reference mAb 107G3.

[0103] For example, the present disclosure provides functional variant antibodies of reference mAb 101G5, the CDR sequences of which, i.e., the six CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, comprise variable heavy chain (VH) and variable light chain (VL) sequences that share at least 60, 70, 90, 95, or 100 percent sequence identity to the corresponding CDR sequences of reference antibody mAb 101G5, as set forth in SEQ ID NOs: 21-26, and which specifically bind to BTN2A and have the following functional properties i)-iv): i. the property of inhibiting the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. The ability to induce direct activation of NK cells; iv. Properties that enhance NK cell-mediated cytotoxicity The present invention relates to a functional variant antibody that exhibits at least one of the following characteristics:

[0104] Preferably, the antibody exhibits at least one of properties i) and ii) and at least one of properties iii) and iv), and most preferably the antibody exhibits properties i) to iv).

[0105] The present disclosure also provides functional variant antibodies of reference mAb 107G3, comprising variable heavy (VH) and variable light (VL) sequences that share at least 60, 70, 90, 95, or 100 percent sequence identity to the corresponding CDR sequences of reference antibody mAb 107G3, the CDR sequences, i.e., the six CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as set forth in SEQ ID NOs: 3-8, and that specifically bind to BTN2A1 and have the following functional properties: i. the property of inhibiting the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. The ability to induce direct activation of NK cells; iv. The property of enhancing NK cell-mediated cytotoxicity; v. The property of activating the production of IFNγ or TNFα by Vγ9 / Vδ2 T cells; vi. The property of activating the cytolytic function of Vγ9 / Vδ2 T cells; vii.Properties that activate Vγ9 / Vδ2 T cell proliferation The present invention also relates to functional variant antibodies that exhibit at least one of the following:

[0106] Preferably, said functional variant exhibits at least one of the functional activities i) to iv) (preferably at least activities i) and / or ii), and iii) and / or iv)), and at least one of the functional activities v) to vii).

[0107] The present disclosure relates to functional variant antibodies of reference mAb 101G5, comprising heavy and light chain variable regions that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99%, or 100% identical to the corresponding heavy and light chain variable regions of said reference antibody, mAb 101G5, as defined in SEQ ID NOs: 19 and 20, respectively, and that specifically bind to BTN2 and have the following functional properties: i. the property of inhibiting the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. The ability to induce direct activation of NK cells; iv. Properties that enhance NK cell-mediated cytotoxicity The present invention further relates to a functional variant antibody which exhibits at least one of the following:

[0108] Preferably, a functional variant antibody exhibits at least one of properties i) and ii) and at least one of properties iii) and iv), most preferably the antibody exhibits properties i) to iv).

[0109] The present disclosure relates to an antibody of a reference mAb, 107G3, comprising heavy and light chain variable regions that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99%, or 100% identical to the corresponding heavy and light chain variable regions of said reference antibody, mAb 107G3, as defined in SEQ ID NOs: 1 and 2, respectively, and which specifically binds to BTN2A and has the following functional properties: i. the property of inhibiting the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. the ability to induce direct activation of NK cells; and / or iv. The property of enhancing NK cell-mediated cytotoxicity; v. The property of activating the production of cytolytic molecules (IFNγ or TNFα) by Vγ9 / Vδ2 T cells; vi. The property of activating the cytolytic function of Vγ9 / Vδ2 T cells, and / or vii.Properties that activate Vγ9 / Vδ2 T cell proliferation The present invention further relates to a functional variant antibody which exhibits at least one of the following:

[0110] In some embodiments, the functional variant exhibits at least one of the functional activities i) to iv) (in particular at least activities i) and / or ii), and iii) and / or iv)), and at least one of the functional activities v) to vii).

[0111] In some embodiments, the functional variant exhibits one or more of the functional activities i)-iv) or one or more of the functional activities v)-vii).

[0112] Typically, the functional properties of a functional variant of the reference mAb 101G5 or 107G3 according to points (i) to (iv) above are substantially equivalent to or superior to the corresponding functional properties of the corresponding reference antibody, mAb 101G5 or 107G3, described above. By "substantially equivalent" herein is intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding functional properties of the reference mAb 107G3.

[0113] In various embodiments, the antibody may exhibit one or more of the desired functional properties discussed above.

[0114] The antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody. Typically, the antibody or protein is a humanized antibody, more particularly a humanized silenced antibody.

[0115] As used herein, the term "silent" antibody refers to an antibody that exhibits no or low ADCC activity as measured in an in vitro ADCC activity assay, which measures cytolysis of target cells.

[0116] In one embodiment, the term "no or low ADCC activity" means that the silenced antibody exhibits less than 50%, e.g., less than 10%, of the ADCC activity observed with a corresponding wild-type (non-silenced) antibody, e.g., a wild-type human IgG1 antibody. Typically, no detectable ADCC activity is observed in an in vitro ADCC activity assay with the silenced antibody compared to a control Fab antibody.

[0117] Silencing of effector functions is obtained by mutations in the Fc constant part of the antibody and is described in the art: Strohl, 2009 (LALA and N297A); Baudino, 2008, D265A (Baudino et al., J. Immunol. 2008, Strohl, CO Biotechnology, 20, 2009). An example of a silent IgG1 antibody contains mutations at positions 234, 235 and / or 331 (EU numbering) of the amino acid sequence of IgG1 Fc that reduce ADCC. Another silent IgG1 antibody contains the N297A mutation, resulting in a glycosylated or non-glycosylated antibody.

[0118] The sequence of a CDR variant may differ from the sequence of a CDR of a parent antibody sequence by conservative substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the variant are conservative replacements of amino acid residues. In the context of this disclosure, conservative substitutions are defined as follows: Aliphatic residues: I, L, V, and M Cycloalkenyl associated 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 residues: A, C, D, G, N, P, S, T, and V Very small residues: A, G, and S Residues involved in turn formation: 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. Conservative substitutions may be defined by substitutions within amino acid classes reflected as follows: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. In the variant CDRs, the hydrophobic / hydrophilic properties and the conservation of residue weight / size are also substantially retained compared to the CDRs of any one of mAb1 to mAb6. The importance of the hydrophobic index of amino acids in conferring interactive biological function to a protein is generally understood in the art. It is accepted that the relative hydrophobic character of amino acids contributes to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge characteristics: 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); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues may also, or alternatively, be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8, available from NCBI, using standard settings: BLOSUM62, Open Gap=11, and Extended Gap=1). Suitable variants typically exhibit at least about 80% identity to the parent peptide.In accordance with the present disclosure, a first amino acid sequence having at least about 70% identity to a second amino acid sequence means that the first sequence has 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% identity with the second amino acid sequence. In accordance with the present disclosure, a first amino acid sequence having at least about 50% identity to a second amino acid sequence means that the first sequence has 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% identity with the second amino acid sequence.

[0119] In some embodiments, the antibody of the present disclosure is a chimeric antibody, typically a mouse / human chimeric antibody. The term "chimeric antibody" refers to a monoclonal antibody that comprises the VH and VL domains of an antibody derived from a non-human animal and the CH and CL domains of a human antibody. Any animal can be used as the non-human animal, such as mouse, rat, hamster, rabbit, etc. In particular, the mouse / human chimeric antibody can comprise the VH and VL domains of any one of the reference antibodies mAb 101G5 or mAb 107G3.

[0120] In some embodiments, the antibody of the present disclosure is a humanized antibody. In a detailed embodiment, the antibody of the present disclosure is a humanized antibody that includes six CDRs of any one of the reference antibodies mAb 101G5 or mAb 107G3. As used herein, the term "humanized antibody" refers to an antibody whose framework regions (FRs) are modified to include FRs derived from a donor immunoglobulin of a different species (e.g., a human species) compared to the FRs of a parent immunoglobulin (e.g., a mouse CDR).

[0121] In some embodiments, the antibody of the present disclosure is selected from the group consisting of Fab, F(ab')2, Fab', and scFv. As used herein, the term "Fab" refers to an antibody fragment obtained by treating IgG with papain, a protease, that has a molecular weight of about 50,000 and antigen-binding activity, and in which about half of the N-terminal side of the H chain and the entire L chain are bound together via a disulfide bond. The term "F(ab')2" refers to an antibody fragment obtained by treating IgG with pepsin, a protease, that has a molecular weight of about 100,000 and antigen-binding activity, and is slightly larger than Fab, which is bound via a disulfide bond in the hinge region. The term "Fab'" refers to an antibody fragment obtained by cleaving the disulfide bond in the hinge region of F(ab')2, that has a molecular weight of about 50,000 and antigen-binding activity. Single-chain Fv ("scFv") polypeptides are covalently linked VH:VL heterodimers that are typically expressed by a gene fusion comprising genes encoding VH and VL linked by a peptide-encoding linker. Human scFv fragments of the present disclosure typically contain the CDRs held in the proper conformation by using recombinant gene methods.

[0122] Functional variant antibodies with variant amino acid sequences may be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of the encoding nucleic acid molecule, followed by testing of the encoded altered antibodies for retained function (i.e., the functions set forth above) using the functional assays described herein.

[0123] Antibodies that cross-compete with reference 101G5 mAb or reference 107G3 mAb Additional antibodies with similar advantageous properties to the reference mAb, 101G5, or reference mAb, 107G3, disclosed herein, can be identified based on their ability to statistically significantly cross-compete (e.g., competitively inhibit the binding of) the reference mAb, 107G3, described above, in a standard BTN2A1 binding assay.

[0124] Test antibodies may first be screened for their binding affinity to BTN2A1, typically using, for example, phage display technology, from human recombinant antibody libraries or from transgenic mice expressing human variable region antibodies immunized with a BTN2A1 antigen, as assessed in the Examples (see the Materials and Methods section).

[0125] In another embodiment, the present disclosure provides an antibody that binds to the same epitope as at least the reference mAb 107G3 or the reference mAb 101G5, described above. As illustrated in the Examples section, the reference mAbs 101G5 and 107G3 do not bind to the same epitope on BTN2A1.

[0126] The ability of a test antibody to cross-compete with or inhibit the binding of an antibody of the present disclosure to human BTN2A1 supports the belief that the test antibody may compete with this antibody for binding to human BTN2A1, and, according to non-limiting theory, such an antibody binds to the same or a similar (e.g., structurally similar or spatially proximal) epitope on human BTN2A1 as the antibody it competes with.

[0127] For example, the following test can be used to screen anti-BTN2A1 antibodies for their ability to cross-compete with the reference antibody, mAb 107G3, and / or to screen anti-BTN2A1 antibodies for their ability to bind to the same epitope as the reference antibody: BTN2KO cells transfected with human BTN2A1 (typically HEK293T, as described in the Examples) can be stained with a saturating concentration (e.g., 10 μg / mL) of the reference antibody, mAb 107G3. Different doses of the test anti-BTN2A1 mAb can then be examined for their potential competitiveness with the reference antibody, mAb 107G3. In the presence of such a reference antibody, the mAb competing with the reference antibody will not be able to recognize BTN2A1. Data can be expressed as mean fluorescence intensity. Alternatively, the competition assay can be performed in a binning assay, as described in the Examples section. Typically, a binning experiment can be performed by immobilizing recombinant human BTN2A1 on a Biosensor and presenting a reference antibody followed by a competing antibody.

[0128] Selected antibodies may be further examined for the advantageous properties of mAb 101G5 or mAb 107G3, in particular those detailed supra.

[0129] Thus, in one embodiment, the disclosure provides an isolated antibody that competes with reference mAb 101G5 or reference mAb 107G3 for binding to BTN2A1, i. have specificity for BTN2A1, in particular bind to human BTN2A1 expressed in a cell line, e.g., HEK-293T BTN2 KO cells transfected with human BTN2A1 as described in the Examples, more particularly an EC of less than 50 μg / mL, more particularly less than 40 μg / mL 50 or a K of 10 nM or less as measured by surface plasmon resonance (SPR), or by Luminex assay (exemplified in the Examples) or Octet® assay. Dand / or has a ratio of binding affinity to BTN2A1 relative to non-specific binding of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1, or more (see also above for further details); and / or ii. inhibiting the polarization of monocytes into M2 macrophages; iii. Induce reversion of M2 macrophages to antitumor M1 macrophages; iv. Inducing direct activation of NK cells; and / or v. Enhancement of NK cell-mediated cytotoxicity An isolated antibody is presented.

[0130] Alternatively, or in addition, the antibody comprises vi. activates the production of cytolytic molecules (e.g., IFNγ or TNFα) by Vy9 / V52 T cells, typically as assessed as illustrated in the Examples; and / or vii. activates the cytolytic function of Vy9 / V52 T cells, typically assessed as illustrated in the Examples; and / or viii. Activating the proliferation of Vy9 / V52 T cells, typically assessed as illustrated in the Examples.

[0131] In such embodiments, more particularly, the antibody does not cross-react with BTN3.

[0132] Typically, the functional properties according to points (iii)-(v) above of an antibody that competes with the reference mAb 101G5 or 107G3 for binding to BTN2A1 are substantially equivalent to or better than the corresponding functional properties of the reference antibody mAb 101G5 or mAb 107G3 described above. By "substantially equivalent" herein is intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the corresponding functional property of the reference mAb 101G5 or 107G3.

[0133] Typically, an antibody that competes with the reference mAb, 107G3, for binding to BTN2A1 according to the present disclosure will still retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of the affinity and / or selectivity relative to the reference antibody (e.g., mAb 107G3), and in some cases may be associated with greater affinity, selectivity, and / or specificity than the reference antibody (e.g., mAb 107G3).

[0134] In certain embodiments, the cross-blocking antibody, or an antibody that competes with the reference mAb, 101G5 or 107G3, for binding to BTN2A1 is a chimeric antibody, a humanized antibody, or a human recombinant antibody.

[0135] Generation of transfectomas producing monoclonal antibodies The antibodies of the present disclosure may be produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic method, alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can easily produce said antibodies by standard techniques for producing polypeptides. For example, the antibodies of the present disclosure may be synthesized using well-known solid-phase methods, typically using a commercially available peptide synthesizer (such as a peptide synthesizer from Applied Biosystems, Foster City, California) following the manufacturer's instructions. Alternatively, the antibodies of the present disclosure may be synthesized by recombinant DNA methods well known in the art. For example, the antibodies may be obtained as DNA expression products following incorporation of a DNA sequence encoding the antibody into an expression vector and introduction of such vector into a suitable eukaryotic or prokaryotic host, which allows the desired antibody to be expressed, and isolated from these hosts using well-known techniques.

[0136] Thus, a further object of the present disclosure relates to a nucleic acid molecule encoding an antibody disclosed herein. More particularly, the nucleic acid molecule encodes a heavy or light chain of the antibody of the present disclosure. More particularly, the nucleic acid molecule comprises a VH or VL encoding region having at least 70%, 80%, 90%, 95%, or 100% identity to the corresponding nucleic acid encoding the heavy chain variable region (VH region) or light chain variable region (VL) of the reference antibody, mAb 107G3.

[0137] Typically, the 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. As used herein, the terms "vector", "cloning vector", and "expression vector" refer to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Thus, a further object of the present disclosure relates to a vector comprising the nucleic acid disclosed herein. Such a vector can include regulatory elements, such as a promoter, enhancer, terminator, etc., to cause or direct expression of the antibody upon administration to a subject. Examples of promoters and enhancers that can be used in expression vectors for animal cells include SV40 early promoter and early enhancer, Moloney murine leukemia virus LTR promoter and LTR enhancer, immunoglobulin H chain promoter and enhancer, etc. Any expression vector for animal cells can be used as long as the gene encoding the C region of human antibody can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1 beta d2-4, etc. Examples of other plasmids include replicative plasmids containing a replication origin, or integrative plasmids such as pUC, pcDNA, pBR, etc. Examples of other viral vectors include adenoviral vectors, retroviral vectors, herpes virus vectors, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection of helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO 94 / 19478.

[0138] A further object of the present disclosure relates to host cells transfected, infected or transformed with the above described nucleic acids and / or vectors. As used herein, the term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed".

[0139] The nucleic acids disclosed herein can be used to produce the antibodies of the present disclosure in a suitable expression system. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by, for example, the foreign DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host 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, etc.). Particular examples include E. coli, yeast of the genera Kluyveromyces or Saccharomyces, mammalian cell lines (e.g., HEK-293 cells, Vero cells, CHO cells, 3T3 cells, COS cells, etc.), as well as primary or established mammalian cell cultures (e.g., cultures made from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "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 the like.

[0140] The present disclosure also relates to a method of making a recombinant host cell expressing an antibody disclosed herein, comprising the steps of (i) introducing, in vitro or ex vivo, a recombinant nucleic acid or vector as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, the resulting recombinant host cell, and (iii) optionally selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used to make the antibodies of the present disclosure.

[0141] The antibodies of this disclosure are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0142] In some embodiments, the human chimeric antibody of the present disclosure can be produced by obtaining nucleic acid sequences encoding the VL and VH domains described above, inserting them into an expression vector for animal cells having genes encoding the CH and CL of a human antibody, constructing a human chimeric antibody expression vector, and expressing the coding sequences by introducing the expression vector into an animal cell. The CH domain of the human chimeric antibody can be any region belonging to human immunoglobulin, but the CH domain of the IgG class is suitable, and any one of the subclasses belonging to the IgG class, such as IgG1, IgG2, IgG3, and IgG4, can be used. The CL domain of the human chimeric antibody can also be any region belonging to Ig, but the CL domain of the kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection methods well known in the art (see Morrison SL. et al. (1984), and patent documents U.S. Pat. Nos. 5,202,238 and 5,204,244).

[0143] The humanized antibody of the present disclosure can be produced by obtaining nucleic acid sequences encoding the CDR domains described above, inserting them into an expression vector for animal cells having genes encoding (i) a heavy chain constant region and a heavy chain variable framework region identical to those of a human antibody, and (ii) a light chain constant region and a light chain variable framework region identical to those of a human antibody, constructing a humanized antibody expression vector, and expressing the genes by introducing the expression vector into an appropriate cell line. The humanized antibody expression vector may be of a type in which a gene encoding the antibody heavy chain and a gene encoding the antibody light chain are present in separate vectors, or of a type in which both genes are present in the same vector (tandem type). In terms of ease of construction of the humanized antibody expression vector, ease of introduction into a cell line, and balance of expression levels between the antibody H chain and L chain in the cell line, a tandem type humanized antibody expression vector is preferred. Examples of tandem type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, etc.

[0144] Methods for humanizing antibodies based on conventional recombinant DNA and gene transfection methods are well known in the art (see, for example, Riechmann L. et al., 1988; Neuberger MS. et al., 1985). Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see EP 125023 and WO 96 / 02576).

[0145] The Fab of the present disclosure can be obtained by treating an antibody that specifically reacts with anti-Mullerian hormone with the protease papain. The Fab can also be produced by inserting DNA encoding the Fab of an antibody into a vector for a prokaryotic expression system or a vector for a eukaryotic expression system, and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express the Fab.

[0146] The F(ab')2 of the present disclosure can be obtained by treating an antibody specifically reactive with anti-Müllerian hormone with the protease pepsin. F(ab')2 can also be produced by linking Fab' via a thioether bond or a disulfide bond, as described below.

[0147] The Fab' of the present disclosure can be obtained by treating F(ab')2 specifically reactive with anti-Mullerian hormone with a reducing agent, dithiothreitol. Fab' can also be produced by inserting DNA encoding the Fab' fragment of an antibody into an expression vector for prokaryotic cells or an expression vector for eukaryotic cells, and introducing the vector into a prokaryotic or eukaryotic cell (as appropriate) to express it.

[0148] The scFvs of the present disclosure can be produced by obtaining cDNA encoding the VH and VL domains as described supra, constructing DNA encoding the scFv, inserting the DNA into an expression vector for prokaryotic cells or an expression vector for eukaryotic cells, and then introducing the expression vector into a prokaryotic or eukaryotic cell (as appropriate) to express the scFv.

[0149] To generate humanized scFv fragments, a well-known technique called CDR grafting can be used, which involves selecting complementarity determining regions (CDRs) from a donor scFv fragment and grafting them onto a framework of known three-dimensional structure from a human scFv fragment (see, for example, WO 98 / 45322; WO 87 / 02671; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,585,089; U.S. Pat. No. 4,816,567; EP 0173494).

[0150] The engineered antibodies of the present disclosure further include antibodies in which modifications have been made to framework residues within VH and / or VL, e.g., to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues may be identified by comparing the framework sequence of the antibody to the germline sequence from which the antibody is derived. To return the sequences of the framework regions to their germline configuration, the somatic mutations may be "backmutated" to the germline sequence, e.g., by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present disclosure. Another type of framework modification involves mutating one or more residues in the framework regions and / or one or more residues in the CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail by Carr et al. in U.S. Patent Publication No. 20030153043.

[0151] Fc operation The antibodies disclosed herein may be characterized by one or more of the functional or structural features in the aspects described above, or by any combination of selected functional and structural features.

[0152] The antibodies disclosed herein can be of any isotype. The choice of isotype will typically be guided by the desired effector function, such as silencing ADCC. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the kappa region or the lambda region of the human light chain constant region may be used. If desired, the class of the antibody of the present disclosure may be switched by known methods. A typical class switching method may be used to convert one IgG subclass to another IgG subclass, for example, converting IgG1 to IgG2. Thus, the effector function of the antibody of the present disclosure may be changed by isotype switching, for example, to an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgD antibody, an IgA antibody, an IgE antibody, or an IgM antibody for various therapeutic uses. In some embodiments, the antibodies disclosed herein are full-length antibodies. In some embodiments, the full-length antibody is an IgG1 antibody. In some embodiments, the full-length antibody is an IgG4 antibody. In some embodiments, the BTN2A-specific IgG4 antibody is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4 antibodies are those in which the arginine at position 409 of the heavy chain constant region of human IgG4, as indicated in the EU index, is replaced with lysine, threonine, methionine, or leucine, typically lysine (described in WO2006033386), and / or the hinge region comprises the Cys-Pro-Pro-Cys sequence, as described in WO2008145142. Other suitable stabilized IgG4 antibodies are disclosed in WO2008145142.

[0153] In some embodiments, the antibodies of the present disclosure do not comprise an Fc portion that induces antibody-dependent cell-mediated cytotoxicity (ADCC). The terms "Fc domain", "Fc portion", and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of a human gamma heavy chain, or other types of antibody heavy chains (e.g., α, δ, ε, and μ chains for human antibodies), or the corresponding sequences in naturally occurring allotypes thereof. Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) "Sequences of Protein of Immunological Interest", 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD). In some embodiments, the antibodies of the present disclosure do not comprise an Fc domain capable of substantially binding to an FcgRIIIA (CD16) polypeptide. In some embodiments, the antibody of the disclosure lacks an Fc domain (e.g., lacks a CH2 domain and / or a CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype. In some embodiments, the antibody of the disclosure consists of or comprises a Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, single chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the antibody of the disclosure is not linked to a toxic moiety. In some embodiments, one or more amino acids selected from the amino acid residues may be replaced with a different amino acid residue such that the antibody has altered binding to C2q and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Pat. No. 6,194,551.

[0154] Another modification of the antibodies herein that is contemplated herein is PEGylation. Antibodies may be PEGylated, for example, to extend the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are conjugated to the antibody or antibody fragment. PEGylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxypolyethylene glycol or aryloxypolyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be PEGylated is a non-glycosylated antibody. Methods for PEGylating proteins are known in the art and may be applied to the antibodies of the present disclosure. See, for example, European Patent No. 0154316 to Nishimura et al., and European Patent No. 0401384 to Ishikawa et al.

[0155] Another modification of the antibodies contemplated herein is the conjugation or protein fusion of at least the antigen-binding region of an antibody of the present disclosure to a serum protein, such as human serum albumin or a fragment thereof, to extend the half-life of the resulting molecule.

[0156] In some embodiments, the present disclosure also provides multispecific antibodies. Exemplary formats for the multispecific antibody molecules of the present disclosure include: (i) two antibodies crosslinked by chemical heteroconjugation, one antibody with specificity for BTN2A and another antibody with specificity for a second antigen; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, e.g., two scFvs, linked in tandem by an additional peptide linker; (iv) a dual variable domain antibody (DVD-Ig), in which each light chain and each heavy chain contains two variable domains in tandem via a short peptide linkage (Wu et al., "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule": "Antibody Engineering", Springer Berlin, 1999). Heidelberg, 2010); (v) chemically linked bispecific (Fab')2 fragments; (vi) Tandabs, which are fusions of two single chain diabodies resulting in tetravalent bispecific antibodies with two binding sites for each of the target antigens; (vii) Flexibodies, which are combinations of scFvs with diabodies resulting in multivalent molecules; (viii) so-called "dock and lock" molecules based on the "dimer / docking domain" in protein kinase A, which when applied to Fabs can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (ix) so-called Scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (x) diabodies. Another exemplary format for bispecific antibodies is an IgG-like molecule with complementary CH3 domains that force heterodimerization.Such molecules can be prepared using known techniques, such as, for example, the techniques known as Triomab / Quadroma technology (Trion Pharma / Fresenius Biotech), KIH (Knob-into-Hole) technology (Genentech), CrossMAb technology (Roche) and electrostatic match technology (Amgen), LUZ-Y technology (Genentech), Strand Exchange Engineered Domain body technology (SEEDbody) (EMDSerono), Biclonic technology (Merus), and DuoBody technology (Genmab A / S). In some embodiments, bispecific antibodies are obtained or obtainable via controlled Fab arm exchange, typically using DuoBody technology. In vitro methods for generating bispecific antibodies by controlled Fab arm exchange are described in WO2008119353 and WO2011131746 (both by Genmab A / S). In one exemplary method described in WO2008119353, bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (swapping of heavy chains with joined light chains) between two monospecific antibodies, both of which contain an IgG4-like CH3 region, upon incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.In another exemplary method described in WO2011131746, a bispecific antibody of the disclosure can be prepared by the following steps: a) providing a first antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a first CH3 region; and b) providing a second antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a second CH3 region, wherein heterodimeric interaction between the first CH3 region and the second CH3 region is determined by a heterodimeric interaction between the first CH3 region and the second CH3 region. a) a sequence of the first CH3 region differs from a sequence of the second CH3 region such that the homodimeric interaction is stronger than that of the first antibody; b) a step of incubating the first antibody together with the second antibody under reducing conditions; and c) a step of obtaining the bispecific antibody, in which the first antibody is an antibody of the present disclosure and the second antibody has a different binding specificity, or vice versa, and at least one of the first antibody and the second antibody is an antibody of the present disclosure. The reducing conditions can be brought about, for example, by adding a reducing agent selected from, for example, 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) can further include restoring the conditions to non-reducing or less reducing conditions, for example, by removing the reducing agent, for example, by desalting. Typically, the sequence of the first CH3 region and the sequence of the second CH3 region differ only by a small number of conservative, to a large extent, asymmetric mutations, such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region, respectively. Further details about these interactions and how they can be achieved are provided in WO2011131746, which is incorporated herein by reference in its entirety. The following are exemplary embodiments of combinations such as symmetric mutations, optionally with one or both of the Fc regions being of IgG1 isotype:

[0157] Thus, the present disclosure provides bispecific or multispecific antibodies (also named bispecific or multispecific molecules) that comprise the anti-BTN2A antibodies described herein. Thus, the present disclosure includes bispecific molecules that comprise at least one first binding specificity for BTN2A, e.g., an antigen-binding portion of one of the antibodies disclosed herein, and a second binding specificity for a second target epitope. For example, a bispecific molecule according to the present disclosure comprises at least CDR1 is a heavy chain variable region comprising SEQ ID NO:3, CDR2 is a heavy chain variable region comprising SEQ ID NO:4, CDR3 is a heavy chain variable region comprising SEQ ID NO:5, CDR1 is a light chain variable region comprising SEQ ID NO:6, CDR2 is a light chain variable region comprising SEQ ID NO:7, and CDR3 is a light chain variable region comprising SEQ ID NO:8, or CDR1 is a heavy chain variable region comprising SEQ ID NO:21, CDR2 is a heavy chain variable region comprising SEQ ID NO:22, CDR3 is a heavy chain variable region comprising SEQ ID NO:23, CDR1 is a light chain variable region comprising SEQ ID NO:24, CDR2 is a light chain variable region comprising SEQ ID NO:25, and CDR3 is a light chain variable region comprising SEQ ID NO:26. The antigen-binding portion may comprise one antigen-binding portion comprising:

[0158] In one embodiment, the bispecific molecule comprises a second binding specificity for BTN3. More particularly, the bispecific molecule may further comprise an antigen-binding portion of an anti-BTN3A activating antibody that specifically binds to BTN3A and may activate the cytolytic function of Vy9 / V52 T cells.

[0159] Additionally, in embodiments in which the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the first and second target epitopes.

[0160] In one embodiment, the bispecific molecules disclosed herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, a Fab, Fab', F(ab')2, Fv, unibody or single chain Fv. The antibody can also be any minimal fragment thereof, such as a light chain dimer or a heavy chain dimer, or an Fv or single chain construct as described in Ladner et al., U.S. Patent No. 4,946,778.

[0161] Other antibodies that can be used in the bispecific molecules disclosed herein are murine, chimeric, and humanized monoclonal antibodies.

[0162] Pharmaceutical Compositions In another aspect, the present disclosure provides compositions, such as pharmaceutical compositions, that contain at least one antibody as disclosed herein, formulated together with a pharma- ceutically acceptable carrier. Such compositions can contain one or a combination of the antibodies described above (e.g., two or more different antibodies). The pharmaceutical compositions disclosed herein can also be administered in combination therapy, i.e., in combination with other agents.

[0163] For example, the antibodies of the invention will typically be combined with at least one anti-viral agent, an anti-inflammatory agent, or another anti-proliferative agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the antibodies of the present disclosure.

[0164] As used herein, a "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and all of these carriers, such as physiologically compatible carriers. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous routes.

[0165] Depending on the route of administration, the active compound, i.e., the antibody, may be coated with a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. The form of the pharmaceutical composition, the route of administration, the dosage, and the administration regimen will, of course, depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc.

[0166] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and the like.

[0167] Uses and Methods of the Invention The antibodies of the disclosure have in vitro and in vivo diagnostic and therapeutic utilities, for example, these molecules may be administered to cells in culture, e.g., in vitro, ex vivo, or in vivo, or to a subject in vivo, e.g., to treat, prevent, or diagnose various disorders.

[0168] The antibodies of the disclosure are anti-BTN2A1 antibodies that inhibit the differentiation of monocytes into tumor-promoting M2 macrophages in relation to phenotype, cytokine secretion, and / or T cell inhibitory properties.

[0169] Alternatively, or preferably, in addition, the antibodies of the present disclosure directly bind to BTN2A (particularly BTN2A1) on the cell membrane of NK cells, triggering their activation and cytotoxicity against cancer cells.

[0170] In some embodiments, the antibodies of the disclosure may further activate the cytolytic function, cytokine production, and proliferation of Vy9 / V52 T cells.

[0171] Thus, the antibodies of the present disclosure may be used to overcome immune suppressive mechanisms observed in cancer patients and also during chronic infections.

[0172] In some embodiments, the antibodies of the present disclosure may be used to reduce the immunosuppressive effects of the tumor environment.

[0173] The anti-BTN2A antibodies disclosed herein may also potentiate the cytotoxic effects of both NK cells and / or Th1 cells by acting against the tumor microenvironment (via polarization towards M1 and / or inhibition of M2) and also by acting directly on the NK cell compartment.

[0174] In some embodiments, the antibodies of the present disclosure (such as the 107G3 antibody and its variants described herein) further activate the cytolytic function, cytokine production, and proliferation of Vy9 / V52 T cells. Thus, such antibodies have the potential to act in concert on three immune cell compartments: NK cells, macrophages, and γδ T cells, and therefore represent a powerful tool for cancer treatment, especially for the treatment of solid tumors.

[0175] Preclinical studies have firmly supported that NK cells can kill myeloid lineage leukemic cells. However, in CML, for example, NK cells decrease in number with disease progression, become less responsive to stimuli, and exhibit reduced cytolytic activity. In AML, high cytolytic activity of NK cells also predicts good long-term outcomes for patients, both at diagnosis and in remission (Carlsten M, Jaras M, "Natural Killer Cells in Myeloid Malignancies: Immune Surveillance, NK Cell Dysfunction, and Pharmacological Opportunities to Bolster the Endogenous NK Cells", Front Immunol., 2019). Thus, in some embodiments, the antibodies of the present invention that exhibit NK cell activating properties may be used in combination with NK cell therapy, such as adoptively transferred NK cell therapy to restore the function of NK cells and / or induce or improve their cytotoxicity. In particular, the antibodies of the present application may be used in the treatment of solid tumors that are typically resistant to killing by NK cells. As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells having an abnormal stage or condition characterized by a capacity for self-renewal, i.e., rapid proliferative cell proliferation. Hyperproliferative and neoplastic disease states may be categorized as pathological states, i.e., conditions that characterize or constitute a disease state, or as non-pathological states, i.e., conditions that are deviations from the normal state but are not associated with a disease state. The terms are intended to include all types of cancerous growths or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of invasive histopathological type or histopathological stage.

[0176] The term "cancer" or "neoplasm" includes malignancies of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, gastrointestinal, and genitourinary tract, as well as adenocarcinomas, including most colon cancers, squamous cell lung carcinoma, skin or vaginal carcinoma, renal cell carcinoma, prostate and / or testicular cancer, non-small cell lung carcinoma, small cell lung carcinoma, endometrial cancer, ovarian cancer, cervical adenocarcinoma, pancreatic cancer, small intestine cancer, and esophageal cancer, and generally any cancer that can be treated by in vivo stimulation of activation and / or proliferation of γδ T cells in a subject suffering from said cancer.

[0177] Examples of cancer include, but are not limited to, hematological malignancies such as B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, non-Hodgkin's lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK-cell lymphoid neoplasms, and myeloid lineage neoplasms including acute myeloid leukemia.

[0178] Examples of non-hematological cancers include, but are not limited to, colon cancer, breast cancer, lung cancer, ovarian cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, testicular cancer, and skin cancer.

[0179] Examples of chronic infections include, but are not limited to, viral, bacterial, parasitic, or fungal infections, such as chronic hepatitis, lung infections, lower respiratory tract infections, bronchitis, influenza, pneumonia, and sexually transmitted diseases.

[0180] Thus, the present disclosure relates to a method for treating one of the above-disclosed disorders in a subject in need thereof, comprising a therapeutically effective amount of an anti-BTN2A1 antibody, as disclosed above.

[0181] The antibodies for the above disclosed uses may be administered as the sole active ingredient or may be administered together, e.g., as an adjuvant thereto, or in combination with other drugs, e.g., cytokines, antiviral, anti-inflammatory, or cytotoxic, antiproliferative, chemotherapeutic or antitumor agents, cell therapy products (e.g., γδ T cell compositions or NK cell compositions), e.g., for the treatment or prevention of the aforementioned diseases.

[0182] For example, the antibodies for the uses disclosed above may be used in combination with cell therapy, in particular γδ T cell therapy, NK cell therapy, chemotherapy, anti-neoplastic agents, or immunotherapeutic agents.

[0183] As used herein, the term "cell therapy" refers to a therapy comprising administration in vivo of at least a therapeutically effective amount of a cell composition to a subject in need thereof. The cells administered to the patient may be allogeneic or autologous. The term "γδ T cell therapy" refers to a cell therapy in which the cell composition comprises, as an active ingredient, γδ T cells, in particular Vγ9 / Vδ2 T cells (such as adoptive γδ T cell transfer, or chimeric antigen receptor-expressing γδ T cells). The term "NK cell therapy" refers to a cell therapy in which the cell composition comprises, as an active ingredient, NK cells, such as adoptive NK cell transfer, or chimeric antigen receptor-expressing NK cells (CAR-NK).

[0184] A cell therapy product refers to a cell composition administered to the patient for therapeutic purposes, the cell therapy product comprising a therapeutically effective dose of cells and, optionally, additional excipients, adjuvants, or other pharma- ceutically acceptable carriers.

[0185] Suitable antineoplastic agents include, but are not limited to, alkylating agents (such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosoureas, temozolomide, etc.), anthracyclines (such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, etc.), taxanes (such as paclitaxel, docetaxel, etc.), epothilones, inhibitors of type I topoisomerase (such as irinotecan or topotecan), inhibitors of type II topoisomerase (such as etoposide, teniposide, or tafluposide), nucleotide analogs and analogs of nucleotide precursors (such as azacitidine, azathioprine, capecitabine, cytarabine, fluorouracil, gefitinib ... mucitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine), peptide antibiotics (such as carboplatin, cisplatin, and oxaliplatin), retinoids (such as tretinoin, alitretinoin, bexarotene), vinca alkaloids and derivatives (such as vinblastine, vincristine, vindesine, vinorelbine), kinase inhibitors (such as ibrutinib, idelalisib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib), proteasome inhibitors (such as bortezomib, carfilzomib), histone deacetylase inhibitors (such as vorinostat or romidepsin).

[0186] Examples of immunotherapeutic agents include, but are not limited to, pAg (phosphoantigen) (e.g., zoledronic acid or other bisphosphonates), anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and cytokines (such as interleukin 2 (IL-2) (Choudhry H et al., 2018, Biomed Res Int.), interleukin 15 (IL-15) (Patidar M et al., Cytokine Growth Factor Rev., 2016), interleukin 21 (IL-21) (Caccamo N. et al., PLoS ONE., 2012), or interleukin 33 (IL-33) (Duault C et al., J Immunol., 2016)), or recombinant forms and derivatives thereof, or any cytokine capable of inducing lymphocyte activity (e.g., proliferation or changes in cytokine production or metabolism). The term "derivative" is used for any cytokine modification that may rely on PEGylation (e.g., conjugation to polyethylene glycol (PEG) chains), mutations such as amino acid deletions, substitutions, or insertions, or association with enhancers (e.g., an IL15 / IL15Ra complex fused to IgG1 Fc, in which, in addition, IL-15 is mutated (asn72asp) to further increase the biological activity of this complex, making it a superagonist of IL-2 and IL-15Rβγ) (Rhode PR et al., Cancer Immunol Res., 2016) (Barroso-Sousa R et al., Curr Oncol Rep., 2018).

[0187] The term "IL-2" has its conventional meaning and refers to human interleukin 2. IL-2 primarily regulates lymphocyte activity by binding to the IL-2 receptor.

[0188] The term "IL-15" has its conventional meaning and refers to human interleukin 15. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 regulates the activation and proliferation of T and natural killer (NK) cells.

[0189] The term "IL-21" has its general meaning and refers to human interleukin 21. IL-21 has been implicated in pleiotropic properties including, but not limited to, enhancing cytotoxicity by NK cells and CD8+ T cells, modulating plasma cell differentiation, and inhibiting Treg cells.

[0190] The term "IL-33" has its general meaning and refers to human interleukin 33. IL-33, believed to be an alarmin released upon tissue stress or injury, is a member of the IL-1 family and binds to the ST2 receptor. IL-33 inhibits T H 1 immune cells, natural killer (NK) cells, iNKT cells, and CD8 T lymphocytes.

[0191] The term "PD-1" has its common meaning in the art and refers to the programmed death 1 receptor. The term "PD-1" also refers to a type I transmembrane protein that belongs to the CD28-B7 signaling family of receptors, which includes CD28, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), ICOS (inducible costimulator), and BTLA (B- and T-lymphocyte attenuator) (Greenwald RJ et al., 2005; Riley JL et al., 2005).

[0192] The term "anti-PD-1 antibody" or "anti-PD-L1" has its general meaning in the art and refers to an antibody with binding affinity for PD-1 or PD-L1, respectively, and antagonist activity for PD-1, i.e., inhibiting the signaling cascade associated with PD-1 and inhibiting the binding of PD-1 to its ligands (PD-L1; PD-L2). Such anti-PD-1 or anti-PD-L1 antibodies preferentially inactivate PD-1 with greater affinity and potency than its interaction with other subtypes or isoforms of the CD28-B7 signaling family of receptors (CD28; CTLA-4; ICOS; BTLA), respectively. Tests and assays for determining whether a compound is a PD-1 antagonist are well known to those of skill in the art, such as those described in Greenwald et al., 2005; Riley et al., 2005.

[0193] Examples of such anti-PD1 antibodies include, but are not limited to, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

[0194] In accordance with the foregoing, in a still further aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: The method as defined above, comprising co-administration, e.g., contemporaneous or sequential, of a therapeutically effective amount of an anti-BTN2A1 antibody of the present disclosure and at least one second drug substance, said second drug substance being, e.g., an antiviral or antiproliferative or immunotherapeutic agent, or a cytokine or cell therapy product (such as γδ T cells), as indicated above. Present the following.

[0195] In one embodiment, the antibodies of the present disclosure can be used to detect levels of soluble BTN2A1 or levels of cells expressing BTN2A1. This can be accomplished, for example, by contacting a sample (such as an in vitro sample) and a control sample with an anti-BTN2A1 antibody under conditions that allow the formation of a complex between the antibody and BTN2A1 (BTN2A1 expressed on the surface of a cell, or soluble BTN2A1 in, for example, a blood sample). Any complexes formed between the antibody and BTN2A1 in the sample and the control are detected and compared. Standard detection methods well known in the art can be performed using the compositions of the present disclosure, such as, for example, ELISA assays and flow cytometry assays.

[0196] Thus, in one aspect, the disclosure further provides a method for detecting the presence or amount of BTN2A1 (e.g., human BTN2A1 antigen) in a sample, comprising contacting the sample and a control sample with an antibody or protein of the disclosure, or an antigen-binding region thereof, that specifically binds BTN2A1, under conditions that allow the formation of a complex between the antibody or portion thereof and BTN2A1. The formation of the complex is then detected, where a difference in complex formation in the sample compared to the control sample indicates the presence of BTN2A1 in the sample.

[0197] Also within the scope of this disclosure are kits consisting of the compositions disclosed herein (e.g., humanized antibodies, conjugated antibodies, and multispecific molecules) and instructions for use. The kits may further contain at least one additional reagent, or one or more additional antibodies or proteins. The kits typically include labeling indicating the intended use of the contents of the kit. The term labeling includes any written or recorded material supplied in or with the kit, or which otherwise accompanies the kit. The kits may further include a tool for diagnosing whether a patient belongs to the group that will respond to anti-BTN2A1 antibody treatment, as defined above.

[0198] Another therapeutic strategy is based on the use of the humanized antibodies disclosed herein as drugs that selectively activate NK cells isolated from a sample from a human subject.

[0199] Accordingly, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) isolating blood cells, e.g., PBMCs, comprising NK cells from a blood sample of a subject; (b) expanding the NK cells, and optionally other tumor cells or accessory cells, in vitro in the presence of anti-BTN2A1 as disclosed herein; (c) harvesting the expanded NK cells; (d) optionally formulating the expanded NK cells and administering a therapeutically effective amount of the NK cells to the subject; The present invention relates to a method comprising the steps of:

[0200] The present disclosure further relates to the use of the humanized antibodies disclosed herein as agents to selectively activate chimeric antigen receptor (CAR) NK cells. CAR NK cells and their use in adoptive NK cell cancer immunotherapy are described, for example, in Rezvani, K et al., "Adoptive cell therapy using engineered natural killer cells" (Bone Marrow Transplant, 2019).

[0201] The present disclosure also relates to anti-BTN2A1 antibodies disclosed herein for use as potentiating agents in NK cell therapy in vivo, typically in a subject in need thereof suffering from cancer.

[0202] As used herein, the term NK cell therapy refers to a treatment that includes administration of at least an effective amount of NK cells to a subject in need thereof. Such NK cells may be allogeneic or autologous. In particular embodiments, the NK cells may be genetically engineered by deletion or knockout, or by insertion or knockin of a specific gene. In particular embodiments, the NK cells include NK cells expressing a chimeric antigen receptor. The NK cells may be ex vivo expanded and / or purified. Alternatively, the NK cells may also be incorporated into a cell composition that includes other blood cells, e.g., other cells of the immune system. For references regarding γδ T cell therapy, see Rezvani, K, Bone Marrow Transplant, 2019.

[0203] Accordingly, the present disclosure provides a method of treating a subject suffering from cancer, e.g., a hematological malignancy, particularly a leukemia, such as acute myeloid leukemia, and having tumor cells, e.g., hematological tumor cells, comprising: (i) administering in the subject an effective amount of an anti-BTN2A1 antibody disclosed herein; (ii) administering in said subject an effective amount of an NK cell composition; wherein the effective amount of an anti-BTN2A1 antibody is capable of enhancing anti-tumor cytolysis mediated by the NK cell composition against the tumor cells. The present disclosure also relates to a method for treating a subject in need thereof comprising the combined (concurrent or sequential) administration of NK cells, e.g., CAR NK cells, and a humanized antibody disclosed herein.

[0204] In alternative or further embodiments, therapeutic strategies may also be based on the use of the humanized antibodies disclosed herein as drugs to selectively expand and / or activate Vy9 / V52 T cells isolated from a sample of a human subject.

[0205] Accordingly, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) isolating blood cells, e.g., PBMCs, comprising Vy9 / V52 T cells from a blood sample of a subject; (b) expanding Vy9 / V52 T cells, and optionally other tumor cells or accessory cells, in vitro in the presence of anti-BTN2A1 as disclosed herein; (c) harvesting the expanded Vy9 / V52 T cells; (d) optionally formulating the expanded Vy9 / V52 T cells and administering a therapeutically effective amount of said Vy9 / V52 T cells to the subject; The present invention relates to a method comprising the steps of:

[0206] The present disclosure further relates to the use of the humanized antibodies disclosed herein as agents to selectively expand chimeric antigen receptor (CAR) Vy9V52 T cells. CAR γδ T cells and their use in adoptive T cell cancer immunotherapy are described, for example, in Mirzaei et al., Cancer Lett, 2016.

[0207] The antibodies of the present disclosure may also be used to prepare artificial T cell receptors, also known as chimeric T cell receptors, or chimeric antigen receptors (CARs). For example, the variable regions of an antibody may be linked via a spacer to the transmembrane and signaling intracellular domains of a TCR and used to form Fabs or scFvs that are produced on the surface of T cells. Such CARs may be used in adoptive transfer therapy, for example, to treat proliferative disorders.

[0208] The present disclosure also relates to anti-BTN2A1 antibodies for use as potentiating agents in γδ T cell therapy in vivo, typically in a subject in need thereof suffering from cancer.

[0209] As used herein, the term γδ T cell therapy refers to a treatment comprising administration of at least an effective amount of γδ T cells to a subject in need thereof. Such γδ T cells may be allogeneic or autologous. In particular embodiments, the γδ T cells may be genetically engineered by deletion or knock-out, or by insertion or knock-in of a specific gene. In particular embodiments, the γδ T cells comprise γδ T cells expressing a chimeric antigen receptor. The γδ T cells may be ex vivo expanded and / or purified. Alternatively, the γδ T cells may also be incorporated into a cell composition comprising other blood cells, e.g., other cells of the immune system. For references regarding γδ T cell therapy, see Pauza CD. et al., Front Immunol., 2018; J Saudemont A. et al., Frontiers Immunol, 2018.

[0210] Indeed, without being bound by any particular theory, the proposed mode of action for the anti-BTN2A1 antibodies of the present disclosure is that their binding to BTN2A1 expressed on the surface of tumor cells induces a conformational change that enables their signaling to their counter-receptor in Vy9V52 T cells.

[0211] Accordingly, the present disclosure provides a method of treating a subject suffering from cancer, e.g., a hematological malignancy, particularly a leukemia, such as acute myeloid leukemia, and having tumor cells, e.g., hematological tumor cells, comprising: (i) administering in the subject an effective amount of an anti-BTN2A1 antibody disclosed herein; (ii) administering in the subject an effective amount of a γδ T cell composition; wherein said effective amount of an anti-BTN2A1 antibody is capable of enhancing anti-tumor cytolysis mediated by said γδ T cell composition against said tumor cells. The present disclosure also relates to a method for treating a subject in need thereof comprising the combined (concurrent or sequential) administration of a CAR T cell, e.g., a CAR γδ T cell, and a humanized antibody disclosed herein.

[0212] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present disclosure in any way. [Brief description of the drawings]

[0213] [Figure 1] Figure 1. Anti-BTN2A1 107G3 mAb identification. A. Anti-BTN2A1 mAb screening cascade from mouse immunization to mAb sequencing. B. Bar graph shows the number of clones per affinity (KD) range measured in Luminex during primary hit selection. C. Stacked bar graph shows the number of clones classified as neutral (grey), antagonist (white), or agonist (black) according to their ability to modulate IFN-γ production by Vy9 / V52 T cells during primary hit screening (1st round) and secondary hit screening (2nd round). [Diagram 2]Figure 2: Anti-BTN2A1 107G3 mAb enhances the cytolytic function of Vy9 / V52 T cells. Vy9 / V52 T cells were expanded from PBMCs (see Materials and Methods) of three healthy donors and co-cultured at 37°C with target cells using an effector:target (E:T) ratio of 1:1 in the presence of anti-CD107a / b antibodies and Golgi stop with or without the indicated antibodies. After 4 hours, cells were harvested, fixed and analyzed in flow cytometry. In A, different target cell lines were used, including Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), L-IPC (pancreatic adenocarcinoma), and MDA-MB-134 (breast adenocarcinoma), with or without anti-BTN2A1 107G3 supernatant or control hybridoma culture medium. Bar graphs show the percentage of CD107+ cells depicting Vy9 / V52 T cell degranulation. In B, Daudi cells were used as target cells in the presence of the indicated concentrations of purified anti-BTN2A1 107G3 mAb or irrelevant mouse IgG1 as isotype control. Graphs show dose-response curves allowing calculation of EC50. [Diagram 3] Anti-BTN2A1 107G3 mAb recognizes BTN2A1 but not BTN3. HEK-293T BTN2 KO cells were transiently transfected with a plasmid encoding a BTN2A1-CFP fusion protein. A. Histograms show overlays for the indicated cells and transfected cells stained with purified anti-BTN2A1 107G3 mAb (top, black line), purified anti-BTN3 103.2 mAb (bottom, black line), or mIgG1 or IgG2a controls (dashed lines). For transfected cells, staining is shown after gating on CFP+ cells. B. Graph shows dose-response curves for binding of purified anti-BTN2A1 107G3 mAb in HEK-293T BTN2 KO cells transfected with a plasmid encoding BTN2A1-CFP. All staining was analyzed after gating on CFP+ cells. [Figure 4A]Figure 1 shows the effect of reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb on BTN2A expression in NK cells and monocytes, and monocyte polarization to M2 macrophages: (A) Representative histograms of BTN2A1 and BTN2A2 expression (white) versus control isotype (grey) in NK cells and monocytes derived from unstimulated HD-PBMCs, as assessed by flow cytometry. [Figure 4B] (B) Representative CD14 / CD163 dot plot profiles of M1 / M2 macrophages or 101G5 and 107G3 mAb-induced macrophages in the presence of M-CSF in vitro. After 5 days of differentiation, CD14 and CD163 dot plots were generated by flow cytometry analysis. [Figure 5-1] FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb inhibit M2 macrophage polarization in a dose-dependent manner. M1, M2, GM-CSF and IFNγ-reverted M2, and M-CSF-induced macrophages were polarized for 5 days in the presence of different concentrations of 101G5 mAb or 107G3 mAb (or their isotype controls) and stimulated or left unstimulated with LPS for an additional 2 days. Expression of CD14 (A) and CD163 (B) was analyzed in unstimulated cells by flow cytometry. Results are expressed as median fluorescence intensity (MFI) minus their corresponding isotype controls. [Figure 5-2] Expression of PDL1 (C) and CD86 (D) was analyzed by flow cytometry in unstimulated (C) or LPS-stimulated (D) cells. Results are expressed as median fluorescence intensity (MFI) subtracted from their corresponding isotype controls. [Figure 5-3] IL-10 (E) and TNFα (F) were quantified in LPS-stimulated macrophage supernatants by ELISA. Results are expressed in pg / mL. [Figure 6-1]FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb inhibit "M2+IL-4" induced polarization from monocytes. M1, M2, "M2+IL4", reverted M2 with GM-CSF and IFNγ, and M-CSF+IL-4 induced macrophages were generated in the presence of 10 μg / mL of 101G5 mAb or 107G3 mAb (or their isotype controls) for 5 days and stimulated or left unstimulated with LPS for an additional 2 days. Expression of CD14 (A), CD163 (B), PDL1 (C), DC-SIGN (D) was analyzed in unstimulated cells (A-D) by flow cytometry. Results are expressed as median fluorescence intensity (MFI) minus their corresponding isotype controls. [Figure 6-2] FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb inhibit "M2+IL-4" induced polarization from monocytes. M1, M2, "M2+IL4", reverted M2 with GM-CSF and IFNγ, and M-CSF+IL-4 induced macrophages were generated in the presence of 10 μg / mL of 101G5 mAb or 107G3 mAb (or their isotype controls) for 5 days and stimulated or left unstimulated with LPS for an additional 2 days. Expression of CD86 (E) was analyzed in LPS stimulated cells (E) by flow cytometry. Results are expressed as median fluorescence intensity (MFI) subtracted from their corresponding isotype controls. IL-10 (F) and TNFα (G) were quantified by ELISA in LPS stimulated macrophage supernatants. Results are expressed in pg / mL. [Figure 7]FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb inhibit cancer cell-induced M2 polarization. M1, M2, PANC-1 conditioned medium-induced M2, reverted M2 with GM-CSF and IFNγ, and PANC-1 conditioned medium-induced macrophages were developed in the presence of 10 μg / mL 101G5 mAb or 107G3 mAb (or their isotype controls) for 5 days and stimulated or left unstimulated with LPS for an additional 2 days. Expression of CD14 (A), CD163 (B) was analyzed in unstimulated cells by flow cytometry. Results are expressed as median fluorescence intensity (MFI) subtracted from their corresponding isotype controls. IL-10 (C) and TNFα (D) were quantified by ELISA in LPS-stimulated macrophage supernatants. Results are expressed in pg / mL. [Figure 8-1] FIG. 1: Reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb revert M2 macrophages to pro-inflammatory M1 macrophages: phenotype and cytokine secretion. M1, M2 were generated from monocytes for 5 days. After 5 days, 10 μg / mL of 101G5 mAb or 107G3 mAb (or isotype control) or IFNγ was added to M2 macrophages for 2 days and stimulated or left unstimulated with LPS for another 2 days. Expression of CD14 (A), CD163 (B), PDL1 (C) was analyzed in unstimulated cells (A-C) by flow cytometry. Results are expressed as median fluorescence intensity (MFI) minus their corresponding isotype control. [Figure 8-2]FIG. 13. Reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb revert M2 macrophages to pro-inflammatory M1 macrophages: phenotype and cytokine secretion. M1, M2 were generated from monocytes for 5 days. After 5 days, 10 μg / mL of 101G5 mAb or 107G3 mAb (or isotype control), or IFNγ was added to M2 macrophages for 2 days and stimulated or left unstimulated with LPS for another 2 days. Expression of CD86 (D) was analyzed in LPS-stimulated cells (D) by flow cytometry. Results are expressed as median fluorescence intensity (MFI) subtracted from their corresponding isotype controls. IL-10 (E) and TNFα (F) were quantified by ELISA in LPS-stimulated macrophage supernatants. Results are expressed in pg / mL. [Figure 8-3] FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb revert M2 macrophages to pro-inflammatory M1 macrophages: phenotype and cytokine secretion. [Figure 9-1] FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb release M2-mediated inhibition of T cell proliferation and IFNγ secretion. Differentiated M1, M2, or macrophages induced in the presence of 101G5 and 107G3 mAbs (or their isotype controls) were co-cultured with allogeneic OKT3-activated CTV-labeled CD3+ T cells for 5 days. After co-culture, cells were stimulated with PMA / ionomycin and GolgiStop protein inhibitors for 5 hours, and the number of CD3+ T cells (A and B) and intracellular IFNγ production (C and D) were quantified by flow cytometry. Proliferation was quantified by the dilution rate of CTV dye (CTV signal at day 0 as baseline). Results are absolute numbers of CD3+ T cells (B) or percentages of CD3+ T cells (C and D) calibrated in CountBright absolute counting beads. [Figure 9-2]FIG. 1 shows that reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb relieve M2-mediated inhibition of T cell proliferation and IFNγ secretion. Differentiated M1, M2, or macrophages induced in the presence of 101G5 and 107G3 mAbs (or their isotype controls) were co-cultured with allogeneic OKT3-activated CTV-labeled CD3+ T cells for 5 days. After co-culture, cells were stimulated with PMA / ionomycin and GolgiStop protein inhibitors for 5 hours and intracellular IFNγ production (E and F) and proliferation (CellTrace Violet, CTV dim) (G-J) were quantified by flow cytometry. Proliferation was quantified by dilution of CTV dye (CTV signal at day 0 as baseline). Results are absolute numbers of CD3+ T cells (E, F, I, and J) or percentages of CD3+ T cells (G and H) calibrated in CountBright absolute counting beads. [Figure 10-1] Figure 1 shows the effect of reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb on purified NK cell activation and cytotoxicity. (A-B) Purified NK cells were cultured with reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb (or control isotype) with stimulation with IL-2 or IL-2 / IL-15 for 5 days. NK cell activation was assessed by evaluating the expression (MFI) of CD69 (A) and CD25 (B) in unstimulated and IL-2 / IL-15 stimulated NK cells in the presence of the indicated mAbs or control isotype. [Figure 10-2]Figure 1 shows the effect of reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb on the activation and cytotoxicity of purified NK cells. (C-D) Purified NK cells were preincubated overnight with reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb (or control isotype) in the presence or absence of IL-2 / IL-15 stimulation and then co-cultured with human tumor cell lines for 4 hours. NK cell degranulation was assessed by flow cytometry as the percentage of CD107αβ in unstimulated NK cells (C) and in IL-2 / IL-15 stimulated NK cells (D) for each tumor cell line in the presence of the indicated mAb or control isotype. [Figure 10-3] Figure 1 shows the effect of reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb on purified NK cell activation and cytotoxicity (E) Degranulation of NK cells against A549 cell line when reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb (or control isotype) were added to NK cells or target cells and pre-incubated 4 hours prior to co-culture compared to mAbs added to co-culture without pre-incubation. [Figure 11]Reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb enhance NK cell degranulation and killing against adenocarcinoma cell lines. (A) Purified NK cells were pre-incubated overnight with reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb (or control isotype) in the presence or absence of IL-2 / IL-15 stimulation, and then co-cultured with DU-145 cell line for 4 hours. NK cell degranulation was assessed by flow cytometry as the percentage of CD107αβ+ cells. EC50 for enhancement of NK cell degranulation was calculated for the indicated mAbs using a 4-parameter dose-response curve in Prism software. (B) Purified NK cells were preincubated overnight with reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb (or control isotype or IL-2 / IL-15 stimulation) and then co-cultured with HL-60 and A549 cell lines for 4 hours. NK cell-mediated cancer cell death was assessed by evaluating the percentage of caspase 3 / 7+ cells in the presence of the indicated mAbs or control isotypes. [Figure 12-1] Figure 2 shows binning experiments for anti-BTN2A1 101G5 mAb, a reference mAb against BTN2A1, and anti-BTN2A1 107G3 mAb. The binning experiments were performed on the Octet Red96 platform, a system based on Biolayer Interference (BLI) technology. 107G3 and 101G5 were investigated in a paired combinatorial manner against rhBTN2A1-His protein. A. 107G3 is a saturating mAb and 101G5 is a competitive mAb. [Figure 12-2]Figure 2 shows a binning experiment for anti-BTN2A1 101G5 mAb, a reference mAb against BTN2A1, and anti-BTN2A1 107G3 mAb. The binning experiment was performed on the Octet Red96 platform, a system based on Biolayer Interference (BLI) technology. 107G3 and 101G5 were examined in a paired combinatorial manner against rhBTN2A1-His protein. B: 101G5 is a saturating mAb and 107G3 is a competitive mAb. C: Arbitrary unit measurement of binding and self-blocking pairs of mAbs. [Figure 13-1] Figure 1 shows BTN2A1 peptides by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin. 96.37% of the sequence is covered by the identified peptides. [Figure 13-2] Figure 1 shows BTN2A1 peptides by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin. 96.37% of the sequence is covered by the identified peptides. [Figure 14] Figure 1 shows the interaction of reference mAbs 107G3 and 101G5 with human BTN2A1. A. 107G3 / BTN2A1. B. 101G5 / BTN2A1. [Figure 15] Figure 1. BTN2A1 / 107G3 interaction. The PDB structure of BTN2A1, 4F9P, is colored in gray at the epitope site. The amino acids of BTN2A1 colored in blue correspond to 65-78 (RWFRSQFSPAVFVY) and 84-100 (RTEEQMEEYRGRTTFVS) of the proposed BTN2A1 sequence. A, B, C, D, E: Ribbon / surface representations of front view (A); back view (B), side view 1 (C), side view 2 (D), and top view (E). F, G, H, I, J: Ribbon representations of front view (F); back view (G), side view 1 (H), side view 2 (I), and top view (J). [Figure 16]Figure 1. BTN2A1 / 101G5 interaction. The PDB structure of BTN2A1, 4F9P, is colored in gray at the epitope site. The amino acids of BTN2A1 colored in blue correspond to 212-229 (KSVRNMSCSINNTLLGQK) of the proposed BTN2A1 sequence. A, B, C, D, E: Ribbon / surface representations of front view (A); back view (B), side view 1 (C), side view 2 (D), and top view (E). F, G, H, I, J: Ribbon representations of front view (F); back view (G), side view 1 (H), side view 2 (I), and top view (J). [Figure 17] Figure 1 shows the evaluation of cross-reactivity of anti-BTN2A1 101G5 mAb and anti-BTN2A1 107G3 mAb, reference mAbs against the cynomolgus BTN2A1 orthologue. ELISA-based measurements of 107G3 and 101G5 binding to recombinant human BTN2A1-Fc fusion protein or recombinant cynomolgus BTN2A1-Fc fusion protein coated on ELISA plates. The graph depicts dose-response curves allowing calculation of EC50 by nonlinear regression using a variable slope model. EXAMPLES

[0214] Materials and Methods Cell culture, monocyte and NK cell sorting: Peripheral blood mononuclear cells (PBMCs) were obtained from EDTA (ethylenediaminetetraacetic acid) buffy coats from healthy donors (HDs) provided by a local blood bank (Etablissement Francais du Sang (EFS), Marseille, France) and isolated by centrifugation in density gradients (Eurobio). Freshly collected PBMCs were cultured at 37°C in 5% CO2 in Roswell Park Memorial Institute medium 1640 (RPMI; Lonza) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S).

[0215] Natural killer (NK) cells were isolated from freshly harvested PBMCs by negative selection using the EasySep™ Human NK Cell Enrichment kit (StemCell Technologies) according to the manufacturer's instructions. Human CD14+ monocytes were isolated by using a CD14+ microbead kit (Miltenyi) according to the manufacturer's instructions. 10 cells per mL were cultured in RPMI supplemented with 1% L-glutamine, 100 U / mL penicillin / streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mM non-essential amino acids, and 10% FBS (all from Thermofisher). 6 Monocytes were cultured at a density of 10x for 5 days at 37°C. Pancreatic adenocarcinoma cell line, PANC-1, was cultured in RPMI supplemented with 10% FBS. Upon growth to 90% confluency, the culture medium was discarded and cells were rinsed with 1x PBS. PANC-1 cells were then cultured for an additional 24 hours in RPMI supplemented with 5% FBS (175cm to concentrate the supernatant). 2 PANC-1 conditioned medium was then collected, filtered (0.2 μM) and stored at −20° C. until use. The other human cell lines and their corresponding culture media are summarized in Table 1 below.

[0216] [Table 1]

[0217] The following human cell lines were obtained from the American Type Culture Collection: Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), MDA-MB-134 (ductal carcinoma), and HEK-293T (embryonal kidney). The human pancreatic adenocarcinoma cell line L-IPC (PDAC087T) was a kind gift from Dr. Juan IOVANNA. Daudi and Jurkat cells as well as PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% sodium pyruvate, and 1% L-glutamine (all from Life Technologies). HEK-293T BTN2 KO cells were generated by CRISPR-Cas9-mediated inactivation of all isoforms of BTN2 (data not shown). MDA-MB-134, L-IPC, HEK-293 cells, and HEK-293T BTN2 KO cells were cultured in DMEM medium (Life Technologies) with 10% FCS. Hybridomas were cultured in DMEM / Ham's F12 (1:1) (ThermoFisher Scientific), 4% FetalClone I (Hyclone), Chemically Defined Lipid Concentrate (1:250), 1% glutamine, 1% sodium pyruvate, and 100 μg / mL PenStrep (all from ThermoFisher Scientific). To collect hybridoma supernatants, hybridomas were cultured for 4-5 days without Fetalclone.

[0218] To assess the specificity of the anti-BTN2A1 mAb, HEK-293T BTN2 KO cells were independently transfected with pcDNA3-Zeo-BTN2A1-CFP plasmids encoding BTN2A1 and BTN2A2 CFP(Nter) fusion proteins using Lipofectamine 3000 reagent (Thermofisher Scientific) according to the manufacturer's instructions.

[0219] Identification of the reference anti-BTN2A1 mAb 107G3 Forty-eight mice bearing six different MHC combinations were immunized with recombinant human BTN2A1-Fc fusion protein to generate mouse anti-human BTN2A1 antibodies. After 21 days, the mice were bled and serum titers of BTN2A1-specific polyclonal antibodies were determined via Luminex assay. Mice exhibiting the highest BTN2A1-specific antibody titers were euthanized. Splenic B cells were isolated via positive selection and subjected to PEG-induced fusion with myeloma cells to generate hybridomas.

[0220] Hybridomas were cloned by limiting dilution and hybridoma supernatants were subjected to two rounds of screening for target specificity and their ability to induce degranulation of Vy9V52- T cells (Figure 1C and Figure 2), leading to the identification of the reference mAb, 107G3. The VH and VL regions of these subclones were sequenced (see Table 1).

[0221] Expansion of Vγ9Vδ2 T cells Effector Vy9 / V52-T cells were established by culturing PBMCs derived from HV in the presence of zoledronate (Sigma, 1 μM) and recombinant human (rh) IL-2 (Proleukin, 200 IU / mL) beginning on day 0. From day 5, rhIL-2 was supplemented every other day, maintaining a cell density of 1.15 × 10 per mL for a total of 15 days. 6 On the final day, the purity of Vy9 / V52 T cells was assessed by flow cytometry. Only cell cultures that reached a Vy9 / V52 T cell purity of greater than 80% were selected for use in functional studies. Purified Vy9 / V52 T cells were frozen until use.

[0222] Luminex Assay COOH magnetic beads (Biorad) were conjugated to rhBTN2A1 protein (R&D) according to the manufacturer's instructions, and the beads were stored in storage buffer (Biorad) at -20°C until use. For titration of mouse sera, a serum dilution series was made in Luminex assay buffer (Nanotool) starting from 1:50 with a dilution step of 1:4, 100 μL of bead suspension was mixed with 100 μL of serum dilution and incubated for 1 h at RT, after which the beads were washed three times in wash buffer, incubated with 1 μg / mL biotinylated goat anti-mouse IgG-Fc in Luminex assay buffer, and washed three more times in Luminex assay buffer. Finally, the beads were incubated with 1 μg / mL streptavidin PE in Luminex assay buffer for 1 h, followed by three final washes in Luminex read buffer (Nanotool). The beads were resuspended in Luminex read buffer and data was collected on a Luminex 100 / 200 system. For hit identification, 30 μL of the supernatant was transferred to a 96-well plate and 90 μL of Luminex assay buffer was added. 100 microliters of the bead suspension was mixed with 100 μL of the supernatant dilution and incubated at RT for 16 hours before proceeding with the protocol described above. For hit identification, the bead suspension with the highest affinity to the target and the lowest affinity to an unrelated control protein (Rank-Fc) was selected. For affinity / Kd calculation, serial dilutions starting at 40.000 pM with a dilution step of 1:4 in Luminex assay buffer were applied to the hybridoma supernatant and analyzed as described above. Kd corresponds to the midpoint of the corresponding binding curve.

[0223] Flow cytometry PBMCs, purified Vy9V52-T cells (T cells or cell lines) were incubated with the indicated mAbs prior to analysis on BD LSRFortessa (BD Biosciences), CytoFlex LX, or CytoFlex S (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo). Antibodies used for Vy9V52-T cell degranulation assays were anti-CD107a-FITC (BD Biosciences), anti-CD107b-FITC (BD Biosciences), anti-CD3-PeVio700 (Miltenyi), anti-PanTγδ-PE (Miltenyi), live / dead near IR (Thermofisher). All immunostaining was performed using purified mAbs at 10 μg / mL in the presence of FcR Block reagent (Miltenyi), goat anti-mouse-PE 1:100 (Jackson Immunoresearch), and live / dead near IR (Thermofisher). Mouse anti-human CD277 (also known as BTN3A; clone 103.2 with IgG2a isotype) was previously described (WO 2012 / 080351). To evaluate the specificity of anti-BTN2A1 mAb, HEK-293T BTN2 KO cells (5 × 10 per sample) were transfected with mAb 24 h after transfection. 4 ) were harvested and stained with anti-human BTN2A1 107G3 mAb at the indicated concentrations (5 ng / mL to 75 μg / mL) as described above. Mouse IgG1 antibody (Miltenyi) was used as an isotype control for staining.

[0224] Functional assays for Vγ9 / Vδ2-T cells Vy9 / V52-T cells purified from HV were cultured overnight in rhIL-2 (200UI / mL). Vy9 / V52-T cells were then co-cultured at 37°C with the indicated target cell lines (at an effector:target (E:T) ratio of 1:1) with or without the following mAbs (50 μL of hybridoma supernatant or 10 μg / mL of purified mAbs as indicated): anti-BTN2A1 mAb, mIgG1 (isotype control antibody) or hybridoma culture medium. Phorbol 12-myristate 13-acetate (PMA, 20 ng / mL) with ionomycin (1 μg / mL) was used as a positive control for activation of Vy9 / V52-T cells. For the first round of hybridoma supernatant screening, culture supernatants were harvested after 4 hours and examined for their IFNγ content as an indicator of Vγ9 / Vδ2-T cell activation using the Human IFNγ ELISA set (BD Biosciences). For the characterization of the second round of hybridoma supernatants, Vγ9 / Vδ2-T cell degranulation was assessed by incubation for 4 hours in the presence of GolgiStop (BD Biosciences) and soluble CD107(a&b)-FITC. After 4 hours, cells were harvested, fixed in 2% paraformaldehyde in PBS, and analyzed in CytoFlex LX (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo).

[0225] Expansion of Vγ9 / Vδ2 T cells Vy9 / V52-T cells were isolated from PBMCs of healthy donors using an anti-TCR γδ microbead kit (Miltenyi Biotec). The purity of γδ-T cells was greater than 80%, as assessed by flow cytometry. γδ-T cells were labeled with CellTrace Violet for 20 min at 37°C. Then, 5 × 10 CellTrace-labeled cells were added to the PBMCs of healthy donors. 5Cells were cultured in 96-well round-bottom plates in the presence of IL-2 (200UI / ml) with or without pAg and with or without purified anti-BTN2A1 107G3 antibody (10 μg / ml). After 5 days of culture, CellTrace dilutions were assessed by flow cytometry on a CytoFlex LX (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo).

[0226] Statistical analysis: For Vγ9 / Vδ2-T cell degranulation, results are expressed as mean ± SEM. EC of purified anti-BTN2A1 mAb on BTN2A1-transfected HEK-293T BTN2 KO cells. 50 was determined based on log(dose)-response curves after nonlinear regression according to a variable slope model. All analyses were performed using GraphPad Prism 7.04 software (GraphPad).

[0227] Identification of reference anti-BTN2A mAb 101G5 After VH and VL sequencing, 23 anti-BTN2A mAbs were generated in chimeric IgG1 format, derived from the generation of mouse hybridomas described above. Briefly, VH and VK sequences of mouse anti-BTN2A mAbs were synthesized in vitro and amplified by PCR using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). PCR products were cloned into heavy and light chain expression vectors (MI-mAb) using the In Fusion system (Clontech), and the plasmids were transformed into Stellar competent cells (Clontech). To validate the anti-BTN2A mAbs, vector sequencing (MWG Eurofins) was performed, followed by large-scale (maxi) preparation of the plasmids for further transfection. For each anti-BTN2A clone, vectors encoding matched light and heavy chains were transiently transfected into HEK-293 cells (2.9 × 10 cells per mL) at a heavy / light chain ratio of 1:1.2. 6100 ml) and 18 hours later the medium was changed. Seven days after transfection the culture supernatant was harvested for purification of the mAb. Affinity purification of the antibody was performed overnight at 44°C with Protein A Sepharose Fast Flow (GE Healthcare). The binding buffer was 0.5 M glycine, 3 M NaCl, pH 8.9. Elution was performed with the following buffer: 0.1 M citric acid pH 3. Immediately after elution with 1 M Tris-HCl, pH 9 (10% v / v), the sample was neutralized. Finally, the chimeric anti-BTN2A mAb was dialyzed into 1x PBS and filtered through a 0.22 μM filter (Millex GV hydrophilic PVDF, Millipore). The chimeric anti-BTN2A mAb concentration was determined in a Nanodrop 2000 Spectrophotometer (ThermoScientific) taking into account the extinction coefficient of the antibody. Purity, as defined by the mAb monomer fraction, was determined by UPLC-SEC using an Acquity UPLC-HClass Bio (Waters) with an Acquity UPLC Protein-BEH-200A, 1.7 μm, 4.6×50 mm column (Waters). Antibody masses were determined on a Xevo G2-S Q-Tof mass spectrometer (Waters) using a reversed-phase column (PLRP-S 4000 A, 5 μm, 50 × 2.1 mm (Agilent technologies). All samples were analyzed after deglycosylation with PNGase F glycosidase (New England Biolabs) at 37 °C. If unexpected masses were found, the primary amino acid sequence was analyzed using bioinformatics tools to identify putative glycosylation sites within the Fab region. SDS-PAGE of purified antibodies allowed the detection of fragmentation and / or aggregation of the final material with an unstained Mini protean TGX gel 4-15 (Biorad).Endotoxin levels were determined using the Chromogenic LAL Limulus Amebocyte lysate kinetic assay (Charles River Endosafe) using a ClarioStar spectrophotometer (BMG Labtech).

[0228] In vitro macrophage polarization assay: M1 or M2 macrophages were polarized from monocytes sorted from healthy donors. For this purpose, sorted monocytes were cultured in the presence of GM-CSF or M-CSF (40 ng / mL; Miltenyi) to generate M1 or M2 macrophages, respectively. After 5 days, the resulting macrophages were either harvested for phenotypic analysis or stimulated with LPS (200 ng / mL) for another 2 days. In some experiments, IL-4 (20 ng / mL) was added to M2 macrophages on day 4, which resulted in the generation of "M2+IL-4" macrophages. In some experiments, M2 macrophages were generated by culturing monocytes in the presence of PANC-1 cancer cell-conditioned medium (diluted 30% v / v in culture medium on days 0 and 3) without supplementation of M-CSF. In this application, the resulting M2 macrophages are referred to as "Tum-ind-M2". To screen anti-BTN2A mAbs for their ability to modulate M2 differentiation, M2 macrophages were generated from monocytes with or without chimeric anti-BTN2A mAbs or their isotype control (human IgG1; Sigma) at the indicated concentrations as described above. All mAbs were wet coated (1x in PBS, overnight at RT). As a control for inhibition of M2 differentiation, GM-CSF (40 ng / mL) and IFNγ (100 ng / mL, BioTechne) were added to monocytes during M-CSF-induced M2 polarization. M1 macrophages polarized in the presence of GM-CSF were used as a phenotypic control. After polarization, the resulting macrophages and their culture supernatants were harvested and the expression of M1- and M2-associated markers on the cell membrane was assessed by flow cytometry.In addition, the cytokine content in the culture supernatants was quantified using an IL-10 and TNFα ELISA kit (ThermoFisher Scientific) according to the manufacturer's instructions.

[0229] In vitro M2 macrophage reversion assay: M2 macrophages were generated from monocytes in the presence of M-CSF in the absence of reference mAb as described above. M2 macrophages were harvested and cultured for 2 days in pre-wet-coated culture wells overnight with 10 μg / mL of reference antibodies or their control isotype mAbs (human IgG1 from Sigma) with or without LPS. As a control for M2 reversion, GM-CSF (40 ng / mL) and IFNγ (100 ng / mL) were added to M2 macrophage cultures for 2 days. M1 macrophages polarized in the presence of GM-CSF were used as phenotypic controls. After reversion experiments, macrophages reverted without the addition of LPS were harvested for phenotypic analysis by flow cytometry. Culture supernatants from LPS-stimulated reverted macrophages were harvested for cytokine quantification using ELISA.

[0230] In vitro assay for M2 macrophage-mediated inhibition of T cell proliferation and IFNγ production M1 and M2 macrophages were generated as described above with or without the addition of reference antibodies or their isotype control mAbs. CD3+ T cells were sorted from healthy donor PBMCs by using a CD3+ MicroBead Kit (Miltenyi) according to the manufacturer's instructions and frozen until co-culture. Activated CD3+ T cells were generated as follows: CD3+ T cells were stained with 5 μM CellTrace Violet dye (ThermoFisher Scientific) and then 10 such cells were cultured at 4 °C for 1 h. 5 10 cells were incubated with 20 U / mL IL-2 (Miltenyi), LPS (200 ng / mL), and CountBright absolute counting beads (5 × 10 cells per well) in a 96-well flat-bottom plate precoated with 1 μg / mL anti-CD3 mAb (clone OKT3, BD biosciences). 3For coculture with macrophages, 2 × 10 allogeneic M1 macrophages, M2 macrophages, or macrophages polarized in the presence of M-CSF and reference mAbs or their control isotypes were cultured in X-Vivo 10 medium (ThermoFisher Scientific, Thermo ... 4 1000 ng / mL of PMA and 0.5 μg / mL of ionomycin were added to the co-cultures for 5 hours in the presence of GolgiStop protein transport inhibitor to enhance cytokine production after 5 days of co-culture. Cells were then harvested for phenotypic analysis by flow cytometry. Dilution of CellTrace was used as an index of CD3+ T cell proliferation. Phenotypic and proliferation results were expressed as percentage or absolute cell number per mL (after calibration with absolute counting beads).

[0231] Natural Killer (NK) challenge with reference anti-BTN2A1 mAb: Natural killer (NK) cells sorted from healthy donors were labeled and then cultured in RPMI supplemented with 10% FBS and 1% P / S, IL-2 (50UI / mL) at 5% CO2 and 37°C with or without IL-15 (10ng / mL) stimulation. On day 0, reference anti-BTN2A mAb or control isotype (10μg / mL) was added to the cultures. After 5 days, NK cells were subjected to extracellular phenotyping for expression of activation markers. NK activation was assessed by induction of CD69 and CD25 expression (percentage and median fluorescence intensity MFI). The gating strategy for NK cells is shown in Figure 4. For measurement of NK cell cytotoxicity, NK cells isolated from three healthy donors were cultured in RPMI, 10% FBS, and 1% P / S with or without IL-2 (50 UI / mL) and IL-15 (10 ng / mL) at 37°C with 5% CO2. Reference anti-BTN2A mAb or control isotype (10 μg / mL) was added to unstimulated NK cells or IL-2 / IL-15 stimulated NK cells overnight. The next day, NK cells were co-cultured with the indicated hematological or carcinoma cell lines at a 1:1 ratio, and FITC-labeled anti-CD107a and anti-CD107b mAbs (all from BD Biosciences) were added to the co-cultures and incubated for 4 hours. NK cell degranulation was assessed by flow cytometry as the percentage of CD107αβ+ cells in unstimulated NK cells or IL-2 / IL-15 stimulated NK cells. 50For calculation of , reference anti-BTN2A mAbs and their isotype control mAbs were used at concentrations ranging from 0.005 nM to 300 nM. To evaluate NK cell-mediated killing in cancer cells, purified NK cells were pre-incubated overnight with 10 μg / mL of reference mAbs, 101G5 mAb and 107G3 mAb, or the corresponding IgG1 control, in RPMI supplemented with 10% FBS and 1% P / S at 37°C with 5% CO2. IL-2 (50 UI / mL) and IL-15 (10 ng / mL) were used as positive controls. The next day, NK cells were co-cultured with the indicated CellTrace-labeled cancer cell lines at a 1:1 ratio for 4 h. Cancer cell death was assessed in tumor cell lines by assessing the percentage of caspase-3 / 7+ cells using CellEvent™ Caspase-3 / 7 Green Detection reagent (Thermofisher Scientific).

[0232] Flow cytometry: Prior to staining, PBMC / NK cells and monocytes / macrophages were incubated for 10 min with human Fc block (Miltenyi) or human IgG1 (Sigma) to saturate Fc receptors. The labeled mAbs used were as follows: CD14-FITC and CD14-APC-Vio770 (Miltenyi), CD163-VioBlue (Miltenyi), DC-SIGN-PE-Vio770 (Miltenyi), CD80-PE (BD Biosciences), PDL1-APC (BD Biosciences), CD3-PE-CF594 (BD Biosciences) and CD3-BV605 (Biolegend), CD56-PE-Vio770 (Miltenyi) and CD56-BV605 (BD Biosciences), CD69-BV421 (BD Biosciences), CD25-APC (BD Biosciences). Cells were incubated with the antibody cocktail for 30 min at 4°C. Dead cells were excluded using live / dead near IR dye (ThermoFisher Scientific) to define the "live" gate. For intracellular IFNγ staining, extracellular staining cells were fixed and permeabilized using Intracellular Fixation & Permeabilization Buffer Set (eBioscience) and incubated with APC-labelled anti-IFNγ (BD Biosciences). Acquisition was performed on a Fortessa flow cytometer (BD Biosciences) using FlowJo 10 software. For phenotyping for BTN2A1 and BTN2A2, purified anti-BTN2A1 specific antibodies (mAb5) and anti-BTN2A2 specific antibodies (mAb17) were used at 10 μg / mL and phenotyped with PE-labelled anti-IgG(H+L) (Jackson Immunoresearch). NK cells were CD45+ CD14- CD3- CD56+ cells in the "live" gate after single cell selection.Monocytes were CD45+ CD19- CD3- CD56- CD14+ cells in the "live" gate after single cell selection. Collection was performed on a Cytoflex LS flow cytometer (Beckman Coulter), an iQue Screener flow cytometer (Intellicyt) or a Fortessa flow cytometer (BD Biosciences) and data were analyzed using FlowJo 10 software. Results are expressed as median fluorescence intensity (MFI) after subtraction of values ​​obtained with the corresponding staining controls.

[0233] Octet-based affinity measurement for the BTN2A1 epitope and binning assay: After the generation of a reference anti-BTN2A antibody in a chimeric IgG1 format, the affinity of this target to two different isoforms (BTN2A1 and BTN2A2) was assessed and a competition assay was performed to determine whether these mAbs recognized the same epitope region of BTN2A1. Affinity and binning experiments were performed on the Octet Red96 platform (Fortebio / PALL), a system based on biolayer interference (BLI) technology. For affinity experiments, recombinant human (rh)BTN2A1-Fc (GTP) was biotinylated using the EZ-Link™ NHS-PEG4 biotinylation kit according to the manufacturer's instructions, and biotinylated rhBTN2A2-Fc was purchased from R&D Systems. For the BTN2A1 affinity assay, biotinylated rhBTN2A1-Fc was loaded onto a streptavidin (SA) biosensor (ForteBio) and diluted in Kinetic Buffer 1X (ForteBio) to a loading target level of approximately 1 nM, and a chimeric anti-BTN2A antibody was used as the analyte. For the BTN2A2 affinity assay, a chimeric anti-BTN2A antibody was loaded onto a FAB2G sensor (anti-human CH1; Fortebio) as described above, and biotinylated rhBTN2A2-Fc was used as the analyte. In both cases, the analytes were kept in solution and their working concentrations were diluted in Kinetic Buffer 10X (ForteBio). For the first run, standard working concentrations ranged from 200 to 3.125 nM. If necessary for the second run, the working concentration was adjusted from 80 to 1.25 nM. All runs (including loading, equilibration, association / soaking of the sensor with the analyte, dissociation, and regeneration) were performed at 30°C with shaking at 1000 rpm. Analyses were performed using a 1:1 or 2:1 Langmuir model (for BTN2A1 or BTN2A2, respectively) calculated by Octet software, which allows good fit calculations. For binning experiments, His-tagged BTN2A1 (rhBTN2A-His) was purchased from R&D Systems.A reference anti-BTN2A antibody was examined in a pairwise fashion against BTN2A1. Binning experiments were performed in the following "in tandem" format, meaning that rhBTN2A-His was immobilized on a biosensor (anti-Penta-His "HIS1K" biosensor; ForteBio / PALL) and presented to two competing antibodies in successive steps. In this kinetic screening, rhBTN2A-His was loaded onto HIS1K (signal intensity: 1 nm) followed by an association step with 10 μg / mL of the antibody for 3 minutes, then a dissociation step for 3 minutes. rhBTN2A1-His activity was confirmed via a kinetic screening assay performed in the same format as the binning assay (BTN2A1 as the ligand / capture in the sensor and the antibody as the analyte). All antibodies (saturating or competing mAbs) were used at 10 μg / mL diluted in Kinetic Buffer 1X. The kinetic screen involved loading of rhBTN2A1-His onto HIS1K (signal intensity: 1 nm) followed by an association step with the antibody for 3 min, then a dissociation step for 3 min. The assay steps were as follows: baseline→antigen capture→baseline→saturating antibody→baseline→competitive antibody→regeneration following an "in tandem" scheme. Binned data were analyzed using epitope bin calculations using Octet Data Analysis HT 11.1.

[0234] Epitope mapping for reference mAbs, 107G3 and 101G5 The interaction of BTN2A1 with the reference mAbs, 107G3 and 101G5, was assessed by differential evaluation of peptide mass fingerprints of BTN2A1 alone or with 107G3 or 101G5. Prior to commencing epitope mapping, high-mass MALDI analysis was performed on rhBTN2A1-Fc proteins (GTP Technologies) to verify their integrity and aggregation levels using an Autoflex II MALDI ToF mass spectrometer (Bruker) equipped with an HM4 interaction module from CovalX (CovalX), confirming that no non-covalent aggregates or multimers of BTN2A1 were present in the samples. To characterize BTN2A1 and determine the epitopes of BTN2A1 / 107G3 and BTN2A1 / 101G5, we subjected samples to proteolysis with trypsin, chymotrypsin, Asp-N, elastase, and thermolysin followed by nLC-LTQ-Orbitrap MS / MS analysis using a nLC Ultimate 3000-RSLC system coupled with a LTQ-Orbitrap mass analyzer (Thermo Scientific). For the BTN2A1 / 107G3 and BTN2A1 / 101G5 complexes, the protein complexes were incubated with deuterated crosslinkers and then subjected to multienzyme cleavage. After enrichment of crosslinked peptides, samples were assayed and the generated data was analyzed using XQuest 2.0 and Stavrox 3.6 software. For sample preparation, reductive alkylation was performed as follows: BTN2A1 (4.04 μM) was mixed with DSS d0 / d12 (2 mg / mL; DMF) before incubation for 180 min at room temperature. After incubation, the reaction was stopped by adding 1 μL of ammonium bicarbonate (20 mM final concentration) before incubation for 1 h at room temperature. The solution was then dried using a SpeedVac and suspended in 8 M urea in H2O (10 μL). After mixing, 1 μL of DTT (500 mM) was added to the solution. The mixture was then incubated at 37° C. for 1 h.After incubation, 1 μl of iodoacetamide (1M) was added before incubation time of 1 h at room temperature in the dark. After incubation, 100 μl of proteolysis buffer was added. Trypsin buffer contains 50 mM Ambic pH 8.5, 5% acetonitrile, chymotrypsin buffer contains 100 mM Tris HCl, 10 mM CaCl2 pH 7.8, ASP-N buffer contains 50 mM phosphate buffer pH 7.8, elastase buffer contains 50 mM Tris HCl pH 8.0, and thermolysin buffer contains 50 mM Tris HCl, 0.5 mM CaCl2 pH 9.0. For trypsin proteolysis, 100 μL of reduced / alkylated BTN2A1 was mixed with 1 μL of trypsin (Roche Diagnostic) in a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37° C. For chymotrypsin proteolysis, 100 μL of reduced / alkylated BTN2A1 was mixed with 0.5 μL of chymotrypsin (Roche Diagnostic) in a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 25° C. For ASP-N proteolysis, 100 μL of reduced / alkylated BTN2A1 was mixed with 0.5 μL of ASP-N (Roche Diagnostic) in a ratio of 1 / 200. The proteolysis mixture was incubated overnight at 37° C. For elastase proteolysis, 100 μL of reduced / alkylated BTN2A1 was mixed with 1 μL of elastase (Roche Diagnostic) in a ratio of 1 / 100. The proteolysis mixture was incubated overnight at 37° C. For thermolysin proteolysis, 100 μL of reduced / alkylated BTN2A1 was mixed with 2 μL of thermolysin (Roche Diagnostic) at a ratio of 1 / 50. The proteolysis mixture was incubated overnight at 70° C. After digestion, formic acid was added to the solution to a final concentration of 1%.After proteolysis, 10 μL of peptide solution generated by proteolysis was loaded onto a nano liquid chromatography system (Ultimate 3000-RSLC) with the following settings: A: 95 / 05 / 0.1 HO / ACN / HCOOH v / v / v; B: 20 / 80 / 0.1 HO / ACN / HCOOH v / v / v, gradient: 5-40% B in 10 μL injection volume over 35 min, precolumn ID: 300 μm×5 mm C18 PepMap™, precolumn flow rate: 20 μL / min, column ID: 75 μm×15 cm C18 PepMap RSLC, column flow rate: 200 nL / min.

[0235] ELISA assay for cross-reactivity between human BTN2A1 and cynomolgus monkey BTN2A1 After BLAST search, the human BTN2A1 amino acid sequence was used to identify the cynomolgus BTN2A1 ortholog sequence (XM_015448906.1), and its extracellular domain was cloned into the pFUSE-hIgG1FC2 vector (InvivoGen) using EcoRI / EcoRV restriction sites. Recombinant cynoBTN2A1-Fc fusion protein was generated by transfection of the resulting pFUSE-hIgG1FC2-cynoBTN2A1 plasmid into Expi293F™ cells with ExpiFectamine™ 293 (ThermoFisher) according to the manufacturer's instructions. Cell culture supernatants harvested on day 6 were used for purification through affinity purification columns. Purified cynoBTN2A1-Fc protein was analyzed by SDS-PAGE and Western blotting to determine molecular weight and purity. The concentration of cynoBTN2A1-Fc protein was determined by Bradford assay with BSA as standard. For ELISA, plates were coated with cynoBTN2A1-Fc protein or recombinant human BTN2A1-Fc (huBTN2A1-Fc; GTP Technologies) (5 μg / mL in 1× PBS) overnight at 4° C. After three washes with PBS, plates were saturated with 2% v / v BSA in PBS for 1 h at room temperature, and then the saturation mAb buffer was discarded. Reference mAbs 101G5 and 107G3, or control human IgG1, were diluted in 2% BSA in PBS in a 10-fold dilution cascade starting from 1 μM to 1 pM, 100 μL of each dilution was added per well, and incubated for 90 min at room temperature on a plate shaker. All wells were washed three times with PBS before addition of Goat anti-mouse IgG HRP (Jackson ImmunoResearch; 1:10,000 dilution in 2% BSA in PBS) and incubation for 1 hour at room temperature.All wells were then washed three times with PBS and a 1-step ABTS solution (ThermoFisher) was added to reveal binding as assessed by absorbance at 405 nm in a Spark spectrometer (Tecan). All samples were assessed in duplicate.

[0236] result: Identification of the reference antibody, anti-BTN2A1 107G3 The reference anti-BTN2A1 107G3 antibody was identified as follows: mice were immunized with BTN2A1-Fc antigen, and spleen cells from the mice exhibiting the highest titers of BTN2A1-specific serum were collected and fused with a myeloma cell line to obtain hybridomas. Hybridoma culture supernatants exhibiting the highest affinity for BTN2A1 were selected for a first round of screening based on their ability to modulate IFN-γ secretion by Vγ9 / Vδ2-T cells. Clones selected from this first round of screening were subcloned and examined for their ability to induce IFN-γ secretion and Vγ9 / Vδ2-T cell degranulation, especially their ability to induce Vγ9Vδ2-T cell degranulation (Figure 1C and Figure 2), leading to the identification of the reference mAb, 107G3. Sequencing of the VH and VL regions of these subclones was performed (see Table 2 below).

[0237] Anti-BTN2A1 107G3 antibody induces degranulation of Vγ9Vδ2 T cells against different cancer cell line targets Purified Vy9 / V52 T cells were expanded from PBMCs of healthy donors and co-cultured with different cancer cell lines, including Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), L-IPC (pancreatic adenocarcinoma), and MDA-MB-134 (breast adenocarcinoma) cells as target cells, with or without anti-BTN2A1 107G3 hybridoma culture supernatant. As shown in Figure 2 and Table 3, the addition of anti-BTN2A1 107G3 hybridoma supernatant results in an induction of the cytolytic function of Vy9 / V52 T cells, as measured by the percentage of CD107+ degranulated cells, compared to co-cultures with target cells alone or in the presence of control hybridoma culture medium. Treatment of Vy9 / V52 T cells with PMA / ionomycin resulted in maximal induction of their cytolytic function, as expected, independent of the target cell.

[0238] The percentage of CD107+ cells induced by anti-BTN2A1 107G3 hybridoma supernatants ranged from 71.1±7.4% in Daudi cells to 17.1±2.9% in MDA-MB-134 cells, compared with 24.9±4.7% and 4.9±0.4%, respectively, in cocultures with control hybridoma culture medium. In cocultures of Vγ9 / Vδ2 T cells with all of the tested cancer cell lines as targets, anti-BTN2A1 107G3 induced between 2- and 8-fold Vγ9 / Vδ2 T cell degranulation compared to the same cocultures in the presence of control hybridoma culture medium.

[0239] Purified anti-BTN2A1 mAb 107G3 had an EC of 0.77 μg / mL (95% IC: 0.32–13.22 μg / mL) for induction of Vγ9 / Vδ2 T cell degranulation, depicted by the percentage of CD107+ cells co-cultured with Daudi cells as targets in the presence of increasing concentrations (0–18 μg / mL) of anti-BTN2A1 107G3 mAb. 50 The patient presented with a stenosis (Figure 2B).

[0240] [Table 2]

[0241] Anti-BTN2A1 107G3 antibody recognizes BTN2A1 but not BTN3 To establish the specificity of anti-BTN2A1 mAb 107G3 for only the BTN2A1 isoform, HEK-293T BTN2 KO cells carrying CRISPR-Cas9-mediated inactivation of both BTN2 isoforms were transiently transfected with a plasmid encoding BTN2A1 as a CFP fusion protein. As shown in Figure 3A, staining with purified anti-BTN2A1 mAb 107G3 was detected only in HEK-293T BTN2 KO cells transfected with a plasmid encoding BTN2A1, but not in HEK-293T BTN2 KO cells alone.

[0242] Anti-BTN3 mAb 103.2, which recognizes all BTN3 isoforms, readily detected BTN3 expression in HEK-293T BTN2 KO cells, and anti-BTN2A1 107G3 is specific for the BTN2A1 isoform and does not cross-react with BTN3.

[0243] Intracellular affinity of anti-BTN2A1 107G3 mAb for BTN2A1 To measure the affinity of anti-BTN2A1 107G3 mAb for its target, HEK-293T BTN2 KO cells transfected with a plasmid encoding BTN2A1 were stained with increasing concentrations (5 ng / mL-75 μg / mL) of purified anti-BTN2A1 107G3 mAb or control mIgG1 (Figure 3B). Nonlinear regression analysis of the mean fluorescence intensity data revealed an EC of 0.32 μg / mL (95% IC: 0.21-0.46 μg / mL) for anti-BTN2A1 107G3 mAb. 50 found.

[0244] Generation of chimeric anti-BTN2A mAbs, determination of affinity, and specificity for BTN2A isoforms: Twenty-three monoclonal antibodies were transiently produced in HEK-293T cells, achieving different ranges of productivity. Most anti-BTN2A antibodies were produced at high levels (>100 mg / L and up to 430 mg / L). One antibody, anti-BTN2A mAb3 (Table 3), exhibited very low production in HEK-293T cells, ranging from 6 to 8 mg / L. Amino acid sequence analysis revealed an N-glycosylation site in the Fab portion, in CDR1 of its VH. Two other antibodies, anti-BTN2A mAb9 and anti-BTN2A mAb11, also exhibited N-glycosylation sites in their Fab regions (in CDR1_VH for mAb9 and CDR1_VL for mAb11). Six antibodies exhibited low purity levels (<95% monomeric), with only anti-BTN2A mAb1 and anti-BTN2A mAb3 exhibiting purity levels <90% (86% and 75%, respectively). All final purified anti-BTN2A mAbs exhibited extremely low levels of intracellular toxins (in the range of 0.1 EU / mg). Only mAb3 had a higher intracellular toxin level (0.73 EU / mg) than the other antibodies, but was still within the acceptance criteria (<1 EU / mg). The affinity constants (K D , k on , and k off ) was determined for the BTN2A1 and BTN2A2 isoforms. Table 4 shows the K D The constants are listed again. For mAb6 and mAb9, K D The calculation of is based on the absence of dissociation observed during the measurement (k off <10 -7mAb16, mAb21, mAb22, and mAb23), and seven mAbs were found to bind to both isoforms (mAb1, mAb7, mAb11, mAb12, mAb13, mAb14, and mAb15).

[0245] [Table 3] JPEG0007652705000004.jpg97149

[0246] BTN2A1 and BTN2A2 expression on the plasma membrane of monocytes and NK cells: We sought to determine whether anti-BTN2A mAbs could therapeutically target non-Vy9V52 T cell compartments of peripheral blood, namely monocytes and NK cells. To this end, we used mAb5 and mAb17, which were found to bind only BTN2A1 or BTN2A2 in our octet assay, respectively, to phenotype monocytes and NK cells derived from peripheral blood. As shown in Figure 4, only anti-BTN2A1 mAb5 stained the cell membrane of both monocytes and NK cells, although a stronger signal was observed in monocytes. Thus, BTN2A1, but not BTN2A2, was detected on the cell membrane of monocytes and NK cells, providing a rationale for screening mAbs recognizing BTN2A1 for their ability to modulate the immune functions of these immune cell compartments.

[0247] Screening of anti-BTN2A mAbs for their ability to modulate monocyte-to-macrophage polarization In response to signals from their microenvironment, monocytes can be polarized into M1 or M2 macrophages. M1 macrophages are associated with pro-inflammatory and anti-tumor properties, whereas M2 macrophages have anti-inflammatory properties and are associated with tumor development. Given that only the BTN2A1 isoform is found on the cell membrane of monocytes, anti-BTN2A mAbs that recognize only the BTN2A1 isoform or both the BTN2A1 / BTN2A2 isoforms were assessed for their ability to interfere with the polarization of monocytes into M2 macrophages in the presence of M-CSF in vitro. M1 macrophages generated in the presence of GM-CSF (CD14+ / - CD163-) and M2 (CD14+ CD163+) macrophages generated in the presence of M-CSF, both without mAb, were used as controls for macrophage polarization. After 5 days of in vitro polarization, the expression of CD14 and CD163 on the plasma membrane of M1, M2, and M-CSF-induced macrophages polarized in the presence of anti-BTN2A mAbs or their control IgG1 was evaluated by flow cytometry (Table 4). As expected, M1 cells exhibited low CD14 expression and undetectable CD163 expression (Table 5 and Figure 4), whereas M2 macrophages exhibited high expression of both markers. Interestingly, anti-BTN2A mAb1, referred to below as 101G5, induced the most potent reduction in CD14 and CD163 expression in the presence of M-CSF, skewing M-CSF-induced macrophage polarization towards an M1-like phenotype (Table 4 and Figure 4B). The second best inhibitor of M-CSF-induced M2 macrophage polarization was 107G3, a mAb2 (Table 5 and FIG. 4B). This is in contrast to the phenotype of macrophages obtained in the presence of M-CSF and control IgG1, which resembled untreated M2 macrophages.

[0248] [Table 4]

[0249] Figure 5 shows the dose-dependence of the M2 inhibitory effect of the reference 101G5 and reference 107G3 anti-BTN2A mAbs compared to the isotype control in relation to the inhibition of CD14 and CD163 expression (Figures 5A and 5B), as well as the increased expression of PDL1 and CD86, characteristic of the M1 phenotype (Figures 5C and 5D). The cytokine secretion profile is also a distinguishing feature between M2 and M1 macrophages. Thus, the secretion of IL-10 (anti-inflammatory, M2-associated) and TNFα (pro-inflammatory, M1-associated) from M-CSF-induced macrophage culture supernatants with or without the reference anti-BTN2A mAb was assessed by ELISA after LPS stimulation. As shown in Figures 5E and 5F, the reference anti-BTN2A mAb dose-dependently inhibited IL-10 secretion and dose-dependently increased TNFα secretion in contrast to the isotype control. These observations confirm that 101G5 and 107G3 inhibit M-CSF-induced polarization of monocytes into M2 macrophages in relation to phenotype and cytokine secretion by skewing towards an M1-like phenotype. Furthermore, these effects of 101G5 and 107G3 are dose-dependent. The IC of each mAb 50 and E.C. 50 The results are shown in Table 6. For all parameters except PD-L1, 101G5 achieved the lowest IC compared to 107G3. 50 and E.C. 50 It is noteworthy that was obtained.

[0250] [Table 5]

[0251] Other stimuli from the tumor microenvironment have also been described to induce M2 macrophage polarization (Mosser and Edwards, Nat Rev Immunol, 2008; Mantovani and Allavena, J Exp Med, 2015). In addition to M-CSF, one of the most commonly used stimuli to induce M2 polarization is IL-4. We determined the effect of 101G5 and 107G3 on the differentiation of so-called tumor-promoting "M2+IL-4" macrophages that arise from monocytes after stimulation with M-CSF and IL-4. Under these conditions, after 5 days of culture, 101G5 and 107G3 inhibited the expression of "M2+IL-4"-associated markers (CD14, CD163, and DC-SIGN; Figures 6A, 6B, and 6D) and IL-10 secretion (Figure 6F), while increasing the expression of M1-associated markers (CD86, PDL1) and TNFα secretion (Figures 6C, 6E, and 6G). Thus, in a tumor-promoting environment (M-CSF and IL-4), 101G4 and 107G3 inhibit "M2+IL-4" differentiation and enhance pro-inflammatory M1 macrophage differentiation.

[0252] In addition, the effect of 101G5 and 107G3 on cancer cell-induced M2 polarization from monocytes was assessed by culturing sorted monocytes in the presence of culture supernatant conditioned with PANC-1 (a pancreatic adenocarcinoma cell line). Addition of 101G5 or 107G3 in this context inhibited M2 polarization, as shown by decreased expression of M2-associated markers (CD14, CD163) and secretion of IL-10 (Figures 7A-7C), and increased expression of M1-associated pro-inflammatory TNFα (Figure 7D).

[0253] Effect of reference mAbs, anti-BTN2A mAb 101G5 and anti-BTN2A mAb 107G3, on reprogramming of M2 macrophages to M1 The potential of the reference mAbs 101G5 mAb and 107G3 mAb to revert M2-polarized macrophages to an M1 phenotype was evaluated. For this purpose, the above M2 macrophages polarized in the presence of M-CSF for 5 days were seeded on the above wells coated with 101G5 mAb and 107G3 mAb and further cultured for 2 or 4 days. Treatment of M2 macrophages with IFNγ was used as a positive control for M2→M1 reversion. As shown in FIG. 8, M2 macrophages cultured in the presence of 101G5 and 107G3 acquired an M1-like phenotype similar to IFNγ treatment. Indeed, treatment of M2 macrophages with the reference mAbs 101G5 mAb and 107G3 mAb resulted in a decrease in CD14 (FIG. 8A) and CD163 (FIG. 8B) expression, and an increase in CD86 expression (FIG. 8C). After treatment with 101G5 or 107G3, respectively, a weak to absent upregulation of PDL1 was observed (FIG. 8D). Furthermore, treatment of M2 macrophages with 101G5 and 107G3 inhibited the secretion of IL-10 (FIG. 8E) and enhanced the secretion of TNFα (FIG. 8F), pointing to an M1 phenotype.

[0254] Figures 8G-8I show the dose-dependence of the effect of reference mAbs, 101G5 mAb and 107G3 mAbon M2 macrophages on reprogramming to M1 macrophages compared to isotype control in relation to inhibition of CD163 expression (Figure 8G), reduction of IL-10, and enhancement of TNFα secretion (Figures 8H and 8I). The IC of each mAb 50 and E.C. 50 are shown in Table 7 for relevant specific M1 / M2 markers, where anti-BTN2A 101G5 mAb shows the best activity for reprogramming M2 macrophages to M1 macrophages.

[0255] [Table 6]

[0256] Reference anti-BTN2A mAb 101G5 and mAb 107G3 relieve M2-mediated inhibition of T cell proliferation The ability of the reference mAbs 101G5 mAb and 107G3 mAb to affect the function of M2 macrophages, i.e., M2-mediated inhibition of T cell proliferation, was explored. For this purpose, preactivated allogeneic CD3+ T cells were co-cultured with M1, M2, or M2 generated in the presence of mAbs. As expected, co-culture with conventional M2 macrophages resulted in a decrease in the number of CD3+ T cells as well as a decrease in T cell proliferation (assessed by the dilution of CTV) and IFNγ production compared to M1 macrophages (Figures 9A, 9C, 9G, 9E, and 9I). In contrast, M2 macrophages generated in the presence of 101G5 and 107G3 did not appear to inhibit T cell proliferation, as shown by the higher percentage and absolute number of CD3+ T cells compared to cultures of M2 macrophages generated in the presence of control IgG1 (Figures 9B, 9H, and 9J). Furthermore, the percentage and number of IFNγ-producing T cells were also higher in co-cultures containing macrophages developed in the presence of 101G5 and 107G3 compared to control IgG1 (FIGS. 9D and 9F).

[0257] Thus, in contrast to M2 macrophages induced by M-CSF alone, macrophages generated in the presence of M-CSF plus 101G5 or 107G3 enable allogeneic CD3+ T cell proliferation and Th1 function (IFNγ production) similar to M1 macrophages.

[0258] Reference anti-BTN2A mAb 101G5 and mAb 107G3 induce NK cell activation and cytotoxicity Since BTN2A1 was found on the plasma membrane of NK cells, we explored the potential ability of 101G5 and 107G3 to modulate NK cell activation. Purified NK cells from healthy donors were cultured for 5 days in the presence of 101G5 or 107G3 with or without additional activation (IL-2 and IL-15, or IL-2 alone, respectively). As shown in Figure 10, both 101G5 and 107G3 enhanced the expression of CD69 on the plasma membrane of NK cells under all tested conditions (Figure 10A). In addition, 101G5 and 107G3 also enhanced the expression of CD25 induced by treatment of NK cells with IL-2 and IL-15 (Figure 10B). Since 101G5 and 107G3 were able to activate purified NK cells, we explored whether these mAbs could also enhance cytotoxicity by NK cells. Therefore, NK cell degranulation (% of CD107+ cells) against cancer cell lines HL-60 (myeloid leukemia), HT-29 (colon cancer), MDA-MB-231 (breast adenocarcinoma), and A549 (lung adenocarcinoma) was evaluated in the presence of 101G5 or 107G3 with or without stimulation with IL-2 and IL-15. As expected, in the presence of control IgG1, only HL-60 cells induced NK cell degranulation (Figure 10C), which was enhanced by stimulation with IL-2 and IL-15 (Figure 10D). Moderate NK cell degranulation against solid tumor cell lines HT-29, MDA-MB-231, and A549 was also observed in the presence of control IgG1 and IL2+IL-15. Table 8 summarizes the NK cell degranulation against these and other tested cancer cell lines (Raji, HCT116, DU-145). Interestingly, the reference mAbs 101G5 and 107G3 enhanced the NK cell degranulation against solid tumor cell lines MDA-MB-231 and A549, and to a lesser extent against HT-29, without stimulation with IL-2+IL-15. The addition of IL-2 and IL-15 accentuated the effect of 101G5 and 107G3 in MDA-MB-231, A549, and DU145 cells (Table 8).No such enhancement was observed with the addition of the reference 101G5 mAb or the reference 107G3 mAb for the HL-60 and Raji hematological cancer cell lines (Table 8 and Figures 10C and 10D). In addition, the reference mAbs 101G5 and 107G3 were able to induce NK cell degranulation against A549 cells when preincubated with NK cells before co-culture without further addition of mAbs to the co-culture (Figure 10E). This suggests a direct effect of the reference mAbs 107G3 mAb and 101G5 mAb by direct binding to NK cells inducing cytotoxicity against cancer cells. Furthermore, the dose-dependence of 101G5 and 107G3 in enhancing NK cell degranulation against the prostate adenocarcinoma DU-145 cell line was evaluated. Indeed, 101G5 and 107G3 enhanced NK cell degranulation in response to DU-145 cells in a dose-dependent manner (EC for 101G5 and 107G3, respectively). 50(no stim) = 0.14 and 0.54 nM; EC 50(IL-2+IL-15) =0.08 and 0.2 nM).

[0259] [Table 7]

[0260] Finally, we investigated the ability of 101G5 and 107G3 to enhance NK cell-mediated killing of cancer cells by assessing the percentage of caspase 3 / 7 cells after co-culture of purified NK cells with the leukemia cell line HL-60 or the lung adenocarcinoma cell line A459. As shown in Figure 11, 101G5 and 107G3 enhanced NK cell-mediated killing of adenocarcinoma A549 cells (approximately 2-fold), but not HL-60 leukemia cells. Taken together, these observations indicate that 101G5 and 107G3 preferentially enhance the cytotoxic effect of NK cells against cancer cells derived from solid tumors.

[0261] Reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb recognize different epitopes of BTN2A1 Both 101G5 and 107G3 share the ability to bind to BTN2A1, inhibit M2 macrophage polarization, and enhance NK cell activation and cytotoxicity. Therefore, we explored whether these mAbs recognized the same epitope region in the BTN2A1 protein. Therefore, we performed an octet-based binning experiment in which 101G5 and 107G3 competed for binding to BTN2A1 using the "indantem" context. As shown in Figure 12, 101G5 and 107G3 did not block each other's binding to BTN2A1, indicating that these two mAbs do not bind to the same epitope region in BTN2A1.

[0262] Epitope mapping for reference mAbs, 101G5 and 107G3 To characterize BTN2A1, we subjected samples to trypsin, chymotrypsin, Asp-N, elastase, and thermolysin proteolysis followed by nLC-LTQ-Orbitrap MS / MS analysis. After trypsin proteolysis, 32 peptides were identified in the BTN2A1 sequence, covering 79.84% of the sequence; after chymotrypsin proteolysis, 27 peptides were identified, covering 94.76% of the BTN2A1 sequence; after Asp-N proteolysis, 2 peptides were identified, covering 12.50% of the BTN2A1 sequence; after elastase proteolysis, 33 peptides were identified, covering 89.11% of the BTN2A1 sequence; after thermolysin proteolysis, 29 peptides were identified, covering 78.23% of the BTN2A1 sequence. Based on the obtained results, overlapping mapping of peptides by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin was designed ( FIG. 13 ). The combination of proteolytic peptides by trypsin, chymotrypsin, Asp-N, elastase, and thermolysin covered 96.37% of the BTN2A1 sequence. To determine the epitopes of the BTN2A1 / 107G3 and BTN2A1 / 101G5 complexes at high resolution, the protein complexes were incubated with deuterated crosslinkers and then subjected to multienzyme cleavage. After proteolysis of the protein complex BTN2A1 / 107G3 by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-orbitrap MS / MS analysis detected 17 crosslinked peptides between BTN2A1 and the antibody 107G3.

[0263] [Table 8] JPEG0007652705000010.jpg207149

[0264] Thus, our analysis indicates that the interaction between BTN2A1 and 107G3 mAb involves the following amino acids in BTN2A1: 65, 68, 69, 72, 78, 84, 85, 95, 97, 100. These results are illustrated in FIG. 14A and FIG.

[0265] After proteolysis of the protein complex BTN2A1 / 101G5 with trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-orbitrap MS / MS analysis detected 14 cross-linked peptides between BTN2A1 and the antibody 101G5.

[0266] [Table 9] JPEG0007652705000012.jpg58149

[0267] Thus, our analysis indicates that the interaction between BTN2A1 and 101G5 involves the following amino acids in BTN2A1: 212, 213, 218, 220, 224, 229. These results are illustrated in FIG. 14B and FIG.

[0268] Reference anti-BTN2A 101G5 mAb and reference anti-BTN2A 107G3 mAb exhibit cross-reactivity with the cynomolgus monkey BTN2A1 orthologue BTN2A1 orthologues exist in most non-human primates, including cynomolgus monkeys (Macaca fascicularis). To determine the cross-reactivity of reference mAbs 101G5 and 107G3 with the cynomolgus BTN2A1 orthologue (cynoBTN2A1; NCBI reference number: XM_015448906.1, 93.31% identity to human BTN2A1), we generated a recombinant Fc fusion protein containing the extracellular domain of cynoBTN2A1 (cynoBTN2A1-Fc) and performed an ELISA assay to evaluate the binding of the reference mAbs to this protein. We also performed ELISAs using recombinant human BTN2A1-Fc protein to compare the affinity of reference mAbs between the human and cynomolgus BTN2A1 orthologues. As shown in Figure 13, both 101G5 and 107G3 bound to the extracellular domain of cynoBTN2A1 with the corresponding EC20 binding sites obtained for huBTN2A1 (0.82 and 0.56 nM, respectively). 50 EC of 0.60 and 0.57 nM, respectively. 50 It was possible to combine them with

[0269] [Table 10] JPEG0007652705000014.jpg100149 JPEG0007652705000015.jpg179149 JPEG0007652705000016.jpg132149 JPEG0007652705000017.jpg163149

[0270] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. Further embodiments are as follows. [Embodiment 1] The following features: i. Inhibits the polarization of monocytes into M2 macrophages; ii. The ability to induce reversion of M2 macrophages to antitumor M1 macrophages; iii. The ability to directly induce NK cell activation; iv. Function of enhancing NK cell-mediated cytotoxicity An anti-butyrophilin 2A (BTN2A) antibody characterized in having at least one of the following: [Embodiment 2] Regarding binding to BTN2A1, (i) mAb 107G3, a reference antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2; or mAb 101G5, a reference antibody comprising: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19; and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. 2. The anti-BTN2A antibody of embodiment 1, which competes with [Embodiment 3] 3. The anti-BTN2A antibody of embodiment 1 or 2, which binds to an epitope comprising residues located at positions 65, 68, 69, 72, 78, 84, 85, 95, 97, and 100 of SEQ ID NO:17. [Embodiment 4] The anti-BTN2A antibody of embodiment 1 or 2, which binds to an epitope comprising residues located at positions 212, 213, 218, 220, 224, and 229 of SEQ ID NO:17. [Embodiment 5] 5. An anti-BTN2A antibody according to any one of embodiments 1 to 4, which does not cross-react with human BTN3 isoforms and / or cross-reacts with cynomolgus BTN2A1 orthologues. [Embodiment 6] CDR1 is a heavy chain variable region comprising SEQ ID NO:3, CDR2 is a heavy chain variable region comprising SEQ ID NO:4, CDR3 is a heavy chain variable region comprising SEQ ID NO:5, CDR1 is a light chain variable region comprising SEQ ID NO:6, CDR2 is a light chain variable region comprising SEQ ID NO:7, and CDR3 is a light chain variable region comprising SEQ ID NO:8, or CDR1 is a heavy chain variable region comprising SEQ ID NO:21, CDR2 is a heavy chain variable region comprising SEQ ID NO:22, CDR3 is a heavy chain variable region comprising SEQ ID NO:23, CDR1 is a light chain variable region comprising SEQ ID NO:24, CDR2 is a light chain variable region comprising SEQ ID NO:25, and CDR3 is a light chain variable region comprising SEQ ID NO:26. The anti-BTN2A antibody of any one of embodiments 1 to 5, comprising: [Embodiment 7] a heavy chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:2; or A heavy chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 20. 7. The anti-BTN2A antibody of any one of embodiments 1 to 6, comprising: [Embodiment 8] The following features: i. The ability to activate Vγ9Vδ2 T cells to secrete cytolytic molecules; ii. Activating the cytolytic function of Vγ9Vδ2 T cells; and / or iii. Function of activating the proliferation of Vγ9Vδ2 T cells The anti-BTN2A antibody of any one of embodiments 1 to 7, further comprising at least one of the following: [Embodiment 9] An anti-BTN2A antibody as described in embodiment 8, which competes with mAb 107G3, a reference murine antibody comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, for binding to BTN2A1. [Embodiment 10] CDR1 is a heavy chain variable region comprising SEQ ID NO:3, CDR2 is a heavy chain variable region comprising SEQ ID NO:4, CDR3 is a heavy chain variable region comprising SEQ ID NO:5, CDR1 is a light chain variable region comprising SEQ ID NO:6, CDR2 is a light chain variable region comprising SEQ ID NO:7, and CDR3 is a light chain variable region comprising SEQ ID NO:8, or a heavy chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:2; or A heavy chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2. 10. The anti-BTN2A antibody of embodiment 8 or 9, comprising: [Embodiment 11] The anti-BTN2A antibody of any one of embodiments 8 to 10, further having specificity for the human BTN2A1 isoform. [Embodiment 12] The anti-BTN2A antibody of any one of embodiments 1 to 11, which is a human antibody, a chimeric antibody, or a humanized antibody. [Embodiment 13] A nucleic acid molecule encoding the heavy and / or light chain of the anti-BTN2A antibody of any one of embodiments 1 to 10. [Embodiment 14] A host cell comprising the nucleic acid of embodiment 13. [Embodiment 15] An anti-BTN2A antibody according to any one of embodiments 1 to 12 for use in therapy. [Embodiment 16] 13. An anti-BTN2A antibody according to any one of embodiments 1 to 12 for use in the treatment of cancer or an infectious disease. [Embodiment 17] A pharmaceutical composition comprising an anti-BTN2A antibody according to any one of embodiments 1 to 12, and at least one pharma- ceutically acceptable carrier.

Claims

1. An anti-BTN2A1 antibody that specifically binds to butyrophilin 2A1 (BTN2A1), a heavy chain variable region CDR1 comprising SEQ ID NO:21, a heavy chain variable region CDR2 comprising SEQ ID NO:22, a heavy chain variable region CDR3 comprising SEQ ID NO:23, a light chain variable region CDR1 comprising SEQ ID NO:24, a light chain variable region CDR2 comprising SEQ ID NO:25, and a light chain variable region CDR3 comprising SEQ ID NO:26, or CDR1 of the heavy chain variable region comprising SEQ ID NO:3, CDR2 of the heavy chain variable region comprising SEQ ID NO:4, CDR3 of the heavy chain variable region comprising SEQ ID NO:5, CDR1 of the light chain variable region comprising SEQ ID NO:6, CDR2 of the light chain variable region comprising SEQ ID NO:7, and CDR3 of the light chain variable region comprising SEQ ID NO:

8. An anti-BTN2A1 antibody comprising:

2. a heavy chain variable region comprising a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 20; or A heavy chain variable region comprising a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1, and a light chain variable region comprising a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2. The anti-BTN2A1 antibody of claim 1 .

3. An anti-BTN2A1 antibody described in claim 1 or 2, which is a chimeric antibody or a humanized antibody.

4. A nucleic acid molecule encoding the heavy and / or light chain of the anti-BTN2A1 antibody of any one of claims 1 to 3.

5. A host cell comprising the nucleic acid molecule of claim 4.

6. An anti-BTN2A1 antibody according to any one of claims 1 to 3 for use in therapy.

7. The anti-BTN2A1 antibody of any one of claims 1 to 3 for use in the treatment of cancer or an infectious disease.

8. A pharmaceutical composition comprising an anti-BTN2A1 antibody according to any one of claims 1 to 3 and at least one pharma- ceutically acceptable carrier.

9. 9. The pharmaceutical composition of claim 8 for treating cancer or an infectious disease by inhibiting the polarization of monocytes into M2 macrophages.

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