Antibodies specific for BTN2 and uses thereof

Abstract

The present invention relates to antibodies specific 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, divert macrophage populations toward anti-tumor M1 macrophages, directly activate NK cells, and are cytotoxic to cancer cells. Alternatively, or in combination, the antibodies can activate Vγ9 / Vδ2 T cells. Such antibodies are particularly useful for treating cancer. Background Art

[0002] Macrophages exhibit different phenotypes, ranging from the classically activated M1 to the alternative M2 type. M1 macrophages rapidly differentiate from monocytes after migration and are activated by bacterial-derived products such as LPS (lipopolysaccharide) as well as infection-related signals such as IFNγ. They are highly inflammatory, with high phagocytic and bactericidal potential. They secrete important pro-inflammatory cytokines such as TNFα, IL-1, IL-6 and IL-12, as well as reactive oxygen species. In contrast, M2 macrophages are present in the later stages of the healing process, when granulation tissue formation occurs; they antagonize the inflammatory response, thereby initiating healing. These anti-inflammatory cells recruit fibroblasts and activate them to differentiate into myofibroblasts, which release pro-angiogenic factors to recruit endothelial progenitor cells and enable new blood vessel formation, a process that occurs through the secretion of key anti-inflammatory cytokines IL-4, IL-10 and IL-13, and is also associated with a reduction in the production of ROS, nitric oxide (NO) and TNFα.

[0003] The tumor microenvironment (TME) strongly polarizes macrophages toward an M2-like phenotype, particularly in the setting of tumor recovery from cancer therapy. This polarization is not only highly pro-angiogenic but also immunosuppressive. Consequently, increased tumor-associated macrophage (TAM) infiltration has long been associated with poor patient prognosis in most solid cancers, highlighting their value as potential diagnostic and prognostic biomarkers in cancer.

[0004] Therefore, reprogramming and selective killing of M2 macrophages is considered a promising therapeutic strategy (Zhu Yet al. Cancer Res 2014).

[0005] Furthermore, as innate immune cells, natural killer (NK) cells play a key role in cancer immune surveillance. NK cells can eliminate a variety of abnormal or stressed cells without prior sensitization, and even preferentially kill stem-like cells or cancer stem cells. Once an immune synapse is formed with a target cell, NK cells release preformed cytolytic granules, including perforins and granzymes, which function to induce cell lysis.

[0006] Based on this hypothesis, several studies have successfully used adoptive transfer of NK cells to fight various tumors, especially hematological malignancies. However, cancers use various strategies to delay, alter, or even stop anti-tumor immunity, resulting in failure to control tumor growth. The anti-tumor response of NK cells also faces many limitations. In particular, the tumor microenvironment (TME) remains a major obstacle to the effectiveness 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, may interfere with the activation of NK cells by secreting immunosuppressive cytokines or interfering with receptor expression. For example, in the TME, TGF-β (especially secreted by M2 polarized macrophages) is considered to be the main inhibitory cytokine of NK cells, which limits the number and anti-metastatic function of NK cells.

[0007] Therefore, from a therapeutic perspective, it would be extremely valuable to have tools that can stimulate antitumor activity by i) suppressing the immunosuppressive effects of the tumor environment and ii) directly triggering NK cell activation and mediated cytotoxicity.

[0008] Butyrophilins constitute a family of transmembrane proteins that include butyrophilin (BTN), BTN-like (BTNL), and the selection and maintenance of intraepithelial T cells (SKINT) protein (Arnett and Viney, Nat Rev Immunol 2014). Their extracellular portions contain IgV-like and IgC-like domains that show homology to the corresponding domains of the B7 costimulatory molecule (Arnett and Viney, 2014), and therefore butyrophilins are considered members of the extended B7 or Ig superfamily.

[0009] The butyrophilin (BTN) gene family consists of 13 genes in humans, organized into eight distinct groups (Abeler-Dorner et al. Trends Immunol 2012; Afrache et al. Immunogenetics 2012). Seven human BTN genes are clustered in the MHC class I region of chromosome 6 and are divided into three subfamilies that form phylogenetically related groups: BTN1, BTN2, and BTN3. The BTN1 subfamily contains only the prototype single-copy BTN1A1 gene, while the BTN2 and BTN3 subfamilies each contain three genes, BTN2A1, BTN2A2, and BTN2A3 (which are pseudogenes), namely BTN3A1, BTN3A2, and BTN3A3, respectively. Several polymorphisms have been described in members of the BTN gene family, which are associated with various 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 & 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 variants have been described in BTNL2, BTN2A1, BTN3A2, and BTN3A3, as well as deleterious copy number variations involving BTNL3 and BTNL8 (Aigner et al. BMC Genet, 2013).

[0010] BTN1A1, the earliest identified milk fat protein, is required for the formation, secretion, and stabilization of milk fat globules (Ogg et al. Proc Natl Acad Sci, 2004). Subsequently, it was proposed that the B7 gene and MHC class I and II genes may share a common ancestral gene and encode proteins involved in similar functions, such as T cell activation (Rhodes et al. Genomics, 2001; Harly C et al. Blood, 2012). Similar to BTN3A1 and BTN3A3, the BTN2A1 and BTN2A2 protein isoforms display IgV and IgC extracellular domains, a transmembrane domain, and a characteristic intracellular B30.2 domain, but BTN3A2 does not. In mice, BTN2A2 is a single-copy gene and an ortholog of the human BTN2A2 gene. Recombinant human BTN2A1-Fc protein showed that specific glycoforms of BTN2A1 bind to DC-SIGN, a lectin molecule found on dendritic cells (DCs). The binding of BTN2A1 to DC-SIGN depends on high mannose glycosylation of the protein when expressed in tumor cells (Malcherek et al. J Immunol, 2007).

[0011] Subsequently, increasing evidence suggested that casein plays diverse roles in the immune system. RNA-seq data from 53 human tissue samples from the Genotype Tissue Expression Project (The GTEx Consortium, 2013) revealed ubiquitous expression of BTN2A1 transcripts in normal tissues. Using gene expression profiling cross-analysis (Tang, Z. et al., Nucleic Acids Res, 2017), a comparison of RNA-seq data from GTEx with data from The Cancer Genome Atlas (TCGA) database revealed that BTN2A1 transcript expression is regulated in several cancers, including cervical squamous cell carcinoma and adenocarcinoma, small cell lung carcinoma, ovarian cancer, pancreatic cancer, and endometrial cancer.

[0012] Antibodies that recognize two subtypes of BTN2A have been reported previously (WO2019057933), but the antibodies inhibit the production of IFN-γ and / or TNF-α by activated Vγ9 / Vδ2 T cells, and / or inhibit the cytolytic function of activated Vγ9 / Vδ2 T cells, and / or inhibit the proliferation of activated Vγ9 / Vδ2 T cells.

[0013] Vγ9 / Vδ2 T cells are important effectors of immune defense. They directly lyse infected pathogens or abnormal cells. Furthermore, they modulate immune responses by inducing dendritic cell (DC) maturation, isotype switching, and immunoglobulin production. This crucial cellular platform of the immune system is tightly regulated by surface receptors, chemokines, and cytokines. Vγ9 / Vδ2 T cells are activated by non-peptide phosphorylated isoprenoid pathway metabolites, known as phosphoagonists (PAgs).

[0014] The development of antibodies that target BTN2A1 and activate immune cells, especially more than one immune cell compartment, such as macrophages, NK and / or γδT cells, especially Vγ9 / Vδ2T cells, may be particularly useful in the treatment of cancer and infectious diseases. SUMMARY OF THE INVENTION

[0016] The present disclosure provides the first antibodies that bind to BTN2A (particularly to the BTN2A1 isoform—e.g., human BTN2A1 polypeptide) and exhibit at least one of the following properties:

[0017] i. Inhibit the polarization of monocytes to M2 macrophages,

[0018] ii. Inducing the reversal of M2 macrophages to anti-tumor M1 macrophages,

[0019] iii. Directly trigger NK cell activation,

[0020] iv. Enhance NK cell-mediated cytotoxicity.

[0021] 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), more particularly, the antibodies of the present disclosure exhibit properties i)-iv).

[0022] Thus, such antibodies according to the present disclosure may

[0023] - Favoring an anti-tumor microenvironment by shifting macrophage phenotype and function toward pro-inflammatory M1 macrophages, leading to the secretion of pro-inflammatory cytokines, and / or

[0024] - Directly trigger NK cell activation, thereby further enhancing their cytolytic activity.

[0025] Thus, such antibodies according to the present disclosure represent powerful tools that can be used in various strategies for cancer treatment.

[0026] In specific embodiments, the anti-BTN2A antibodies of the present disclosure compete for binding to BTN2A with any of:

[0027] - the reference murine antibody mAb 101G5, which comprises (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, or

[0028] - the reference murine antibody mAb 107G3, comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0029] In some embodiments, an anti-BTN2A antibody of the present disclosure binds to an epitope comprising an amino acid residue located at:

[0030] - position 65, 68, 69, 72, 78, 84, 85, 95, 97, 100 of SEQ ID No. 17, or

[0031] - positions 212, 213, 218, 220, 224, 229 of SEQ ID N° 17.

[0032] In specific embodiments, the anti-BTN2A antibodies of the present disclosure comprise:

[0033] - a heavy chain variable region CDR1 comprising SEQ ID NO: 3, a heavy chain variable region CDR2 comprising SEQ ID NO: 4, a heavy chain variable region CDR3 comprising SEQ ID NO: 5, a light chain variable region CDR1 comprising SEQ ID NO: 6, a light chain variable region CDR2 comprising SEQ ID NO: 7, and a light chain variable region CDR3 comprising SEQ ID NO: 8, or

[0034] - 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.

[0035] In specific embodiments, the anti-BTN2A antibodies of the present disclosure comprise:

[0036] - a heavy chain variable region comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, or

[0037] - a heavy chain variable region comprising a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

[0038] In a specific embodiment, the antibodies of the present disclosure further exhibit at least one of the following properties:

[0039] - activates the secretion of cytolytic molecules from Vγ9Vδ2 T cells,

[0040] - Activation of the cytolytic function of Vγ9Vδ2 T cells, and / or

[0041] -Activates the proliferation of Vγ9Vδ2 T cells.

[0042] Most particularly, the anti-BTN2A antibodies according to such embodiments can typically compete for BTN2A binding with the reference murine antibody mAb 107G3, which comprises (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.

[0043] More specifically, such anti-BTN2A antibodies may comprise:

[0044] - a heavy chain variable region CDR1 comprising SEQ ID NO: 3, a heavy chain variable region CDR2 comprising SEQ ID NO: 4, a heavy chain variable region CDR3 comprising SEQ ID NO: 5, a light chain variable region CDR1 comprising SEQ ID NO: 6, a light chain variable region CDR2 comprising SEQ ID NO: 7, and a light chain variable region CDR3 comprising SEQ ID NO: 8, or

[0045] - a heavy chain variable region comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, or

[0046] - a heavy chain variable region comprising a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2.

[0047] In some embodiments of the disclosure, the antibodies of the disclosure are specific for BTN2A1.

[0048] In specific embodiments, the anti-BTN2A antibodies of the present disclosure are human, chimeric, or humanized antibodies.

[0049] The present disclosure also encompasses nucleic acid molecules encoding the heavy and / or light chains of the above-mentioned anti-BTN2A antibodies.

[0050] The present disclosure also relates to host cells comprising such nucleic acids, particularly for producing any of the above-mentioned anti-BTN2A antibodies.

[0051] Another aspect of the present disclosure relates to an anti-BTN2A as described above for use in therapy, in particular for the treatment of cancer or infectious diseases.

[0052] Typically, cancers according to the present disclosure include blood cancers and solid cancers, including 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 cancer; prostate cancer; colon cancer; pancreatic cancer and endometrial cancer, more specifically: squamous cell carcinoma and cervical adenocarcinoma, squamous cell carcinoma, ovarian cancer, small cell lung cancer, prostate cancer, colon cancer, pancreatic cancer and endometrial cancer.

[0053] The present disclosure also relates to a pharmaceutical composition comprising the anti-BTN2A antibody as described above and at least a pharmaceutically acceptable carrier.

[0054] The present disclosure also provides methods for activating an immune response in a subject, comprising administering to the subject an effective amount of an anti-BTN2A disclosed herein. Detailed Description of the Invention

[0056] definition

[0057] As used herein, the term "BTN2" has the 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.

[0058] SEQ ID NO: 17: BTN2A isoform 1 precursor (Homo sapiens):

[0059] MESAAALHFSRPASLLLLLLSLCALVSAQFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVIIRDKSVRNMSCSINNTLLGQKKESVIFIPESFMPSVSPCAVALPIIVVILMIPIAVCIYWINKLQKEKKILSGEKEFERETREIALKELEKERVQKEEELQVKEKLQEELRWRRTFLHAVDVVLDPDTAHPDLFLSEDRRSVRRCPFRHLGESVPDNPERFDSQPCVLGRESFASGKHYWEVEVENVIEWTVGVCRDSVERKGEVLLIPQNGFWTLEMHKGQYRAVSSPDRILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAFSVPVRPFFRLGCEDSPIFICPALTGANGVTVPEEGLTLHRVGTHQSL

[0060] SEQ ID NO:18: Precursor of BTN2A Subtype 2 (Homo sapiens):

[0061] MEPAAALHFSLPASLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCY FQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVTTAVIIRDKYVRNVSCSVNNTLLGQEKETVIFIPESFMPSASPWMVALAVILTA SPWMVSMTVILAVFIIFMAVSICCIKKLQREKKILSGEKKVEQEEKEIAQQLQEELRWRRTFLHAADVVLDPDTAHPELFLSEDRRSVRRGPYRQRVPDNPERFDSQPCVLGWESFASGKHYWEVEVENVM VWTVGVCRHSVERKGEVLLIPQNGFWTLEMFGNQYRALSSPERILPLKESLCRVGVFLDYEAGDVSFYNMRDRSHIYTCPRSAFTVPVRPFFRLGSDDSPIFICPALTGASGVMVPEEGLKLHRVGTHQSL

[0062] As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and will be used equivalently in this disclosure.

[0063] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. Thus, the term antibody encompasses not only complete antibody molecules but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments.

[0064] As used herein, the term "antibody" also includes bispecific or multispecific molecules. The antibody can be derivatized or connected to another functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. In fact, the antibody can be derivatized or connected to more than one other functional molecule to produce a multispecific molecule that binds to two or more different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed in the term "bispecific molecule" used herein. In order to produce bispecific molecules, the antibodies of the present invention can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent binding or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, thereby producing bispecific molecules. In addition, for embodiments in which the bispecific molecule is multispecific, in addition to the first and second target epitopes, the molecule can also include a third binding specificity. In one embodiment, the bispecific molecule disclosed herein comprises at least one antibody or its antibody fragment as binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, Unibody or single-chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as a Fv or single chain construct as described in Ladner et al., US Patent No. 4,946,778.

[0065] Other antibodies that can be used in the bispecific molecules disclosed herein are murine, chimeric, and humanized monoclonal antibodies (mAbs).

[0066] The bispecific molecules of the present disclosure can be prepared by combining component binding specificities using methods known in the art. For example, each is bound to each other. When the binding specificity is a protein or peptide, a variety of coupling agents 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-phthalenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester (sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). Other methods include Brennan et al., 1985; Glennie et al., 1987; Paulus, the method described in 1985. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. When the bispecific molecule is mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein, this method is particularly useful. The bispecific molecule of the present invention can be a single-chain molecule comprising a single-chain antibody and a binding determinant, or a single-chain bispecific molecule comprising two binding determinants. The combination of the bispecific molecule and its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (such as growth inhibition and apoptosis) or Western blot assay. Each of these assays is typically detected by using a labeling agent (e.g., antibody) specific for the target complex to detect the presence of a specific target protein-antibody complex.

[0067] In natural antibodies, two heavy chains are interconnected by disulfide bonds, and each heavy chain is connected 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), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity for the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain binding, secretion, translocation, complement fixation, and binding to Fc receptors (FcRs).

[0068] The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and is composed of the variable portion of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed primarily of residues from the hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) may participate in the antibody binding site or influence the overall domain structure and, therefore, the binding site. The complementarity determining regions, or CDRs, refer to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically includes six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions. The framework regions (FRs) refer to the amino acid sequences interposed between the CDRs. The variable regions of the light and heavy chains typically contain four framework regions and three CDRs with the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0069] The residues in the antibody variable domain are generally numbered according to the system designed by Kabat et al. This system is described 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 in this specification. The Kabat residue nomenclature does not always directly correspond to the linear numbering of the amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or more amino acids than in the strict Kabat numbering, which corresponds to shortening or insertion of structural components of the basic variable domain structure (whether framework regions or complementary determining regions (CDRs)). The correct Kabat numbering of the residues of a given antibody can be determined by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. According to the Kabat numbering system, 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).

[0070] In a specific embodiment, the antibodies provided herein are antibody fragments, more particularly any protein comprising the antigen binding domain of an antibody as disclosed herein. Antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies.

[0071] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds BTN2A1 is substantially free of antibodies that specifically bind 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). In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0072] Antibody affinity refers to the strength with which an antibody binds to an epitope presented on an antigen (e.g., BTN2A1 of the present disclosure) through its antigen binding site (paratope). Affinity can be assessed based on an evaluation of the Kd value.

[0073] As used herein, the term "K D ” means the equilibrium dissociation constant, which is given by K off With K on The ratio (K off / K on ) and expressed as molar concentration (M). K D The value is related to the antibody concentration (the amount of antibody required for a specific experiment), so K D The lower the value (the lower the concentration), the higher the affinity of the antibody. The K value of an antibody can be determined using methods well known in the art. D The K values ​​for mAbs can be found in the following references. d Preferred methods for determining K values ​​for antibodies are: Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92: 589-601 (1983), which are incorporated herein by reference in their entirety. D The method is to use surface plasmon resonance, or to use a method such as Biosensor system for the system (see also for details on affinity assessment Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using .Anal Biochem.2001) or system. The platform is based on biolayer interferometry (BLI) technology. The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces - a layer of immobilized protein and an internal reference layer. The binding between the ligand immobilized on the surface of the biosensor tip and the analyte in solution increases the optical thickness of the biosensor tip, which results in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of the biolayer, and when this shift is measured over a period of time and its amplitude is plotted as a function of time, a classical association / dissociation curve can be obtained. This interaction is measured in real time, allowing the monitoring of binding specificity, association and dissociation rates, and concentration (see Abdiche et al. 2008 and Results for details). Affinity measurements are typically performed at 25°C.

[0074] As used herein, the term " assoc ” or “k a ” or “k on " is intended to refer to the on-rate of a particular antibody-antigen interaction, whereas the term "k dis ” or “k d ” or k off It is intended to refer to the off-rate of a specific antibody-antigen interaction.

[0075] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers 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.

[0076] 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 a BTN2A subtype, including BTN2A1 and BTN2A2 in the present disclosure). It is typically intended to refer to a K of 10 nM or less, 5 nM, 1 nM or less, 100 pM or less, or 10 pM or less. D An antibody or protein that binds to human BTN2A, particularly BTN2A1. 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 as defined above. D In some embodiments, specificity can 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., from 5 ng / mL to 75 μg / mL) of a purified anti-BTN2A1 mAb as disclosed herein, or with a negative control such as its control isotype. Nonlinear regression analysis of the mean fluorescence intensity data allows for the EC 50 The concentration of mAb at which 50% of the maximum fluorescence is observed is determined. Typically, according to the present disclosure, the EC of BTN2A1-specific antibody binding to BTN2A1 is 50 Less than 50 μg / mL, in particular less than 40 μg / mL (see Examples section), in particular 0.1 μg / mL-50 μg / mL or 0.5 μg / mL-20 μg / mL.

[0077] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."

[0078] "Selective binding" typically refers to an antibody's binding to a target (e.g., epitope) that is stronger and more specific than its binding to another target. An antibody binds more strongly to a first target than to a second target if its affinity for the first target is higher than its affinity for the second target. Typically, an antibody binds to a target with a specific affinity lower than the equilibrium dissociation constant or EC. 50 The equilibrium dissociation constant (K D ) or EC 50 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 61, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 80, 81, 82, 83, 84, 85, 86, 87, 90, 91, 92, 93, 94, 46, 47, 48, 59, 50, 51, 52, 53, 54, 65, 71, 82, 83, 84, 95, 96, 97, 98, 99, 10 ...

[0079] The selectivity of the antibodies disclosed herein can be tested using a cross-reactivity assay comparing other closely related proteins (e.g., BTN3 isoforms) to the intended target protein (BTN2A isoform). When no such cross-reactivity can be detected, while giving a strong signal for the intended target at the same antibody dilution, the antibody is typically considered selective (see corresponding Figure 3 (The results are detailed in the Examples of the present invention). It is intended that an antibody that "cross-reacts with an antigen" is an antibody that cross-reacts with an antigen with a K of 10 nM or less, 1 nM or less, or 100 pM or less. D An antibody that "does not cross-react with a particular antigen" is intended to mean an antibody that binds to that antigen with a K of 100 nM or greater. D , or K of 1 μM or greater D or K of 10 μM or greater D In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding to these proteins in standard binding assays (see typically Table 3 or Table 4). Figure 3 A).

[0080] In a specific embodiment, the anti-BTN2A1 antibodies of the present disclosure cross-react with the cynomolgus monkey BTN2A1 ortholog (cynoBTN2A1; NCBI reference number XP_015304392.1) of sequence SEQ ID N° 35 defined as follows:

[0081]

[0082] In some of these embodiments, the antibodies disclosed herein are expressed in a manner similar to that obtained on huBTN2A. 50 Equivalent (greater or less than 10%) EC 50 Binds to the cynoBTN2A1 extracellular domain.

[0083] The term "identity" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in the two compared sequences is occupied by the same base or the same amino acid residue, the corresponding molecules are identical at that position. The percent identity between the two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Typically, two sequences are compared when aligned for maximum identity. Identity can be calculated by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) stacking program or any sequence comparison algorithm (such as BLAST, FASTA or CLUSTALW).

[0084] Functional variants of reference molecules according to the present disclosure exhibit functional properties that are substantially equal to or better than the corresponding functional properties of the reference molecule (e.g., 107G3 mAb). Substantially equal herein means 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.

[0085] In one aspect, the present disclosure relates to antibodies specific for BTN2A1 as defined above. Typically, such antibodies are characterized in that they have a K of 10 nM or less as defined above. D Binds to human BTN2A1.

[0086] In some embodiments, the antibodies of the present disclosure do not cross-react with BTN3 isoforms.

[0087] According to the present invention, antibodies specific for BTN2A, in particular BTN2A1, are typically further characterized in that they have at least one of the following properties:

[0088] i. It inhibits the polarization of monocytes to M2 macrophages,

[0089] ii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages,

[0090] iii. It directly triggers NK cell activation,

[0091] iv. It enhances NK cell-mediated cytotoxicity.

[0092] Preferably, the antibody of the present disclosure exhibits at least one of properties i and ii, and at least one of properties iii and iv, most likely it exhibits properties i)-iv).

[0093] Anti-BTN2A antibodies of the present disclosure having such advantageous properties can be screened among anti-BTN2A antibodies using, for example, the assay described in detail in the Examples section. The assay and its implementation are briefly described below.

[0094] In a macrophage polarization assay, as classically performed in the art, M2 macrophages are generated from macrophages in the presence of an anti-BTN2A antibody, particularly an anti-BTN2A1 antibody as previously defined, or a negative control (such as a control isotype thereof). During M2 polarization induced by M-CSF (macrophage colony stimulating factor), GM-CSF (granulocyte-macrophage colony stimulating factor) and IFN-γ can be added to monocytes as controls for inhibition of M2 differentiation. In contrast, M1 macrophages polarized in the presence of GM-CSF can also typically be used as phenotypic controls. After polarization, the expression of M1 and M2 related markers on the plasma membrane can be assessed in the resulting macrophages by flow cytometry.

[0095] According to the present disclosure, M1 markers that can be conveniently detected include, but are not limited to, PDL1, CD86, CD40, CD80, and / or SOCS3. Typically, PDL1 and / or CD86 are detected. According to the present disclosure, M2 markers that can be conveniently detected include, but are not limited to, CD14, CD163, CD206, and CD209. Typically, CD14 and / or CD163 are detected.

[0096] Typically, when:

[0097] - 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 its control isotype; and / or

[0098] - when 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 thereof (such as a control isotype thereof),

[0099] The Anti-BTN2A of the present disclosure induces inhibition of monocyte polarization toward M2 macrophages.

[0100] In addition, typically by using appropriate commercial kits (e.g., ELISA-like kits), the cytokine secretion profile (especially including the M2-related anti-inflammatory IL-10 cytokine and the pro-inflammatory M1-related TNFα cytokine) as a characteristic distinguishing function between M1 and M2 macrophages can be quantified in the culture supernatant. Other cytokines that can also be easily detected to characterize the M1 or M2 macrophage phenotype include IL-1, IL-6, IL-12, and IL-23, which are M1-related cytokines, and TGFβ and IL-10, which are M2-related cytokines.

[0101] Thus, in some embodiments, when:

[0102] - a significant reduction 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), and / or

[0103] - when a significant increase in the secretion of at least one M1-related 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),

[0104] Inhibition of monocyte polarization to M2 macrophages can also be considered as a result of the action of the anti-BTN2A antibodies of the present disclosure.

[0105] Typically, the anti-BTN2A antibodies according to the present disclosure can dose-dependently inhibit the polarization of monocytes to M2 macrophages, as assessed by reduced expression of M2-associated markers (e.g., CD14 and / or CD163) and / or reduced secretion of M2-associated cytokines (e.g., IL-10). The half-maximal inhibitory concentration (IC 500) of such antibodies with respect to the secretion of at least one M2-associated cytokine or the expression of at least one M2-associated marker is 100%. 50 ) can be determined in a dose-response curve, as described in detail in the Examples section. In some embodiments, the anti-BTN2A antibodies of the invention exhibit:

[0106] -IC of M2-related marker expression (typically CD14 and / or CD163) 50 0.05 μg / mL, in particular 0.1 μg / mL-100 μg / mL, in particular 50 μg / mL, and / or IC for M2-related cytokine secretion (typically IL-10) 50 0.01 μg / mL, particularly 0.05 μg / mL-100 μg / mL, particularly 50 μg / mL, most particularly 0.1-20 μg / mL.

[0107] Additionally, or alternatively, despite the presence of M2-promoting stimuli, the anti-BTN2A antibodies according to the present disclosure can dose-dependently reverse the polarization of monocytes to M1 macrophages, as assessed by increased expression of M1-associated markers (e.g., CD86 and / or PDL1) and / or decreased secretion of M2-associated cytokines (e.g., IL-10). The half-maximal effective concentration (EC) of such antibodies with respect to the secretion of the at least one M1-associated cytokine or the expression of the at least one M1-associated marker is 100%. 50 ) can be determined in a dose-response curve, as described in detail in the Examples section. In some embodiments, the anti-BTN2A antibodies of the invention exhibit:

[0108] - ECs expressing M1-related markers (typically CD86 and / or PDL1) 50 0.01 μg / mL, particularly 0.1 μg / mL-100 μg / mL, particularly 50 μg / mL, most particularly 1-50 μg / mL, and / or

[0109] - ECs secreting M1-related cytokines (typically TNFα) 50 0.01 μg / mL, particularly 0.05 μg / mL-100 μg / mL, particularly 50 μg / mL, most particularly 0.1-10 μg / mL.

[0110] In the M2 macrophage reversal assay, M2 macrophages generated from monocytes, typically in the presence of M-CSF, can be cultured with or without lipopolysaccharide (LPS) in the presence of an anti-BTN2A antibody (most particularly an anti-BTN2A1 antibody as defined previously) or with a negative control (such as its control isotype). GM-CSF (granulocyte-macrophage colony stimulating factor) and IFNγ can be added to the M2 culture as a positive control for M2 reversal. M1 macrophages polarized in the presence of GM-CSF are typically used as phenotypic controls. After the reversal experiment, the macrophages that were reversed in the absence of LPS can be analyzed by flow cytometry for the expression of M1 or M2 related markers as described above. Cytokine secretion can also be quantified as described above.

[0111] Typically, when:

[0112] - 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

[0113] - when a significant reduction in M2 markers is observed when M2 macrophages are cultured in the presence of said anti-BTN2A antibody compared to its negative control (such as its control isotype),

[0114] The anti-BTN2A antibodies of the present disclosure induce the reversion of M2 macrophages to M1 macrophages.

[0115] The activation of natural killer (NK) cells can be assessed by culturing NK cells (typically from healthy donors) with an anti-BTN2A antibody as defined herein or a control isotype for a negative control, with or without the addition of IL-2 and / or IL-15. After at least 48 hours, and in particular at least 4 days, the NK cells can then be subjected to extracellular phenotypic analysis for their activation markers such as CD69 and / or CD25. Typically, when a significant increase in NK cell activation markers CD69 and / or CD25 is observed in the presence of the anti-BTN2A (with or without further activation with IL-2 and / or IL-15) compared to a negative control (e.g., an isotype control), NK cell activation is considered to be induced by the anti-BTN2A antibodies of the present disclosure. The results provided herein clearly demonstrate that the antibodies of the present invention trigger direct activation of NK cells.

[0116] The enhancement of the cytotoxicity of NK cells can be further evaluated in vitro by assessing NK cell degranulation. In this functional assay, cancer cell lines (such as leukemia cell lines (myeloid leukemia) or cancer (such as colon cancer, breast cancer or lung adenocarcinoma) cell lines) are co-cultured in the presence of the above-mentioned NK cells (previously activated with anti-BTN2A antibodies or their negative controls as described above) and in the presence or absence of IL-2 and / or IL-15. NK cell degranulation can typically be assessed by flow cytometry as the percentage of CD107 positive NK cells (in the presence or absence of IL-2 and / or IL-15).

[0117] Typically, when NK cell degranulation (e.g., the percentage of CD107-positive NK cells) is significantly increased compared to its negative control (e.g., its control isotype) after NK cell activation with the anti-BTN2A antibody, NK cell cytotoxicity is considered to be induced by the anti-BTN2A antibody. Typically, the anti-BTN2A antibodies of the present disclosure induce NK cell degranulation in a dose-dependent manner (particularly against various cancer cell lines as described above). The half effective concentration (EC50) of such antibodies in terms of NK cell degranulation is 100%. 50 ) can be determined in a dose-response curve as detailed in the Examples section.

[0118] Reference anti-101G5 and / or 107G3 can be used as positive controls in the functional assays described above.

[0119] In some embodiments, as described in detail below, the anti-BTN2A1 antibodies of the present disclosure inhibit the polarization of monocytes to M2 macrophages, induce the reversal of M2 macrophages to anti-tumor M1 macrophages, trigger direct NK cell activation, and / or enhance NK cell-mediated cytotoxicity to a level substantially equal to or better than that of the reference antibodies mAb 101G5 or mAb 107G3. "Inhibit the polarization of monocytes to M2 macrophages, induce the reversal of M2 macrophages to anti-tumor M1 macrophages, trigger direct NK cell activation, and / or enhance NK cell-mediated cytotoxicity to a level substantially equal to or better than that of the reference antibodies" herein means that a change of less than 20%, particularly less than 15%, particularly less than 10%, and typically less than 5% of the tested functional activity of the tested anti-BTN2A1 antibody is observed compared to either of the reference antibodies mAb 107G3 or 101G5.

[0120] The present disclosure also encompasses the following antibodies:

[0121] 1) Antibodies specific for BTN2A as defined above, in particular BTN2A1, in particular having at least one of the following properties:

[0122] - it binds to human BTN2A1, typically expressed in a cell line, such as a HEK293T cell line transfected with a plasmid encoding human BTN2A1, as described in the Examples, more particularly at an EC lower than 50 μg / mL, more particularly lower than 40 μg / mL 50 or it has a K of 10 nM or less D binds to BTN2A1 and / or it binds to BTN2A1 with an affinity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater compared to nonspecific binding;

[0123] -Does not cross-react with BTN3;

[0124] and,

[0125] 2) Additionally or alternatively, it exhibits one or more functional properties:

[0126] – It activates the production of cytolytic molecules (particularly IFN-γ) by Vγ9 / Vδ2 T cells, and / or

[0127] - it activates the cytolytic function of Vγ9 / Vδ2 T cells, and / or

[0128] -It activates the proliferation of Vγ9 / Vδ2 T cells.

[0129] Notably, proliferation of Vγ9 / Vδ2 T cells, cytolytic function, and production of cytolytic molecules (particularly IFN-γ) are achieved by activated Vγ9 / Vδ2 T cells, which are typically achieved by the antibodies disclosed herein.

[0130] Anti-BTN2A1 antibodies of the present disclosure having such favorable properties can be screened from anti-BTN2A1 antibodies using the cellular assays described in the Examples, in particular by evaluating IFNγ secretion by Vγ9 / Vδ2 T cells based on ELISA, and / or CD107 degranulation assays on various cancer cell lines (such as the Daudi cell line, the Jurkat cell line, the L-IPC cell line, or the MDA-MB-134 cell line).

[0131] The cytolytic molecules according to the present disclosure are typically IFNγ or TNFα cytokines.

[0132] As used herein, "production of activating cytolytic molecules (typically in IFNγ and / or TNFα)" refers to a significant increase in the production of at least IFNγ or TNFα by activated Vγ9 / Vδ2T cells when compared to control activated Vγ9 / Vδ2T cells (using IgG1 or hybridoma culture medium as a control), wherein the Vγ9 / Vδ2T cells are activated by co-culturing with target cell lines (Daudi, Jurkat, L-IPC or MDA-MB-134 cell lines) or by phospho-agonists (pAg). Typically, activation of IFNγ or TNFα by activated Vγ9 / Vδ2T cells can be measured in a cell assay by intracellular labeling with antibodies against IFNγ or TNFα assessed by flow cytometry, or by secretion of IFNγ or TNFα by Vγ9 / Vδ2T cells in their culture medium based on an ELISA dose. This assay is described in more detail in the Examples below (see Materials and Methods).

[0133] As used herein, "activation of the cytolytic function of activated Vγ9 / Vδ2T cells" refers to a significant increase in the cytolytic function of activated human Vγ9 / Vδ2T cells observed when compared to control activated human Vγ9 / Vδ2T cells (using IgG1 or hybridoma culture medium as a control), wherein the human Vγ9 / Vδ2T cells are activated by co-culturing with a target cell line (e.g., Daudi, Jurkat, L-IPC or MDA-MB-134 cell line) or by a phospho-agonist (pAg). Typically, the activation of the cytolytic function of activated Vγ9 / Vδ2T cells can be determined based on the measurement of activation of Vγ9 / Vδ2T cell degranulation induced by a standard cell line, using CD107a and CD107b together as degranulation markers for detecting positive degranulating Vγ9 / Vδ2T cells. Phorbol 12-myristate-13-acetate (PMA) and ionomycin treatment of Vγ9 / Vδ2 T cells is typically used as a positive control for Vγ9 / Vδ2 T cell activation. This assay is described in more detail in the Examples below.

[0134] As used herein, "proliferation of activated Vγ9 / Vδ2T cells" refers to a significant increase in the proliferation of activated Vγ9 / Vδ2T cells observed when compared to the proliferation of Vγ9 / Vδ2T cells activated with IgG1 as a control, wherein the Vγ9 / Vδ2T cells are activated by co-culture with a target cell line (e.g., Daudi, Jurkat, L-IPC, or MDA-MB-134 cell line) or by a phospho-agonist (pAg). Typically, the proliferation of activated Vγ9 / Vδ2T cells can be measured in a cell assay by CFSE or Cell Trace purple staining and flow cytometry of purified Vγ9 / Vδ2T cells extracted from peripheral blood and flow cytometry, or by monitoring the expansion of the Vγ9 / Vδ2T cell compartment within peripheral blood mononuclear cells with or without stimulation.

[0135] In some embodiments, as described below, the anti-BTN2A1 antibodies of the present disclosure activate the cytolytic function of activated Vγ9 / Vδ2 T cells to a level that is substantially equal to or better than that of the reference antibody mAb 107G3. "Activates the cytolytic function of activated Vγ9 / Vδ2 T cells to a level that is substantially equal to that of the reference antibody" herein means that a change of less than 15%, particularly less than 10%, and typically less than 5% in the cytolytic function of activated Vγ9 / Vδ2 T cells of the tested anti-BTN2A1 antibody is observed compared to the reference mAb 107G3.

[0136] In some embodiments, as described below, the anti-BTN2A1 antibodies of the present disclosure activate the production of cytolytic molecules (i.e., at least IFNγ or TNFα) by activated Vγ9 / Vδ2 T cells to a level substantially equal to or better than that of the reference antibody mAb 107G3. "Activates at least IFNγ or TNFα activated by Vγ9 / Vδ2 T cells to a level substantially equal to that of the reference antibody" herein means that a change of less than 15%, particularly less than 10%, and typically less than 5%, in the production of cytolytic molecules by activated Vγ9 / Vδ2 T cells of the tested anti-BTN2A1 antibody is observed compared to the reference mAb 107G3.

[0137] In some embodiments, as described below, the anti-BTN2A1 antibodies of the present disclosure activate the cytolytic function of activated Vγ9 / Vδ2 T cells to a level that is substantially equal to or better than that of the reference antibody mAb 107G3. "Activates the cytolytic function of activated Vγ9 / Vδ2 T cells to a level that is substantially equal to that of the reference antibody" herein means that a change of less than 15%, particularly less than 10%, and typically less than 5% in the cytolytic function of activated Vγ9 / Vδ2 T cells of the tested anti-BTN2A1 antibody is observed compared to the reference mAb 107G3.

[0138] Reference antibodies mAb 101G5, 107G3 and their variants

[0139] The antibodies disclosed herein include reference monoclonal antibody mAb 101G5, which comprises corresponding VH and VL regions as defined in SEQ ID NOs: 19 and 20, respectively, and mAb 107G3, which comprises corresponding VH and VL regions as defined in SEQ ID NOs: 1 and 2, respectively.

[0140] Other antibodies of the disclosure include those having at least 90%, particularly at least 95, 96, 97, 98, 99 or 100% identity to the VH and VL regions as defined in SEQ ID NOs: 1 and 2, respectively, or as defined in SEQ ID NOs: 19 and 20, respectively.

[0141] In certain embodiments, an anti-BTN2A antibody according to the present disclosure, typically a humanized anti-BTN2A1, comprises a heavy chain variable region CDR1 comprising SEQ ID NO: 3, a heavy chain variable region CDR2 comprising SEQ ID NO: 4, a heavy chain variable region CDR3 comprising SEQ ID NO: 5, a light chain variable region CDR1 comprising SEQ ID NO: 6, a light chain variable region CDR2 comprising SEQ ID NO: 7, and a light chain variable region CDR3 comprising SEQ ID NO: 8. In specific 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.

[0142] In other specific embodiments, an anti-BTN2A1 antibody according to the present disclosure, typically a humanized anti-BTN2A1, contains 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.

[0143] In specific embodiments of the antibodies disclosed herein, the six CDR regions are 100% identical to the six CDR regions of the reference mAb 101G5 defined in SEQ ID NOs: 21-26. Other antibodies disclosed herein include those having amino acids mutated by amino acid deletion, insertion, or substitution, but having at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% identity in the CDR regions to the CDR regions of the reference mAb 101G5. Typically, according to the present disclosure, an antibody may have 1, 2, 3, or 4 amino acid variations (including deletions, insertions, or substitutions) in one or more CDRs compared to the CDR sequences of the reference antibody mAb 107G3 defined in SEQ ID NOs: 21-26.

[0144] In other specific 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. Other antibodies disclosed herein include those having amino acids mutated by amino acid deletions, insertions, or substitutions, but having at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% identity in the CDR regions to the CDR regions of the reference mAb 107G3. Typically, according to the present disclosure, an antibody may have 1, 2, 3, or 4 amino acid variations (including deletions, insertions, or substitutions) in one or more CDRs compared to the CDR sequences of the reference antibody mAb 107G3 defined in SEQ ID NOs: 3-8.

[0145] In some embodiments, an antibody of the present disclosure is a mutant variant of mAb 101G5 or mAb 107G3, the six CDR regions of which are 100% identical to the corresponding six CDR regions of reference mAb 101G5 or 107G3, respectively, and wherein the mutant variant antibody comprises a mutant amino acid sequence in which no more than 1, 2, 3, 4, or 5 amino acids are mutated by deletion, insertion, or substitution of amino acids in the FR1, FR2, FR3, and FR4 regions, as compared to the corresponding framework regions of the corresponding reference antibody.

[0146] Functional variant antibodies

[0147] As shown by the experimental data provided 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 N° 17, and most particularly comprising amino acid residues located at positions 65, 68, 69, 72, 78, 84, 85, 95, 97, 100 of SEQ ID N° 17, and that have one or more functional properties as previously defined and as further reminded below, in particular having one or more functional properties of the reference mAb 107G3.

[0148] As also shown by the experimental data provided 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 N° 17, and most particularly comprising residues located at positions 212, 213, 218, 220, 224, 229 of SEQ ID N° 17, and that possess one or more functional properties as previously defined and as further reminded below, in particular possess one or more functional properties of the reference mAb 101G5.

[0149] In other embodiments, the functional variant antibodies of the present disclosure have full-length heavy and light chain amino acid sequences; or variable region heavy and light chain amino acid sequences, or all six CDR region amino acid sequences that are homologous or, more specifically, identical to the corresponding amino acid sequences of any of the above-mentioned reference antibodies mAb 101G5 or mAb 107G3, and wherein such functional variant antibodies retain the desired functional properties of the reference antibody.

[0150] Functional variants of the reference mAb 101G5 antibody or the reference antibody mAb 107G3, particularly functional variants of the VL, VH or CDRs used in the context of the monoclonal antibodies of the present disclosure, still allow the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity (typically K as measured by surface plasmon resonance (SPR)) of the parent antibody (e.g., mAb 101G5 or mAb 107G3). D Evaluation, or using biolayer interferometry (BLI) technology The monoclonal antibodies of the present disclosure may be characterized by higher affinity, selectivity and / or specificity than the parent Ab (e.g., mAb 101G5 or mAb 107G3).

[0151] The desired functional properties of the reference mAb 101G5 or 107G3 as disclosed herein, or variants of said reference antibodies, may be selected from the group consisting of:

[0152] i. Specificity for BTN2A1, in particular binding to human BTN2A1 expressed in a cell line, such as HEK-293T BTN2KO cells transfected with a plasmid encoding human BTN2A1, as described in the Examples, more particularly at an EC value of less than 50 μg / mL, more particularly less than 40 μg / mL 50, or as measured by surface plasmon resonance (SPR) (typically at 25° C.) or Luminex (as shown in the Examples) or (Abdiche et al. 2008) measured K values ​​of 10 nM or less D and / or it binds to BTN2A1 with an affinity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10000:1 or greater compared to non-specific binding; and / or

[0153] ii. inhibiting the polarization of monocytes to M2 macrophages, typically assessed as described in the Examples section, and / or

[0154] iii. Inducing the reversion of M2 macrophages to anti-tumor M1 macrophages, typically assessed as described in the Examples section, and / or

[0155] iv. directly triggering NK cell activation, typically assessed as described in the Examples section, and / or

[0156] v. Enhancement of NK cell-mediated cytotoxicity, typically assessed as described in the Examples section.

[0157] In some more specific embodiments, the desired functional properties of the reference mAb 107G3 disclosed herein or a variant of said reference antibody may also be selected from the group consisting of:

[0158] vi. cytolytic molecules (e.g., IFNγ or TNFα) produced by activated Vγ9 / Vδ2 T cells, typically assessed as described in the Examples,

[0159] vii. activating the cytolytic function of Vγ9 / Vδ2 T cells, typically assessed as shown in the Examples; and / or

[0160] viii. Activation of Vγ9 / Vδ2 T cell proliferation, typically assessed as described in the Examples.

[0161] Typically, the functional properties of the functional variants of the reference mAb 101G5 or 107G3 according to points (ii) to (v) above are substantially equal to or better than the corresponding functional properties of the corresponding reference antibodies mAb 101G5 or 107G3 as described above. Substantially equal in this context means 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.

[0162] Typically, the functional properties of the functional variants of the reference mAb 107G3 according to points (vi) to (viii) above are substantially equal to or better than the corresponding functional properties of the corresponding reference antibody mAb 107G3 as described above. Substantially equal in this context means 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.

[0163] For example, the present disclosure relates to functional variant antibodies of reference mAb 101G5 comprising a variable heavy chain (V H ) and variable light chain (V L ) sequence, wherein the CDR sequences, i.e., the six CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, share at least 60, 70, 90, 95, or 100 percent sequence identity with the corresponding CDR sequences of the mAb 101G5 reference antibody defined in SEQ ID NOs: 21-26, wherein the functional variant antibody specifically binds to BTN2A, and the antibody exhibits at least one of the following functional properties i)-iv):

[0164] i. It inhibits the polarization of monocytes to M2 macrophages,

[0165] ii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages,

[0166] iii. It directly triggers NK cell activation,

[0167] iv. It enhances NK cell-mediated cytotoxicity.

[0168] Preferably, it exhibits at least one of characteristics i) and ii) and at least one of characteristics iii and iv), most preferably exhibits characteristics i) to iv).

[0169] The present disclosure also relates to functional variant antibodies of reference mAb 107G3 comprising a variable heavy chain (V H ) and variable light chain (V L ) sequence, wherein the CDR sequences, i.e., the six CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, share at least 60, 70, 90, 95, or 100 percent sequence identity with the corresponding CDR sequences of the mAb 107G3 reference antibody defined in SEQ ID NOs: 3-8, wherein the functional variant antibody specifically binds to BTN2A, and the antibody exhibits at least one of the following functional properties i)-iv):

[0170] i. It inhibits the polarization of monocytes to M2 macrophages,

[0171] ii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages,

[0172] iii. It directly triggers NK cell activation,

[0173] iv. it enhances NK cell-mediated cytotoxicity,

[0174] v. It activates Vγ9 / Vδ2T cells to produce IFNγ or TNFα,

[0175] vi. It activates the cytolytic function of Vγ9 / Vδ2 T cells,

[0176] vii. It activates the proliferation of Vγ9 / Vδ2 T cells.

[0177] Preferably, the 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).

[0178] It also relates to functional variant antibodies of the mAb 101G5 reference antibody, comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding heavy chain and light chain variable regions of said mAb 101G5 reference antibody defined in SEQ ID NOs: 19 and 20, respectively; the functional variant antibodies specifically bind to BTN2 and exhibit at least one of the following functional properties:

[0179] i. It inhibits the polarization of monocytes to M2 macrophages,

[0180] ii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages,

[0181] iii. It directly triggers NK cell activation,

[0182] iv. It enhances NK cell-mediated cytotoxicity.

[0183] Preferably, it exhibits at least one of characteristics i) and ii) and at least one of characteristics iii and iv), most preferably exhibits characteristics i) to iv).

[0184] It also relates to functional variant antibodies of the mAb 107G3 reference antibody, comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding heavy chain and light chain variable regions of said mAb 107G3 reference antibody defined in SEQ ID NOs: 1 and 2, respectively; the functional variant antibodies specifically bind to BTN2 and exhibit at least one of the following functional properties:

[0185] i. It inhibits the polarization of monocytes to M2 macrophages,

[0186] ii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages,

[0187] iii. It directly triggers NK cell activation,

[0188] iv. it enhances NK cell-mediated cytotoxicity,

[0189] v. its activation produces cytolytic molecules (IFNγ or TNFα) through Vγ9 / Vδ2 T cells, vi. its activation of the cytolytic function of Vγ9 / Vδ2 T cells, and / or

[0190] vii. It activates the proliferation of Vγ9 / Vδ2 T cells.

[0191] In some embodiments, the functional variant exhibits at least one of the functional activities i) to iv) (particularly at least activities i and / or ii, and iii and / or iv) and at least one of the functional activities v) to vii).

[0192] In some embodiments, the functional variant exhibits one or more functional activities i to iv or one or more functional activities v to vii.

[0193] Typically, the functional properties of the functional variants of the reference mAb 101G5 or 107G3 according to points (i) to (iv) above are substantially equal to or better than the corresponding functional properties of the corresponding reference antibodies mAb 101G5 or 107G3 as described above. Substantially equal in this context means 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.

[0194] In various embodiments, the antibodies may exhibit one or more of the desired functional properties discussed above.

[0195] For example, the antibody can be a human antibody, a humanized antibody or a chimeric antibody. Typically, the antibody or protein is a humanized antibody, more particularly a humanized silent antibody.

[0196] As used herein, the term "silent" antibody refers to an antibody with no or low ADCC activity as measured in an in vitro ADCC activity assay that measures cell lysis of target cells.

[0197] In one embodiment, the term "no or low ADCC activity" refers to an ADCC activity exhibited by a silent antibody that is less than 50%, for example, less than 10%, of the ADCC activity observed for a corresponding wild-type (non-silent) antibody (e.g., a wild-type human IgG1 antibody). Typically, no detectable ADCC activity is observed in an in vitro ADCC activity assay for the silent antibody compared to a control Fab antibody.

[0198] Silent effector functions can be obtained by mutations in the constant Fc portion of an antibody and have been described in the art: Strohl 2009 (LALA & N297A); Baudino 2008, D265A (Baudino et al., J. Immunol. 2008, Strohl, CO Biotechnology 20 2009). Examples of silent IgG1 antibodies include mutations that reduce ADCC at positions 234, 235, and / or 331 of the IgG1 Fc amino acid sequence (EU numbering). Another silent IgG1 antibody includes the N297A mutation, which results in a glycosylated or non-glycosylated antibody.

[0199] The sequence of a CDR variant may differ from the CDR sequence of the parent antibody sequence by a majority of conservative substitutions, e.g., at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the variant are conservative amino acid residue replacements. In the context of the present disclosure, conservative substitutions can be defined as substitutions within the amino acid class as reflected below:

[0200] Aliphatic residues I, L, V and M.

[0201] Cycloalkenyl related residues F, H, W and Y.

[0202] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0203] Negatively charged residues D and E.

[0204] Polar residues C, D, E, H, K, N, Q, R, S and T.

[0205] Positively charged residues H, K, and R.

[0206] Small residues A, C, D, G, N, P, S, T and V.

[0207] Very small residues A, G and S.

[0208] Residues A, C, D, E, G, H, K, N, Q, R, S, P involved in the turn and residue T involved in the formation.

[0209] Flexible residues Q, T, K, S, G, P, D, E, and R.

[0210] More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Compared to any of the CDRs of mAb1-6, the conservation of hydropathic / hydrophilic properties and residue weight / size is substantially retained in the variant CDRs. The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is generally understood in the art. It is accepted that the relative hydropathicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Based on their hydrophobicity and charge characteristics, each amino acid is assigned a hydropathic index, which are: 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). The retention of similar residues can also or alternatively be measured by a similarity score, such as determined using a BLAST program (e.g., BLAST 2.2.8 available through 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. According to the present disclosure, a first amino acid sequence having at least 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 to the second amino acid sequence. According to the present disclosure, a first amino acid sequence that is at least 50% identical to a second amino acid sequence means that the first sequence is 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% identical to the second amino acid sequence.

[0211] In some embodiments, the antibodies of the present disclosure are chimeric antibodies, typically chimeric mouse / human antibodies. 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. As the non-human animal, any animal such as mouse, rat, hamster, rabbit, etc. can be used. In particular, the mouse / human chimeric antibody can comprise the VH and VL domains of either mAb 101G5 or mAb 107G3 reference antibodies.

[0212] In some embodiments, the antibodies of the present disclosure are humanized antibodies. In specific embodiments, the antibodies of the present disclosure are humanized antibodies comprising the six CDRs of any one of the mAb 101G5 or mAb 107G3 reference antibodies. As used herein, the term "humanized antibody" refers to an antibody in which the framework regions (FRs) have been modified to comprise FRs from a donor immunoglobulin of a different species (e.g., human race) than the parent immunoglobulin (e.g., murine CDRs).

[0213] In some embodiments, the antibodies of the present disclosure are selected from the group consisting of Fab, F(ab')2, Fab', and scFv. As used herein, the term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, wherein in a fragment obtained by treating IgG with a protease (papain), the N-terminal side of about half of the H chain and the entire L chain are bound together by a disulfide bond. The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, which is slightly larger than the Fab bound by a disulfide bond in the hinge region in a fragment obtained by treating IgG with a protease (pepsin). The term "Fab'" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of F(ab')2. Single-chain Fv ("scFv") polypeptides are covalently linked VH:VL heterodimers, which are typically expressed by a gene fusion comprising VH and VL encoding genes connected by a peptide-encoded linker. The human scFv fragments of the present disclosure include CDRs that are typically maintained in proper conformation using genetic recombination techniques.

[0214] Functional variant antibodies having mutant amino acid sequences can be obtained by mutagenesis of the encoding nucleic acid molecule (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) and then testing the encoded altered antibody for retained function (i.e., the function described above) using the functional assays described herein.

[0215] Antibodies that cross-compete with the reference 101G5 or 107G3 mAbs

[0216] Additional antibodies with similar favorable properties as reference mAb 101G5 or reference mAb 107G3 disclosed herein can be identified in standard BTN2A1 binding assays based on their ability to cross-compete (e.g., competitively inhibit binding) with reference mAb 107G3 described above in a statistically significant manner.

[0217] For example, test antibodies can first be screened for binding affinity to BTN2A1 from human recombinant antibody libraries using, for example, phage display technology or from transgenic mice expressing human variable region antibodies immunized with BTN2A1 antigen, as typically assessed in the Examples (see Materials and Methods section).

[0218] In another embodiment, the present disclosure provides antibodies that bind to the same epitope as at least the above-mentioned reference mAb 107G3 or reference mAb 101G5. As shown in the Examples section, reference mAbs 101G5 and 107G3 do not bind to the same epitope on BTN2A1.

[0219] The ability of a test antibody to cross-compete or inhibit binding of an antibody of the disclosure to human BTN2A1 confirms that the test antibody can compete with the antibody for binding to human BTN2A1. According to non-limiting theory, the antibody may bind to the same or related (e.g., structurally similar or spatially proximal) epitope on human BTN2A1 as the antibody with which it competes.

[0220] For example, the following test can be used to screen anti-BTN2A1 antibodies for their ability to cross-compete with the mAb 107G3 reference antibody and / or 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 tested for their competitive potential with the mAb 107G3 reference antibody. In the presence of such a reference antibody, mAbs that do compete with the reference antibody will not 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 providing a reference antibody followed by a competing antibody.

[0221] Selected antibodies can be further tested for the favorable properties of mAb 101G5 or mAb 107G3, particularly as previously detailed.

[0222] Thus, in one embodiment, the present disclosure provides an isolated antibody that competes for binding to BTN2A1 with reference mAb 101G5 or reference mAb 107G3, wherein

[0223] The antibody:

[0224] i. having specificity for BTN2A1, in particular binding to human BTN2A1 expressed in a cell line, such as HEK-293T BTN2 KO cells transfected with a plasmid encoding human BTN2A1, as described in the Examples, more particularly at an EC value of less than 50 μg / mL, more particularly less than 40 μg / mL. 50 , or as determined by surface plasmon resonance (SPR) or Luminex (as shown in the Examples) or (Abdiche et al. 2008) measured K values ​​of 10 nM or less D and / or it binds to BTN2A1 with an affinity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10000:1 or greater compared to non-specific binding (see also above for further details); and / or

[0225] ii. It inhibits the polarization of monocytes to M2 macrophages

[0226] iii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages

[0227] iv. It directly triggers NK cell activation, and / or

[0228] v. It enhances NK cell-mediated cytotoxicity.

[0229] Alternatively or further, the antibody:

[0230] vi. activation of Vγ9 / Vδ2 T cells to produce cytolytic molecules (e.g., IFNγ or TNFα), typically assessed as described in the Examples, and / or

[0231] vii. activating the cytolytic function of Vγ9 / Vδ2 T cells, typically assessed as shown in the Examples; and / or

[0232] viii. Activation of Vγ9 / Vδ2 T cell proliferation, typically assessed as described in the Examples.

[0233] More specifically, in such embodiments, the antibody does not cross-react with BTN3.

[0234] Typically, the functional properties of the antibodies that compete with the reference mAb 101G5 or 107G3 for binding to BTN2A1 according to points (iii) to (v) above are substantially equal to or better than the corresponding functional properties of the corresponding reference antibodies mAb 101G5 or 107G3 as described above. Substantially equal in this context means 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.

[0235] Typically, an antibody that competes for binding to BTN2A1 with the reference mAb 107G3 according to the present disclosure retains at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity and / or selectivity of 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).

[0236] In a certain embodiment, the cross-blocking antibody or the antibody that competes with reference mAb 101G5 or 107G3 for binding to BTN2A1 is a chimeric antibody, a humanized antibody, or a human recombinant antibody.

[0237] Generation of monoclonal antibody-producing transfectomas

[0238] Antibodies of the present disclosure are produced by any technology known in the art, such as, but not limited to, any single or combined chemical, biological, genetic or enzymatic techniques. Typically, given the amino acid sequence of the desired sequence, those skilled in the art can easily produce the antibodies by standard techniques for producing polypeptides. For example, they can be synthesized using well-known solid phase methods, typically using commercially available peptide synthesis equipment (such as manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, antibodies of the present disclosure can be synthesized by recombinant DNA techniques well known in the art. For example, after the DNA sequence encoding the antibody is integrated into an expression vector and these vectors are introduced into a suitable eukaryotic or prokaryotic host that will express the desired antibody, antibodies as DNA expression products can be obtained, which can then be separated using known techniques.

[0239] Therefore, another object of the present disclosure relates to nucleic acid molecules encoding antibodies according to the present disclosure. More particularly, the nucleic acid molecules encode the heavy or light chains of the antibodies disclosed herein. More particularly, the nucleic acid molecules comprise a VH or VL coding region that is at least 70%, 80%, 90%, 95% or 100% identical to the corresponding nucleic acid encoding the heavy chain variable region (VH region) or light chain variable region (VL) of any one of the reference antibodies mAb 107G3.

[0240] Typically, the nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, a cosmid, an episome, an artificial chromosome, a phage or a viral vector. As used herein, the terms "vector," "cloning vector," and "expression vector" refer to a medium that can introduce a DNA or RNA sequence (e.g., an exogenous gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Therefore, another object of the present disclosure relates to a vector comprising the nucleic acid disclosed herein. Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the antibody when administered to a subject. Examples of promoters and enhancers for expression vectors of animal cells include early promoters and enhancers of SV40, LTR promoters and enhancers of Moloney murine leukemia virus, promoters and enhancers of immunoglobulin H chains, etc. Any expression vector for animal cells can be used, as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, etc. Other examples of plasmids include replicating plasmids containing a replication origin, or integrating plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenovirus, retrovirus, herpes virus, 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 with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. For example, detailed protocols for producing such replication-defective recombinant viruses can be found in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056, and WO 94 / 19478.

[0241] A further object of the present disclosure relates to host cells transfected, infected or transformed by the above-mentioned nucleic acids and / or vectors. As used herein, the term "transformation" refers to the introduction of "exogenous" (i.e., external or extracellular) genes, DNA or RNA sequences into a host cell, whereby the host cell will express the introduced genes or sequences to produce a desired substance, typically a protein or enzyme encoded by the introduced genes or sequences. A host cell that accepts and expresses the introduced DNA or RNA is "transformed."

[0242] The nucleic acids disclosed herein can be used to produce 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 example, a protein encoded by an exogenous DNA carried by a vector and introduced into a host cell. Common expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, as well as mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or yeast, mammalian cell lines (such as HEK-293 cells, Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established mammalian cell cultures (for example, produced by lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neurons, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is deficient (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.

[0243] The present disclosure also relates to a method for producing recombinant host cells expressing the antibodies disclosed herein, the method comprising the following steps: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo, (ii) culturing the obtained recombinant host cells in vitro or ex vivo, and (iii) optionally, selecting cells that express and / or secrete the antibody. These recombinant host cells can be used to produce the antibodies disclosed herein.

[0244] The antibodies of the disclosure are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0245] In some embodiments, the human chimeric antibodies of the present disclosure can be produced by obtaining the nucleic acid sequences encoding the VL and VH domains as described above, constructing a human chimeric antibody expression vector by inserting them into an expression vector of an animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of the human chimeric antibody, it can be any region belonging to human immunoglobulin, but those regions of the IgG class are suitable, and any subclass of the IgG class, such as IgG1, IgG2, IgG3, and IgG4, can also be used. In addition, as the CL of the human chimeric antibody, it can be any region belonging to Ig, and regions of the κ class or λ class can also be used. The method for producing chimeric antibodies involves conventional recombinant DNA, and gene transfection technology is well known in the art (see Morrison SL et al. (1984) and patent documents US5,202,238; and US5,204,244).

[0246] Humanized antibodies of the present disclosure can be produced by the following: obtain the nucleic acid sequence of coding CDR domains as aforementioned, construct humanized antibody expression vector by inserting them into the expression vector with the following gene encoding: (i) heavy chain constant region and heavy chain variable framework region identical with human antibody, and (ii) light chain constant region light chain variable framework region identical with human antibody, and express gene by introducing expression vector into suitable cell line.Humanized antibody expression vector can be such type: the gene wherein encoding antibody heavy chain and the gene encoding antibody light chain are present on different vectors, or two kinds of genes are present on the same vector (tandem type).Considering the ease of building humanized antibody expression vector, the ease of introducing cell line, and the balance between the expression level of antibody H and L chain in cell line, preferably tandem type humanized antibody expression vector.The example of tandem type humanized antibody expression vector includes pKANTEX93 (WO 97 / 10354), pEE18 etc.

[0247] Methods for humanizing antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al., 1988; Neuberger MS et al., 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400, PCT Publication WO91 / 09967, U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or remodeling (EP 592,106, EP 519,596, Padlan EA (1991), Studnicka GM et al. (1994), Roguska MA et al. (1994)) and chain replacement (U.S. Patent No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0248] Fab of the present disclosure can be obtained by processing the antibody of AMH specific reaction with protease (papain).In addition, Fab can be produced by following: DNA encoding antibody Fab is inserted into the vector for prokaryotic expression system or for eukaryotic expression system, and the vector is introduced into prokaryotic organism or eukaryotic organism (depending on the situation) to express Fab.

[0249] The F(ab')2 of the present disclosure can be obtained by treating an antibody that specifically reacts with AMH with a protease (pepsin). In addition, F(ab')2 can be produced by binding the following Fab' with a thioether bond or a disulfide bond.

[0250] Fab' of the present disclosure can be obtained by treating F(ab')2 that specifically reacts with AMH with a reducing agent (dithiothreitol). In addition, Fab' can be produced by inserting a DNA encoding the Fab' fragment of an antibody into an expression vector of a prokaryotic organism or an expression vector of a eukaryotic organism, and introducing the vector into a prokaryotic organism or a eukaryotic organism (as the case may be) for expression.

[0251] The scFv of the present disclosure can be produced by obtaining cDNA encoding the VH and VL domains as described above, constructing a DNA encoding the scFv, inserting the DNA into an expression vector for prokaryotes or an expression vector for eukaryotes, and then introducing the expression vector into prokaryotes or eukaryotes (as appropriate) to express the scFv.

[0252] To prepare 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 human scFv fragment framework of known three-dimensional structure (e.g., see WO98 / 45322, WO87 / 02671, US5,859,205, US5,585,089, US4,816,567, EP0173494).

[0253] The engineered antibodies of the present disclosure further include antibodies in which framework residues within VH and / or VL are modified, for example, to improve antibody properties. Typically, the framework modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to "revert" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence of the derived antibody. These residues can be identified by comparing the antibody framework sequence with the germline sequence of the derived antibody. In order to restore the framework region sequence to its germline configuration, the somatic mutation can be "reverted" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "reverted" antibodies are also encompassed in the present disclosure. Another type of framework modification involves mutating one or more residues within the framework region or even within one or more 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 further described in detail in U.S. Patent Publication No. 20030153043 to Carr et al.

[0254] Fc engineering

[0255] The antibodies of the present disclosure may be characterized by one or more of the functional or structural features of the aspects described above, or by any combination of selected functional and structural features.

[0256] The antibodies disclosed herein can be of any isotype. The choice of isotype is typically guided by the desired effector function (such as ADCC silencing). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Any one of the human light chain constant regions, κ or λ, can be used. If necessary, the antibody class of the present disclosure can be converted by known methods. Typically, class switching technology can be used to convert one IgG subclass into another, for example, from IgG1 to IgG2. Therefore, for various therapeutic uses, the effector function of the antibodies disclosed herein can be changed by isotype conversion to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. In some embodiments, the antibodies disclosed herein are full-length antibodies. In some embodiments, the full-length antibodies are IgG1 antibodies. In some embodiments, the full-length antibodies are IgG4 antibodies. In some embodiments, the BTN2A-specific IgG4 antibodies are stabilized IgG4 antibodies. Examples of suitable stabilized IgG4 antibodies are those in which the arginine at position 409 in the heavy chain constant region of human IgG4 (as shown in the EU index of Kabat et al., supra) is substituted with lysine, threonine, methionine or leucine, typically lysine (described in WO2006033386) and / or in which the hinge region comprises a Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO2008145142.

[0257] In some embodiments, the antibodies of the present disclosure do not comprise an Fc portion that induces antibody-dependent cellular cytotoxicity (ADCC). The terms "Fc domain," "Fc portion," and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, for example, a human γ heavy chain from about amino acid (aa) 230 to about aa 450, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε, and μ of a human antibody), or a naturally occurring allotype thereof. Unless otherwise indicated, the generally accepted Kabat amino acid numbering of immunoglobulins is used in 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 that is capable of substantially binding to an FcgRIIIA (CD16) polypeptide. In some embodiments, the antibodies of the present disclosure lack an Fc domain (e.g., lack a CH2 and / or CH3 domain) or comprise an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the antibodies of the present disclosure are composed of or comprise the following: Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, single-chain antibody fragments, or multispecific antibodies comprising a variety of different antibody fragments. In some embodiments, the antibodies of the present disclosure are not connected to a toxic portion. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with different amino acid residues so that the antibody has altered C2q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). U.S. Patent No. 6,194,551 further describes this approach in detail.

[0258] Another modification of the present antibody contemplated herein is pegylation. For example, the antibody can be pegylated to increase the biological (e.g., serum) half-life of the antibody. In order to pegylate the antibody, the antibody or its fragment is typically reacted with polyethylene glycol (PEG) under conditions where one or more PEG groups become connected to the antibody or antibody fragment, such as reactive esters or aldehyde derivatives of PEG. Pegylation can be carried out by acylation reaction or alkylation reaction with reactive PEG molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG for deriving other proteins, such as single (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is an antibody without glycosylation. Methods for pegylating proteins are known in the art and can be applied to antibodies of the present disclosure. For example, see EP 0154316 of Nishimura et al. and EP 0 401 384 of Ishikawa et al.

[0259] Another modification of the antibodies contemplated herein is a conjugate or protein fusion of at least the antigen binding region of an antibody of the disclosure with a serum protein, such as human serum albumin or a fragment thereof, to increase the half-life of the resulting molecule.

[0260] In some embodiments, the present disclosure also provides multispecific antibodies. Exemplary forms of multispecific antibody molecules disclosed herein include, but are not limited to, (i) two antibodies cross-linked by chemical heterologous conjugation, one specific for BTN2A and the other specific 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, for example, two scFvs connected in series by an additional peptide linker; (iv) a dual variable domain antibody (DVD-Ig), in which each light chain and heavy chain contains two variable domains connected in series by a short peptide (Wu et al. Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg 2010); (v) chemically linked bispecific (Fab')2 fragments; (vi) Tandab, which is a fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vii) flexibody, which is a combination of scFv and diabody, resulting in a multivalent molecule; (viii) so-called "dock and lock" molecules based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (ix) so-called Scorpion molecules, which contain, for example, two scFvs fused to the two ends of a human Fab arm; and (x) diabodies. Another exemplary form of a bispecific antibody is an IgG-like molecule with complementary CH3 domains to drive heterodimerization. These molecules can be prepared using known techniques, such as those known as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatic matching (Amgen), LUZ-Y (Genentech), chain exchange engineered domain bodies (SEEDbody) (EMDSerono), Biclonic (Merus) and DuoBody (Genmab A / S) technologies. In some embodiments, bispecific antibodies are typically obtained or obtainable by controlled Fab arm exchange using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab arm exchange have been described in WO2008119353 and WO 2011131746 (both by Genmab A / S). In an exemplary method described in WO 2008119353, bispecific antibodies are formed by "Fab-arm" or "half-molecule" exchange (exchanging the heavy chain and the linked light chain) between two monospecific antibodies, each containing an IgG4-like CH3 region, when incubated under reducing conditions. The resulting product is a bispecific antibody with two Fab arms, which can contain different sequences.In another exemplary method described in WO 2011131746, a bispecific antibody of the present disclosure is prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is an antibody of the present disclosure: a) providing a first antibody comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a second CH3 region; wherein the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between the first and second CH3 regions; c) incubating the first antibody and the second antibody under reducing conditions; and d) obtaining the bispecific antibody, wherein the first antibody is an antibody of the present disclosure and the second antibody has a different binding specificity, or vice versa. For example, reducing conditions can be provided by adding a reducing agent, for example, selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) can further include restoring the conditions to become non-reducing or less reducing, for example, by removing the reducing agent, for example, by desalting. Typically, the first and second CH3 regions differ in sequence, containing only a few relatively conservative asymmetric mutations, such that the heterodimer interaction between the first and second CH3 regions is stronger than the individual homodimer interactions between the first and second CH3 regions. WO 2011131746, incorporated herein by reference in its entirety, provides further details on these interactions and how they are achieved. The following are exemplary embodiments of such asymmetric mutation combinations, optionally wherein one or both Fc regions are of the IgG1 isotype.

[0261] Thus, the present disclosure provides bispecific or multispecific antibodies (also referred to as bispecific or multispecific molecules) comprising the anti-BTN2A antibodies described herein. Thus, the present disclosure includes bispecific molecules comprising at least one first binding specificity for BTN2A (e.g., an antigen-binding portion of an antibody disclosed herein) and a second binding specificity for a second target epitope. For example, a bispecific molecule according to the present disclosure may include an antigen-binding portion comprising at least:

[0262] - a heavy chain variable region CDR1 comprising SEQ ID NO: 3, a heavy chain variable region CDR2 comprising SEQ ID NO: 4, a heavy chain variable region CDR3 comprising SEQ ID NO: 5, a light chain variable region CDR1 comprising SEQ ID NO: 6, a light chain variable region CDR2 comprising SEQ ID NO: 7, and a light chain variable region CDR3 comprising SEQ ID NO: 8, or

[0263] - 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.

[0264] In one embodiment, the bispecific molecule comprises a second binding specificity for BTN3. More specifically, the bispecific molecule may further comprise an antigen-binding portion of an anti-BTN3A activating antibody that specifically binds to BTN3A and activates the cytolytic function of Vγ9 / Vδ2 T cells.

[0265] Furthermore, for embodiments in which the bispecific molecule is multispecific, the molecule can include a third binding specificity in addition to the first and second target epitopes.

[0266] In one embodiment, the bispecific molecules disclosed herein comprise at least one antibody or antibody fragment thereof as a binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, Unibody, or single-chain Fv. The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain construct as described in Ladner et al., U.S. Patent No. 4,946,778.

[0267] Other antibodies that can be used in the bispecific molecules disclosed herein are murine, chimeric, and humanized monoclonal antibodies.

[0268] Pharmaceutical composition

[0269] In another aspect, the present disclosure provides compositions, such as pharmaceutical compositions, comprising at least one antibody as disclosed herein formulated together with a pharmaceutically acceptable carrier. Such compositions may comprise a combination of (e.g., two or more different) antibodies as described above. The pharmaceutical compositions disclosed herein may also be administered in combination therapy, i.e., in combination with other agents.

[0270] For example, the antibodies of the invention are typically combined with at least one antiviral 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.

[0271] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous route.

[0272] Depending on the route of administration, the active compound (i.e., antibody) may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The form of the pharmaceutical composition, route of administration, dosage, and regimen will naturally depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc.

[0273] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, among others.

[0274] Uses and methods of the present invention

[0275] The antibodies disclosed herein have in vitro and in vivo diagnostic and therapeutic utility. For example, these molecules can be administered to cells in culture, e.g., in vitro, ex vivo, or in vivo, or to subjects, e.g., in vivo, to treat, prevent, or diagnose a variety of disorders.

[0276] The antibodies of the present disclosure are anti-BTN2A1 antibodies that inhibit the differentiation of monocytes into tumor-promoting M2 macrophages in terms of phenotype, cytokine secretion and / or T cell suppressive properties.

[0277] Alternatively, or preferably in addition, the antibodies of the present disclosure bind directly to BTN2A (particularly BTN2A1) at the plasma membrane of NK cells and trigger their activation and cytotoxicity towards cancer cells.

[0278] In some embodiments, the antibodies of the present disclosure can further activate the cytolytic function, cytokine production, and proliferation of Vγ9 / Vδ2 T cells.

[0279] Thus, the antibodies of the present disclosure can be used to overcome immunosuppressive mechanisms observed in cancer patients and during chronic infections.

[0280] In some embodiments, the antibodies of the present disclosure can be used to reduce the immunosuppressive effects of the tumor environment.

[0281] The anti-BTN2A antibodies disclosed herein may also enhance the cytotoxic effects of NK and / or Th1 cells by acting on the tumor microenvironment (through M1 polarization and / or M2 suppression) and directly on the NK cell compartment.

[0282] In some embodiments, the antibodies of the present disclosure (e.g., the 107G3 antibody and variants thereof described herein) further activate the cytolytic function, cytokine production, and proliferation of Vγ9 / Vδ2 T cells. Thus, such antibodies have the potential to act in combination on the three cellular compartments of immunity: NK cells, macrophages, and γδ T cells, and therefore represent a powerful tool for cancer treatment, especially for solid tumor treatment.

[0283] Preclinical studies have demonstrated that NK cells can kill myeloid leukemia cells. However, in CML, for example, the number of NK cells decreases as the disease progresses, their response to stimulation weakens, and their cytolytic activity decreases. In AML, higher cytolytic activity of NK cells also predicts better long-term prognosis for patients at diagnosis and during remission (Carlsten M, M. Natural Killer Cells in Myeloid Malignancies: Immune Surveillance, NK Cell Dysfunction, and Pharmacological Opportunities to Bolster the Endogenous NK Cells. Front Immunol. 2019). Therefore, in some embodiments, the antibodies of the present invention that exhibit NK cell activation properties can be used in combination with NK cell therapy (such as adoptive transfer NK cell therapy) to restore NK cell function and / or trigger or improve their cytotoxicity. In particular, the antibodies of the present application can be used to treat solid tumors that are generally resistant to NK cell killing. As used herein, the terms "cancer", "hyperproliferative" and "tumorous" refer to cells with spontaneous growth ability, i.e., abnormal states or conditions characterized by rapidly proliferating cell growth. Hyperproliferation and tumor disease states can be classified as pathological, i.e., characterizing or constituting a disease state, or can be classified as non-pathological, i.e., deviating from normal but unrelated to a disease state. The term is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0284] The terms "cancer" or "tumor" include malignancies of various organ systems, for example, those affecting the lung, breast, thyroid, lymphoid, gastrointestinal and genitourinary tracts, as well as malignancies such as most colon cancers, squamous cell carcinomas of the lung, skin or vagina, renal cell carcinoma, prostate and / or testicular tumors, non-small cell lung cancer, small cell lung cancer, endometrial cancer, ovarian cancer, endocervical adenocarcinoma, pancreatic cancer, small intestine cancer and esophageal cancer, and more generally any cancer that can be treated by stimulating the activation and / or proliferation of γδ T cells in vivo in a subject suffering from such cancer.

[0285] Examples of cancer include, but are not limited to, hematological malignancies, such as B-cell lymphomas, T-cell lymphomas, non-Hodgkin lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK-cell lymphomas, and myeloid neoplasms, including acute myeloid leukemia.

[0286] 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.

[0287] 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.

[0288] Therefore, the present disclosure relates to a method of treating one of the disorders disclosed above in a subject in need thereof, said method comprising a therapeutically effective amount of an anti-BTN2A1 antibody as disclosed above.

[0289] The antibodies disclosed above can be used as the sole active ingredient or in combination with, for example, an adjuvant or with, for example, other drugs for the treatment or prevention of the above-mentioned diseases, such as cytokines, antiviral agents, anti-inflammatory agents or cytotoxic agents, antiproliferative agents, chemotherapeutic agents or anti-tumor agents, cell therapy products (e.g., γδ T cell compositions or NK cell compositions).

[0290] For example, the antibodies used as disclosed above can be used in combination with cell therapy, in particular γδ T cell therapy, NK cell therapy, chemotherapy, anti-tumor agents or immunotherapeutic agents.

[0291] As used herein, the term "cell therapy" refers to a therapy comprising administering to a subject in need thereof at least one therapeutically effective amount of a cell composition. The cells administered to the patient can be allogeneic or autologous. The term "γδT cell therapy" refers to a cell therapy in which the cell composition includes γδT cells as an active ingredient, particularly Vγ9 / Vδ2T cells (e.g., adoptive γδT cell transfer or γδT cells expressing a chimeric antigen receptor). The term "NK cell therapy" refers to a cell therapy in which the cell composition includes NK cells as an active ingredient, such as adoptive NK cell transfer or NK cells expressing a chimeric antigen receptor (CAR-NK).

[0292] Cell therapy products refer to cell compositions administered to the patient for therapeutic purposes, including therapeutically effective doses of cells and optional additional excipients, adjuvants, or other pharmaceutically acceptable carriers.

[0293] Suitable anti-tumor agents may include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosoureas, temozolomide), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), taxanes (e.g., paclitaxel, docetaxel), epothilones, topoisomerase I inhibitors (e.g., irinotecan or topotecan), topoisomerase II inhibitors (e.g., etoposide, teniposide, or taflutoposide), nucleotide analogs and precursor analogs (e.g., azacitidine, azathioprine, capecitabine, cytarabine, fluoroquinolone, cytosine ... uracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, or thioguanine), peptide antibiotics (such as carboplatin, cisplatin, and oxaliplatin), retinoids (such as tretinoin, alitretinoin, and bexarotene), vinca alkaloids and their derivatives (such as vinblastine, vincristine, vinblastine, and vinorelbine), targeted therapies such as kinase inhibitors (such as ibrutinib, idelalisib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib), proteasome inhibitors (such as bortezomib and carfilzomib), and histone deacetylase inhibitors (such as vorinostat or romidepsin).

[0294] Examples of immunotherapeutic agents include, but are not limited to, phosphoantigens (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 thereof and derivatives thereof, or any cytokine capable of inducing lymphocyte activity (e.g., proliferation or cytokine production or metabolic changes). The term derivative is used for any cytokine modification that can rely on PEGylation (e.g., conjugation with polyethylene glycol (PEG) chains), mutations such as amino acid deletions, substitutions, or insertions, or conjugation with enhancers (e.g., with IgG1 The Fc-fused IL15 / IL15Ra complex, in which IL-15 is additionally mutated (asn72asp), further enhances its biological activity, making the complex a super agonist of IL-2 and IL-15Rβγ (Rhode PR et al., Cancer Immunol Res. 2016) (Barroso-Sousa R et al., Curr Oncol Rep. 2018).

[0295] The term "IL-2" has its ordinary meaning and refers to human interleukin-2. IL-2 regulates lymphocyte activity primarily by binding to the IL-2 receptor.

[0296] The term "IL-15" has its ordinary meaning and refers to human interleukin-15. Like IL-2, IL-15 binds to and signals through a complex consisting of the IL-2 / IL-15 receptor β chain (CD122) and the common γ chain (γ-C, CD132). IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells.

[0297] The term "IL-21" has its ordinary meaning and refers to human interleukin-21. IL-21 has been attributed pleiotropic effects including, but not limited to, enhancing NK cell and CD8+ T cell cytotoxicity, regulating plasma cell differentiation, and suppressing Treg cells.

[0298] The term "IL-33" has its ordinary meaning and refers to human interleukin 33. IL-33, which is considered to be an alarmin released when tissues are stressed or damaged, is a member of the IL-1 family and binds to the ST2 receptor. IL-33 is known to be a T H 1 A potent stimulator of immune cells, natural killer (NK) cells, iNKT cells, and CD8 T lymphocytes.

[0299] The term "PD-1" has its ordinary 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 is a member of the CD28-B7 signaling receptor family, which includes CD28, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), inducible costimulator (ICOS), and B and T lymphocyte attenuator (BTLA) (Greenwald RJ et al., 2005; Riley JL et al., 2005).

[0300] The term "anti-PD-1 antibody" or "anti-PD-L1" has the general meaning in the art and refers to an antibody that has binding affinity for PD-1 or PD-L1, respectively, and has antagonistic activity against PD-1, i.e., it inhibits the signal transduction cascade associated with PD-1 and inhibits PD-1 ligand binding (PD-L1; PD-L2). Such anti-PD-1 antibodies or anti-PD-L1 antibodies, respectively, preferentially inactivate PD-1 with greater affinity and potency than their interactions with other subtypes or isoforms of the CD28-B7 signaling family of receptors (CD28; CTLA-4; ICOS; BTLA). Tests and assays for determining whether a compound is a PD-1 antagonist are well known to those skilled in the art and are described, for example, in Greenwald et al. (2005) and Riley et al. (2005).

[0301] Examples of such anti-PD1 antibodies include, but are not limited to, nikolazumab, pembrolizumab, ovelumab, durvalumab, cemiplizumab, or atuzumab.

[0302] In light of the foregoing, the present disclosure provides, in yet another aspect:

[0303] A method as defined above, comprising co-administering (e.g., concomitantly or sequentially) a therapeutically effective amount of an anti-BTN2A1 antibody of the present disclosure, and at least one second drug, which is an antiviral or antiproliferative agent or an immunotherapeutic agent or a cytokine or a cell therapy product (such as γδ T cells), e.g., as described above.

[0304] In one embodiment, the antibodies of the present disclosure can be used to detect the level of soluble BTN2A1 or the level of cells expressing BTN2A1. For example, this can be achieved by contacting a sample (such as an in vitro sample) and a control sample with an anti-BTN3A antibody under conditions that allow the antibody and BTN2A1 to form a complex (e.g., expressed on the cell surface or in soluble BTN2A1, such as a blood sample). Any complex formed between the antibody and BTN2A1 in the sample and control is detected and compared. For example, the compositions of the present disclosure can be used to perform standard detection methods known in the art (such as ELISA and flow cytometry) for determination.

[0305] Thus, in one aspect, the present disclosure further provides a method for detecting the presence of BTN2A1 (e.g., human BTN2A1 antigen) in a sample or measuring the amount of BTN2A1, comprising contacting the sample and a control sample with an antibody or protein of the present disclosure, or an antigen-binding region that specifically binds to BTN2A1, under conditions that allow the antibody or portion thereof to form a complex with BTN2A1, and then detecting the formation of the complex, wherein a difference in complex formation in the sample compared to the control sample indicates the presence of BTN2A1 in the sample.

[0306] Also included within the scope of the present disclosure are kits comprising the compositions disclosed herein (e.g., humanized antibodies, conjugated antibodies, and multispecific molecules) and instructions for use. The kit may further contain at least one other reagent, or one or more other antibodies or proteins. The kit typically includes a label indicating the intended use of the kit contents. The term label includes any text or recorded material on, provided with, or accompanying the kit. As defined above, the kit may further include a tool for diagnosing whether a patient belongs to a population that responds to anti-BTN2A1 antibody treatment.

[0307] Another therapeutic approach is based on the use of the humanized antibodies of the present disclosure as agents for selectively activating NK cells isolated from a sample of a human subject.

[0308] Accordingly, the present disclosure relates to methods for treating a subject in need thereof, comprising:

[0309] (a) isolating blood cells containing NK cells, such as PBMC, from a blood sample of a subject,

[0310] (b) expanding NK cells in vitro in the presence of anti-BTN2A1 disclosed herein, and optionally, other tumor or accessory cells,

[0311] (c) collecting the expanded NK cells,

[0312] (d) Optionally, formulating the expanded NK cells and administering a therapeutically effective amount of the NK cells to a subject.

[0313] The present disclosure further relates to the use of humanized antibodies disclosed herein as reagents that 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).

[0314] The present disclosure also relates to the use of the anti-BTN2A1 antibodies disclosed herein as enhancers in NK cell therapy in a subject in need thereof, typically suffering from cancer.

[0315] As used herein, the term NK cell therapy refers to a therapy comprising administering at least one effective amount of NK cells to a subject in need. Such NK cells can be allogeneic or autologous. In a specific embodiment, NK cells can be engineered by deletion or knockout or insertion or knock-in of specific genes. In a specific embodiment, the NK cells include NK cells expressing chimeric antigen receptors. NK cells may have been expanded and / or purified ex vivo. Alternatively, NK cells may also be contained in a cell composition comprising other blood cells, such as other cells of the immune system. For references on γδT cell therapy, see Rezvani, K. Bone Marrow Transplant 2019.

[0316] Thus, the present disclosure relates to methods of treating a subject having cancer, e.g., a hematological malignancy, particularly a leukemia, such as acute myeloid leukemia, and having tumor cells, e.g., hematological tumor cells, comprising:

[0317] (i) administering to the subject an effective amount of an anti-BTN2A1 antibody disclosed herein, and,

[0318] (ii) administering an effective amount of a NK cell composition to the subject,

[0319] Wherein the effective amount of anti-BTN2A1 antibody has the ability to enhance the anti-tumor cytolysis against the tumor cells mediated by the NK cell composition. The present disclosure also relates to a method for treating a subject in need thereof, comprising administering NK cells, such as CAR NK cells, in combination (simultaneously or sequentially), and a humanized antibody as disclosed herein.

[0320] In alternative or additional embodiments, therapeutic strategies may also be based on the use of humanized antibodies as disclosed herein as reagents that selectively expand and / or activate Vγ9 / Vδ2 T cells isolated from a sample of a human subject.

[0321] Accordingly, the present disclosure relates to methods for treating a subject in need thereof, comprising:

[0322] (a) isolating blood cells, such as PBMCs, containing Vγ9 / Vδ2 T cells from a blood sample of a subject,

[0323] (b) expanding Vγ9 / Vδ2 T cells in vitro in the presence of anti-BTN2A1 disclosed herein, and optionally, other tumor or helper cells,

[0324] (c) collecting the expanded Vγ9 / Vδ2 T cells,

[0325] (d) Optionally, formulating the expanded Vγ9 / Vδ2 T cells and administering a therapeutically effective amount of the Vγ9 / Vδ2 T cells to a subject.

[0326] The present disclosure further relates to the use of humanized antibodies as disclosed herein as reagents that selectively expand chimeric antigen receptor (CAR) Vγ9Vδ2 T cells. CARγδT cells and their use in adoptive T cell cancer immunotherapy are described in, for example, Mirzaei et al, Cancer Lett 2016.

[0327] The antibodies of the present disclosure can also be used to prepare artificial T cell receptors (also known as chimeric T cell receptors, or chimeric antigen receptors (CAR)). For example, the variable region of the antibody can be used to form Fab or scFv, which is connected to the transmembrane domain and signaling domain of the TCR via a spacer and can be produced on the surface of T cells. Such CARs can be used for adoptive transfer therapy, for example, for the treatment of proliferative diseases.

[0328] The present disclosure also relates to the use of anti-BTN2A1 antibodies as tumor cell enhancers in γδ T cell therapy in a subject in need thereof, typically suffering from cancer.

[0329] As used herein, the term γδT cell therapy refers to a therapy comprising administering at least an effective amount of γδT cells to a subject in need. These γδT cells can be allogeneic or autologous. In a specific embodiment, γδT cells can be genetically engineered by deletion or knockout or insertion or knock-in of specific genes. In a specific embodiment, the γδT cells include γδT cells expressing chimeric antigen receptors. γδT cells may have been expanded and / or purified ex vivo. Alternatively, γδT cells may also be contained in a cell composition containing other blood cells, such as other cells of the immune system. For references on γδT cell therapy, see Pauza CD.et al, Front Immunol.2018JSaudemont A.et al, Frontiers Immunol 2018.

[0330] Indeed, without being bound by any particular theory, the proposed mode of action of the anti-BTN2A1 antibodies of the present disclosure is that its binding to BTN2A1 expressed on the surface of tumor cells triggers a conformational change that enables it to signal to its counter-receptor on Vγ9Vδ2 T cells.

[0331] Thus, the present disclosure relates to methods of treating a subject having cancer, e.g., a hematological malignancy, particularly a leukemia, such as acute myeloid leukemia, and having tumor cells, e.g., hematological tumor cells, comprising:

[0332] (i) administering to the subject an effective amount of an anti-BTN2A1 antibody disclosed herein, and

[0333] (ii) administering an effective amount of a γδ T cell composition to the subject,

[0334] Wherein the effective amount of anti-BTN3A antibody has the ability to enhance the anti-tumor cell lysis against the tumor cell mediated by the γδT cell composition. The present disclosure also relates to a method for treating a subject in need thereof, comprising administering a combination (simultaneously or sequentially) of CAR T cells (e.g., CARγδT cells) and a humanized antibody disclosed herein.

[0335] The present invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present disclosure in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0336] Figure 1Identification of anti-BTN2A1 107G3 mAb. A. Cascade of anti-BTN2A1 mAb screening from mouse immunization to mAb sequencing. B. Bar graph showing the affinity (K) of each mAb measured on Luminex during primary hit selection. D C. Stacked bar graph showing the number of clones classified as neutral (grey), antagonist (white), or agonist (black) according to their ability to modulate IFN-γ production by Vγ9 / Vδ2 T cells during primary (first round) and secondary (second round) hit screening.

[0337] Figure 2 Anti-BTN2A1 107G3 mAb enhances the cytolytic function of Vγ9 / Vδ2 T cells. Vγ9 / Vδ2 T cells were expanded from PBMCs of three healthy donors (see Materials and Methods) in the presence of anti-CD107a / b antibodies and Golgi arrest, with or without the indicated antibodies, and co-cultured with target cells at 37°C at an effector:target (E:T) ratio of 1:1. After 4 hours, cells were harvested, fixed, and analyzed on a flow cytometer. In A, different target cell lines, including Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), L-IPC (pancreatic cancer), and MDA-MB-134 (breast cancer), were co-cultured with anti-BTN2A1 107G3 supernatant or control hybridoma culture medium. The bar graph shows the percentage of CD107+ cells, depicting Vγ9 / Vδ2 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 an isotype control. 50 Calculation of dose-response curves.

[0338] Figure 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. Bar graphs show overlays of the indicated cells and cell transfectants stained with purified anti-BTN2A1 107G3 mAb (top, black line), anti-BTN3 103.2 mAb (bottom, black line), or mIgG1 or IgG2a (dashed lines) controls. For transfected cells, staining is shown after gating on CFP+ cells. B. Graph shows a dose-response curve for binding of purified anti-BTN2A1 107G3 mAb to HEK-293T BTN2 KO cells transfected with a plasmid encoding BTN2A1-CFP. All staining was analyzed after gating on CFP+ cells.

[0339] Figure 4 : Expression of BTN2A on NK cells and monocytes and the effect on monocyte-to-M2 macrophage polarization with reference anti-BTN2A 101G5 and 107G3 mAbs. (A) Representative histograms of BTN2A1 and BTN2A2 expression (white) on NK cells and monocytes from unstimulated HD-PBMCs compared to control isotypes (grey), assessed by flow cytometry. (B) Representative CD14 / CD163 dot plots of in vitro M1 / M2 macrophages induced in the presence of M-CSF or macrophages with the 101G5 and 107G3 mAbs. After 5 days of differentiation, CD14 and CD163 dot plots were generated by flow cytometric analysis.

[0340] Figure 5 : Reference anti-BTN2A 101G5 and 107G3 mAbs inhibit M2 macrophage polarization in a dose-dependent manner. M1, M2, M2 restored with GM-CSF and IFNγ, and M-CSF-induced macrophages in the presence of varying concentrations of 101G5 or 107G3 mAb (or their isotype controls) were polarized for 5 days and restimulated with or without LPS for 2 days. The expression of CD14 (A), CD163 (B), PDL1 (C), and CD86 (D) was analyzed by flow cytometry on unstimulated cells (AC) or LPS-stimulated cells (D). Results are expressed as median fluorescence intensity (MFI) minus the corresponding isotype control. IL-10 (E) and TNFα (F) were quantified in LPS-stimulated macrophage supernatants by ELISA. Results are expressed in pg / mL.

[0341] Figure 6 : Reference anti-BTN2A 101G5 and 107G3 mAbs inhibit the polarization induced by "M2+IL-4" from monocytes. M1, M2, "M2+IL4", M2 restored with GM-CSF and IFNγ, and macrophages induced with 10 μg / mL of M-CSF+IL-4 and 101G5 or 107G3 mAb (or its isotype control) were generated for 5 days and restimulated with or without LPS for 2 days. The expression of CD14 (A), CD163 (B), PDL1 (C), DC-SIGN (D), and CD86 (E) was analyzed by flow cytometry on unstimulated cells (AD) or LPS-stimulated cells (E). The results are expressed as median fluorescence intensity values ​​(MFI) minus the corresponding isotype control. IL-10 (F) and TNFα (G) were quantified in LPS-stimulated macrophage supernatants by ELISA. The results are expressed in pg / mL.

[0342] Figure 7: Reference anti-BTN2A 101G5 and 107G3 mAbs inhibit cancer cell-induced M2 polarization. Macrophages induced with M1, M2, PANC-1 conditioned medium, M2 restored with GM-CSF and IFNγ, and 10 μg / mL PANC-1 conditioned medium and 101G5 or 107G3 mAbs (or their isotype controls) were generated for 5 days and restimulated with or without LPS for 2 days. The expression of CD14 (A) and CD163 (B) was analyzed by flow cytometry on unstimulated cells. Results are expressed as median fluorescence intensity (MFI) minus the corresponding isotype control. IL-10 (C) and TNFα (D) were quantified in LPS-stimulated macrophage supernatants by ELISA. Results are expressed in pg / mL.

[0343] Figure 8 : Reference anti-BTN2A 101G5 and 107G3 mAbs restore M2 macrophages to pro-inflammatory M1 macrophages: phenotype and cytokine secretion. M1 and M2 were generated from monocytes for 5 days. After 5 days, 10 μg / mL of 101G5 or 107G3 mAb (or isotype control) or IFNγ was added to M2 macrophages for 2 days and stimulated with or without LPS for another 2 days. The expression of CD14 (A), CD163 (B), PDL1 (C), and CD86 (D) was analyzed by flow cytometry on unstimulated cells (AC) or LPS-stimulated cells (D). Results are expressed as median fluorescence intensity (MFI) minus the corresponding isotype control. IL-10 (E) and TNFα (F) were quantified in LPS-stimulated macrophage supernatants by ELISA. Results are expressed in pg / mL.

[0344] Figure 9 :Reference anti-BTN2A 101G5 and 107G3 mAb releases the inhibition of M2-mediated T cell proliferation and IFNγ secretion. M1, M2 or macrophages induced by differentiation in the presence of 101G5 and 107G3 mAb (or its isotype control) were co-cultured with CD3+T cells labeled with CTV activated by allogeneic OKT3 for 5 days. After co-culture, cells were stimulated for 5 hours with PMA / ionomycin and GolgiStop protein inhibitor, and then the number of CD3+T cells (A and B), intracellular IFNγ production (CF) and proliferation (CellTrace Violet, CTV dim) (GJ) were quantified by flow cytometry. Proliferation was quantified by diluting CTV dye (CTV signal on day 0 was used as baseline). The results are the absolute number (B, E, F, I and J) of CD3+T cells calibrated on CountBright absolute counting beads or the percentage (C, D, G and H) of CD3+T cells.

[0345] Figure 10 Effects of reference anti-BTN2A 101G5 and 107G3 mAbs on activation and cytotoxicity of purified NK cells. (AB) Purified NK cells were cultured with reference anti-BTN2A 101G5 and 107G3 mAbs (or control isotypes) for 5 days in the presence of IL-2 or IL-2 / IL-15. NK cell activation was assessed by evaluating CD69 (A) and CD25 (B) expression (MFI) in unstimulated and IL-2 / IL-15-stimulated NK cells in the presence of the indicated mAbs or control isotypes. (CD) Purified NK cells were preincubated with reference anti-BTN2A 101G5 and 107G3 mAbs (or control isotypes) overnight in the presence or absence of IL-2 / IL-15 stimulation and then cocultured with human tumor cell lines for 4 hours. NK cell degranulation was assessed by flow cytometry as the percentage of CD107αβ in unstimulated (C) and IL-2 / IL-15 stimulated NK cells (D) relative to each tumor cell line in the presence of the indicated mAbs or control isotypes. (E) NK cell degranulation against the A549 cell line when reference anti-BTN2A 101G5 and 107G3 mAbs (or control isotypes) were preincubated on NK cells or target cells prior to coculture for 4 hours, compared to mAbs added to the coculture without preincubation.

[0346] Figure 11 Figure 3: Reference anti-BTN2A 101G5 and 107G3 mAbs enhance NK cell degranulation and killing of adenocarcinoma cell lines. (A) Purified NK cells were preincubated overnight with reference anti-BTN2A 101G5 and 107G3 mAbs (or control isotypes) in the presence or absence of IL-2 / IL-15 stimulation and then co-cultured with the DU-145 cell line for 4 hours. NK cell degranulation was assessed by flow cytometry as the percentage of CD107αβ+ cells. The EC values ​​for NK cell degranulation enhancement for the indicated mAbs were calculated using a four-parameter dose-response curve on Prism software. 50 (B) Purified NK cells were preincubated overnight with reference anti-BTN2A 101G5 and 107G3 mAbs (or control isotypes or IL-2 / IL-15 stimulation) and then cocultured with HL-60 and A549 cell lines for 4 hours. NK cell-mediated cancer cell death was assessed by obtaining the percentage of caspase 3 / 7+ cells in the presence of the indicated mAbs or control isotypes.

[0347] Figure 12Figure 2: Binning experiments of the reference anti-BTN2A1 101G5 and 107G3 mAbs against BTN2A1. Binning experiments were performed on the Octet Red96 platform using biolayer interferometry (BLI) technology. 107G3 and 101G5 were tested in pairwise combinations against rhBTN2A1-His protein. A: 107G3 saturation, 101G5 competition; B: 101G5 saturation, 107G3 competition; C: Measurements of binding and self-blocking mAb pairs in arbitrary units.

[0348] Figure 13 : Trypsin, chymotrypsin, ASP-N, elastase, and thermolysin peptides of BTN2A1. 96.37% of the sequence was covered by the identified peptides.

[0349] Figure 14 : Interactions between reference mAbs 107G3 and 101G5 and human BTN2A1. A. 107G3 / BTN2A1. B. 101G5 / BTN2A1.

[0350] Figure 15 : Interaction BTN2A1 / 107G3. BTN2A1 PDB structure 4F9P is gray at the epitope site. The blue BTN2A1 amino acids correspond to 65-78 (RWFRSQFSPAVFVY) and 84-100 (RTEEQMEEYRGRTTFVS) of the provided BTN2A1 sequence. A, B, C, D, E: front view (A), back view (B), side view (C). Figure 1 (C) Side view Figure 2 (D) and top view (E) of the ribbon / surface. F, G, H, I, J: front view (F), back view (G), side view Figure 1 (H), Side View Figure 2 (I) and ribbon representation of the top view (J).

[0351] Figure 16 : Interaction BTN2A1 / 101G5. BTN2A1 PDB structure 4F9P is gray at the epitope site. The blue BTN2A1 amino acids correspond to 212-229 (KSVRNMSCSINNTLLGQK) of the provided BTN2A1 sequence. A, B, C, D, E: Front view (A), back view (B), side view (C). Figure 1 (C) Side view Figure 2 (D) and top view (E) of the ribbon / surface. F, G, H, I, J: front view (F), back view (G), side view Figure 1 (H), Side View Figure 2 (I) and ribbon representation of the top view (J).

[0352] Figure 17 : Evaluation of cross-reactivity of reference anti-BTN2A1 101G5 and 107G3 mAbs against cynomolgus monkey BTN2A1 orthologs. Binding of 107G3 and 101G5 to recombinant human BTN2A1-Fc fusion protein or recombinant cynomolgus monkey BTN2A1-Fc fusion protein coated on ELISA plates was measured. The graph depicts the EC values ​​calculated by nonlinear regression using a variable slope model. 50 Dose-response curve. DETAILED DESCRIPTION

[0353] Materials and methods

[0354] Cell culture, monocyte and NK cell isolation:

[0355] Peripheral blood mononuclear cells (PBMC) were obtained from EDTA (ethylenediaminetetraacetic acid)-local blood bank (Etablissement Buffy coats from healthy donors (HD) were obtained from E. du Sang (EFS)-Marseille-France and separated by centrifugation on a density gradient (Eurobio). Fresh PBMCs were cultured at 37°C, 5% CO2 in Roswell Park Memorial Institute medium 1640 (RPMI; Lonza) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S).

[0356] Follow the manufacturer's instructions for using EasySep TM Natural killer (NK) cells were isolated from fresh PBMCs by negative selection using a human NK cell enrichment kit (StemCell Technologies). Human CD14+ monocytes were isolated using a CD14+ microbead kit (Miltenyi) according to the manufacturer's instructions. Monocytes were cultured at 10°C for 24 h. 6 The cells were cultured at a density of 10 cells / mL 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) for 5 days. The pancreatic cancer cell line PANC-1 was cultured in RPMI supplemented with 10% FBS. Once grown to 90% confluence, the medium was discarded and the cells were rinsed twice in PBS 1X. PANC-1 cells were then cultured for an additional 24 hours (per 175 cm) in RPMI supplemented with 5% FBS. 230 mL flasks were added to obtain concentrated supernatant). Then, PANC-1 conditioned medium was collected, filtered (0.2 μM) and stored at -20°C until use. Other human cell lines and their corresponding culture media are summarized in Table 1 below:

[0357] Table 1

[0358]

[0359]

[0360] The following human cell lines are from the American Type Culture Collection: Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), MDA-MB-134 (breast ductal carcinoma) and HEK-293T (embryonic kidney). The human pancreatic cancer cell line L-IPC (PDAC087T) was kindly provided by Dr. Juan IOVANNA. Daudi and Jurkat cells and PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% sodium pyruvate, 1% L-glutamine (all from Life Technologies). HEK-293T BTN2 KO cells were generated by CRISPR-Cas9-mediated inactivation of all BTN2 isoforms (data not shown). MDA-MB-134, L-IPC, HEK-293 cells and HEK-293T BTN2 KO cells were cultured in DMEM medium (Life Technologies) containing 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). Hybridomas were cultured for 4-5 days in the absence of Fetalclone for the collection of hybridoma supernatants.

[0361] To evaluate the specificity of anti-BTN2A1 mAbs, 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.

[0362] Identification of reference anti-BTN2A1 mAb 107G3

[0363] Mouse anti-human BTN2A1 antibodies were generated by immunizing 48 mice bearing six different MHC combinations with recombinant human BTN2A1-Fc fusion proteins. Twenty-one days later, the mice were bled, and serum titers of BTN2A1-specific polyclonal antibodies were determined by Luminex assay. The mice displaying the highest BTN2A1-specific antibody titers were euthanized. Splenic B cells were isolated by positive selection and subjected to PEG-induced myeloma cell fusion to generate hybridomas.

[0364] Hybridomas were cloned by limiting dilution and hybridoma supernatants were screened for target specificity and their ability to induce degranulation of Vγ9Vδ2-T cells in two rounds ( Figure 1 c and 2), and identified the reference mAb 107G3. The VH and VL regions of these subclones were sequenced (see Table 1).

[0365] Expansion of Vγ9Vδ2-T cells

[0366] Effector Vγ9 / Vδ2-T cells were established by culturing PBMCs from HV in the presence of zoledronate (Sigma, 1 μM) and recombinant human (rh) IL-2 (Proleukin, 200 IU / mL) starting from day 0. From day 5 onwards, rhIL-2 was refreshed every other day and the cell density was maintained at 1.15 x 10 6 Cells were cultured at 4% cel / mL for a total of 15 days. On the final day, the purity of the Vγ9 / Vδ2-T cells was assessed by flow cytometry. Only cell cultures with a Vγ9 / Vδ2-T cell purity greater than 80% were selected for functional testing. The purified Vγ9 / Vδ2-T cells were frozen until use.

[0367] Luminex assay

[0368] Magnetic COOH beads (Biorad) were conjugated to rhBTN2A1 protein (R&D) according to the manufacturer's instructions and stored in storage buffer (Biorad) at −20°C until use. To titrate mouse serum, serial serum dilutions were performed in Luminex assay buffer (Nanotools) starting at 1:50 and proceeding through 1:4 dilution steps. 100 μL of the bead suspension was mixed with 100 μL of the serum dilution and incubated at room temperature for 1 hour. The beads were then washed three times in wash buffer, incubated with 1 μg / mL biotinylated goat anti-mouse IgG-Fc in Luminex assay buffer, and washed an additional three times in Luminex assay buffer. Finally, the beads were incubated with 1 μg / mL streptavidin-PE in Luminex assay buffer for 1 hour, followed by a final three washes in Luminex read buffer (Nanotools). The beads were resuspended in Luminex read buffer, and data were acquired on a Luminex 100 / 200 system. For hit identification, 30 μL of supernatant was transferred to a 96-well plate and 90 μL of Luminex assay buffer was added. 100 microliters of bead suspension was mixed with 100 μL of supernatant dilution and incubated at room temperature for 16 hours before continuing with the above protocol. For hit identification, those with the highest affinity for the target and the lowest affinity for an irrelevant control protein (Rank-Fc) were selected. For affinity / Kd calculations, hybridoma supernatants were serially diluted in Luminex assay buffer starting from 40.000 pM, in 1:4 dilution steps, and analyzed as described above. Kd corresponds to the midpoint of the corresponding binding curve.

[0369] Flow cytometry

[0370] PBMCs, purified Vγ9Vδ2-T cells, or cell lines were incubated with the indicated mAbs before analysis on a BD LSRFortessa (BD Biosciences), CytoFlex LX, or CytoFlex S (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo). Antibodies used for Vγ9Vδ2-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 10 μg / mL purified mAbs 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 published (WO 2012 / 080351). To evaluate the specificity of anti-BTN2A1 mAb, HEK-293T BTN2 KO cells (5×10 4 Each sample was stained with anti-human BTN2A1 107G3 mAb at the indicated concentrations (5 ng / mL-75 μg / mL) as described above. A mouse IgG1 antibody (Miltenyi) was used as an isotype control for staining.

[0371] Functional assay of Vγ9 / Vδ2-T cells

[0372] Purified Vγ9 / Vδ2-T cells from HV were cultured overnight in rhIL-2 (200 UI / mL). Vγ9 / Vδ2-T cells were then co-cultured with the specified target cell lines (effector: target (E:T) ratio of 1:1) at 37°C in the presence or absence of the following mAbs (50 μL hybridoma supernatant or 10 μg / ml purified mAb, as specified): anti-BTN2A1 mAb, mIgG1 (isotype control antibody) or hybridoma culture medium. 12-Myristate-13-acetic acid phorbol (PMA, 20 ng / mL) and ionomycin (1 μg / mL) were used as positive controls for Vγ9 / Vδ2-T cell activation. For the first round of hybridoma supernatant screening, the culture supernatant was collected 4 hours later and tested for its content of IFNγ using a human IFNγ ELISA set (BD Biosciences) as an indicator of Vγ9 / Vδ2-T cell activation. For the second round of hybridoma supernatant characterization, 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 PBS containing 2% paraformaldehyde, and analyzed using FlowJo 10.5.3 software (FlowJo) on a CytoFlex LX (Beckman Coulter).

[0373] Proliferation of Vγ9 / Vδ2-T cells

[0374] Vγ9 / Vδ2-T cells were isolated from PBMCs of healthy donors using an anti-TCRγδ microbead kit (Miltenyi Biotec). The purity of γδ-T cells assessed by flow cytometry was greater than 80%. γδ-T cells were labeled with CellTraceViolet at 37°C for 20 minutes. 5×10 β-actin-1 cells were then added to the PBMCs in the presence of IL-2 (200 UI / ml), with or without pAg, with or without purified anti-BTN2A1107G3 antibody (10 μg / ml). 5 Cells labeled with CellTrace were cultured in 96-well round-bottom plates. After 5 days of culture, CellTrace dilution was assessed by flow cytometry on a CytoFlex LX (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo).

[0375] statistics:

[0376] For Vγ9 / Vδ2-T cell degranulation, the results are expressed as mean ± SEM. EC values ​​of purified anti-BTN2A1 mAbs on BTN2A1-transfected HEK-293T BTN2 KO cells were determined based on log (dose) response curves after nonlinear regression using a variable slope model. 50 All analyses were performed using GraphPad Prism 7.04 software (GraphPad).

[0377] Identification of reference anti-BTN2A mAb 101G5

[0378] After VH and VL sequencing, 23 kinds of anti-BTN2A mAbs obtained from mouse hybridoma generation as described above were produced under chimeric IgG1 form. In brief, mouse VH and VK anti-BTN2A mAb sequences were synthesized in vitro and amplified by PCR using PrimeSTAR MaxDNA polymerase (Takara). PCR products were cloned into heavy and light chain expression vectors (MI-mAb) using a fusion system (Clontech), and plasmids were transformed into Stellar competent cells (Clontech). Carry out vector sequencing (MWGEurofins) to verify anti-BTN2A mAb, and then prepare the plasmid for further transfection on a large scale (maxi). The vector encoding the matching light chain and heavy chain of each anti-BTN2A clone was transiently transfected into HEK-293 cells (2.9x10 6cells / mL) with a heavy chain / light chain ratio of 1:1.2, and the culture medium was renewed after 18 hours. Seven days after transfection, the culture supernatant was collected for mAb purification. Affinity purification of the antibody was performed using Protein A Sepharose Fast Flow (GE Healthcare) at 44°C overnight. The binding buffer was 0.5M glycine, 3M NaCl, pH8.9. Elution was performed using the following buffer: 0.1M citrate, pH3. The sample was immediately neutralized with 1M Tris-HCl, pH9 (10% v / v) after elution. Finally, the chimeric anti-BTN2AmAb was dialyzed into PBS 1X and filtered through a 0.22μM filter (Millex GV hydrophilic PVDF, Millipore). Taking into account the extinction coefficient of the antibody, the chimeric anti-BTN2AmAb concentration was measured in a Nanodrop 2000 spectrophotometer (ThermoScientific). Purity, defined as the fraction of mAb monomer, was determined by UPLC-SEC using an Acquity UPLC-HClass Bio (Waters) and an Acquity UPLC Protein-BEH-200A, 1.7 μm 4.6 x 50 mm column (Waters). Antibody mass was determined using a reverse phase chromatography column (PLRP-S 4000A, 5 μm, 50 x 2.1 mm (Agilent technologies) in a Xevo G2-S Q-Tof mass spectrometer (Waters). All samples were analyzed after deglycosylation using PNGase F glycosidase (New England Biolabs) at 37°C. When 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 the purified antibodies allowed detection of fragmentation and / or aggregation of the final material without staining 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).

[0379] In vitro macrophage polarization assay:

[0380] M1 or M2 macrophages are polarized from the sorting monocytes of healthy donors. For this reason, the sorted monocytes are cultured in the presence of GM-CSF or M-CSF (40 ng / mL; Miltenyi) to produce M1 or M2 macrophages, respectively. After 5 days, the macrophages produced are collected for phenotypic analysis, or stimulated for 2 more days with LPS (200 ng / mL). In some experiments, IL-4 (20 ng / mL) is added to the M2 macrophages on the 4th day, resulting in the production of "M2+IL-4" or macrophages. In some experiments, M2 macrophages are produced by culturing monocytes under PANC-1 cancer cell conditioned medium (30% v / v diluted in culture medium, on day 0 and day 3) in the absence of M-CSF supplementation. The M2 macrophages thus produced are referred to as "Turn-ind-M2" in this application. In order to screen the ability of anti-BTN2A mAb to regulate M2 differentiation, M2 macrophages were generated from monocytes as described above with or without a specified concentration of chimeric anti-BTN2A mAb or its isotype control (human IgG1; Sigma). All mAbs were wet coated (overnight in PBS 1X at room temperature). As a control for M2 differentiation inhibition, GM-CSF (40 ng / mL) and IFNγ (100 ng / mL, BioTechne) were added to monocytes during M2 polarization induced by M-CSF. M1 macrophages polarized in the presence of GM-CSF were used as phenotypic controls. After polarization, the obtained macrophages and their culture supernatants were collected and the expression of M1 and M2 related markers on the plasma membrane was evaluated by flow cytometry. In addition, the cytokine content in the culture supernatant was quantified using IL-10 and TNFα ELISA kits (ThermoFisher Scientific) according to the manufacturer's instructions.

[0381] In vitro M2 macrophage reversal assay:

[0382] M2 macrophages were generated from monocytes as described above in the presence of M-CSF in the absence of a reference mAb. M2 macrophages were collected and cultured for 2 days in culture wells with or without LPS, which had been wet-coated overnight with 10 μg / mL of a reference antibody or its control isotype mAb (human IgG1 from Sigma). As a control for M2 reversal, GM-CSF (40 ng / mL) and IFNγ (100 ng / mL) were added to the M2 macrophage cultures for 2 days. M1 macrophages polarized in the presence of GM-CSF were used as phenotypic controls. After the reversal experiment, macrophages not reversed with LPS were collected and phenotyped by flow cytometry. ELISA was used to quantitatively collect cytokines in the culture supernatant of the reversal macrophages stimulated by LPS.

[0383] In vitro assay for M2 macrophage-mediated inhibition of T cell proliferation and IFNγ production

[0384] As described above, M1 and M2 macrophages were generated with or without the addition of a reference antibody or its isotype control mAb. CD3+ T cells were sorted from healthy donor PBMCs using a CD3+ microbead kit (Miltenyi) and frozen until co-culture according to the manufacturer's instructions. Activated CD3+ T cells were generated as follows: CD3+ T cells were stained with 5 μM CellTraceViolet dye (ThermoFisher Scientific) and then cultured in 96-well plates coated with 1 μg / mL anti-CD3 mAb (clone OKT3, BD biosciences) in the presence of 20 U / mL IL-2 (Miltenyi), LPS (200 ng / mL) and CountBright absolute counting beads (5x10 per well). 3 ThermoFisher Scientific) X-Vivo 10 medium was used for 10 5 For co-culture with macrophages, 2x10 4 Allogeneic M1, M2, or macrophages polarized in the presence of M-CSF and a reference mAb or its control isotype were added to activated allogeneic CD3+ cells. After 5 days of co-culture, 20 ng / mL PMA and 0.5 μg / mL ionomycin were added to the co-culture in the presence of the GolgiStop protein transport inhibitor to enhance cytokine production for 5 hours. Cells were then recovered by flow cytometry for phenotypic analysis. CellTrace dilution was used as an indicator of CD3+ T cell proliferation. Phenotypic and proliferation results were expressed as percentages or absolute cell counts per mL (after calibration using absolute counting beads).

[0385] Natural Killer (NK) Challenge Using Reference Anti-BTN2A1 mAb:

[0386] Sorted natural killer (NK) cells from healthy donors were labeled and then cultured in RPMI supplemented with 10% FBS and 1% P / S, IL-2 (50 UI / mL) at 37°C, 5% CO2 with or without IL-15 (10 ng / mL) stimulation. Reference anti-BTN2A mAb or control isotype (10 μg / mL) was added to the culture on day 0. After 5 days, NK cells were subjected to extracellular phenotyping analysis for the 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 was as follows: Figure 4As shown. For NK cytotoxicity measurements, sorted NK cells from 3 healthy donors were cultured in RPMI supplemented with 10% FBS and 1% P / S at 37°C, 5% CO2 in the presence or absence of IL-2 (50 UI / mL) and IL-15 (10 ng / mL). Reference anti-BTN2A mAb or control isotype (10 μg / mL) was added to unstimulated or IL-2 / IL-15 stimulated NK cells overnight. The next day, NK cells were co-cultured with the indicated blood or cancer cell lines at a 1:1 ratio, and FITC-labeled anti-CD107a and anti-CD107b mAbs (both from BD Biosciences) were added to the co-culture and incubated for 4 hours. NK cell degranulation was assessed by flow cytometry as the percentage of CD107ab+ cells on unstimulated or IL-2 / IL-15 stimulated NK cells. In order to calculate the EC of NK cell degranulation enhancement 50 , reference anti-BTN2A mAb and its isotype control mAb were used at concentrations of 0.005nM-300nM. For cancer cell NK cell-mediated killing assessment, purified NK cells were pre-incubated with 10μg / mL of reference 101G5 and 107G3 mAbs or corresponding IgG1 controls overnight at 37°C, 5% CO2 in RPMI supplemented with 10% FBS and 1% P / S IL-2 (50UI / mL) and IL-15 (10ng / mL) and 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 hours. By using CellEvent TM Caspase-3 / 7 Green Detection Reagent (Thermofisher Scientific) was used to assess cancer cell death by obtaining the percentage of caspase 3 / 7+ cells in tumor cell lines.

[0387] Flow cytometry:

[0388] Before staining, PBMC / NK cells and monocytes / macrophages were blocked with human Fc (Miltenyi) or human IgG1 (Sigma) and incubated for 10 minutes to saturate Fc receptors. The labeled mAbs used were as follows: CD14-FITC and -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 -BV605 (BD Biosciences), CD69-BV421 (BD Biosciences), CD25-APC (BD Biosciences). Cells were incubated with antibody mixture at 4°C for 30 minutes. Live / dead near-infrared dye (ThermoFisher Scientific) was used to define the "live" gate, while dead cells were excluded. For intracellular IFNγ staining, extracellular stained cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer set (eBioscience) and incubated with APC-labeled anti-IFNγ (BD Biosciences). Collection was performed on a Fortessa flow cytometer (BD Biosciences) using FlowJo 10 software. For BTN2A1 and BTN2A2 phenotypic analysis, 10 μg / mL of purified anti-BTN2A1 specific (mAb5) and anti-BTN2A2 specific (mAb17) were used and displayed with PE-labeled anti-IgG (H+L) (Jackson Immunoresearch). After selecting single cells, NK cells were CD45+CD14-CD3-CD56+ cells within the "live" gate. After selecting single cells, monocytes were CD45+CD19-CD3-CD56-CD14+ cells within the "live" gate. Acquisition was performed on a Cytoflex LS (Beckman Coulter), iQue Screener (Intellicyt), or Fortessa (BD Biosciences) flow cytometer, and data were analyzed using FlowJo 10 software. Results are expressed as median fluorescence intensity (MFI) after subtraction of the value obtained with the corresponding staining control.

[0389] Octet-based BTN2A1 epitope affinity measurement and binning assay:

[0390] After generating a chimeric IgG1 version of the reference anti-BTN2A antibody, the affinity of two different isoforms (BTN2A1 and BTN2A2) to this target was assessed, and competition assays were 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) based on biolayer interferometry (BLI) technology. For affinity experiments, the EZ-Link TMThe NHS-PEG4 biotinylation kit biotinylates recombinant human (rh) BTN2A1-Fc (GTP), and biotinylated rhBTN2A2-Fc is purchased from R&D Systems. In the case of BTN2A1 affinity determination, the biotinylated rhBTN2A1-Fc is loaded into a streptavidin (SA) biosensor (ForteBio) diluted in Kinetic Buffer 1X (ForteBio) with a loading target level of approximately 1 nm, and a chimeric anti-BTN2A antibody is used as the analyte. For BTN2A2 affinity determination, the chimeric anti-BTN2A antibody is loaded into a FAB2G sensor (anti-human CH1; Fortebio) as described above, and biotinylated rhBTN2A2-Fc is used as the analyte. In both cases, the analyte is retained in solution, and its working concentration is diluted in Kinetic Buffer 10X (ForteBio). For the first run, the standard working concentration range was 200-3.125 nM. When required for the second run, the working concentration was adjusted from 80 to 1.25 nM. All runs (including loading, equilibrium, sensor binding / immersion in analyte, dissociation and regeneration) were performed at 30 ° C with a speed of 1000 rpm. Analysis was performed using a 1: 1 or 2: 1 Langmuir model (BTN2A1 or BTN2A2, respectively) calculated by Octet software, which allows for better fitting calculations. For the binning experiment, His-tagged BTN2A1 (rhBTN2A-His) was purchased from R&D Systems. The reference anti-BTN2A antibody was tested in pairs against BTN2A1. The binning experiment was performed by following the "tandem" format, meaning that rhBTN2A-His was fixed on a biosensor (anti-Penta-His <HIS1K biosensor; ForteBio / PALL) and presented to two competing antibodies in consecutive steps. For this kinetic screening, rhBTN2A-His was loaded onto HIS1K (signal intensity: 1 nm), followed by a 3-minute binding step with 10 μg / mL antibody, followed by a 3-minute dissociation step. rhBTN2A1-His activity was confirmed by a kinetic screening assay performed in the same format as the binning assay (BTN2A1 as the ligand / capture on the sensor and the antibody as the analyte). All antibodies (saturated or competing) were used at a concentration of 10 μg / mL, diluted in KineticBuffer 1X. For this kinetic screening, rhBTN2A1-His was loaded onto HIS1K (signal intensity: 1 nm), followed by a 3-minute binding step with the antibody, followed by a 3-minute dissociation step. The assay steps were as follows: baseline -> antigen capture -> baseline -> saturated antibody -> baseline -> competing antibody -> regeneration following a "series" scheme.The binned data were analyzed using Octet Data Analysis HT 11.1 using the epitope binning procedure.

[0391] Epitope mapping of reference mAbs 107G3 and 101G5

[0392] The interaction between BTN2A1 and the reference mAbs 107G3 and 101G5 was assessed by differential evaluation of the peptide mass fingerprints of BTN2A1 alone or with 107G3 or 101G5. Before epitope mapping, high-quality MALDI analysis was performed on rhBTN2A1-Fc protein (GTP Technologies) to verify its integrity and aggregation level (CovalX) using an Autoflex II MALDI ToF mass spectrometer (Bruker) equipped with a CovalX HM4 interaction module, which confirmed the absence of non-covalent aggregates or multimers of BTN2A1 in the samples. To characterize BTN2A1 and identify the epitopes of BTN2A1 / 107G3 and BTN2A1 / 101G5, samples were subjected to trypsin, chymotrypsin, Asp-N, elastase, and thermolysin hydrolysis, followed by nLC-LTQ-Orbitrap MS / MS analysis using an nLCUltimate 3000-RSLC system coupled to an LTQ-Orbitrap mass spectrometer (Thermo Scientific). For the BTN2A1 / 107G3 and BTN2A1 / 101G5 complexes, the protein complexes were incubated with a deuterated cross-linker prior to multienzymatic cleavage. After enrichment of cross-linked peptides, samples and generated data were 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) and incubated at room temperature for 180 minutes. After incubation, the reaction was terminated by adding 1 μL of ammonium bicarbonate (20 mM final concentration) and then incubated at room temperature for 1 hour. The solution was then dried using a speedvac followed by a 10 μL suspension of 8M urea in H2O. After mixing, 1 μL of DTT (500 mM) was added to the solution. The mixture was then incubated at 37°C for 1 hour. After incubation, 1 μL of iodoacetamide (1 M) was added and then incubated at room temperature in the dark for 1 hour. After incubation, 100 μL of proteolysis buffer was added. Trypsin buffer contained 50 mM Ambic pH 8.5, 5% acetonitrile; chymotrypsin buffer contained 100 mM Tris HCl, 10 mM CaCl2, pH 7.8; ASP-N buffer contained 50 mM phosphate buffer, pH 7.8; elastase buffer contained 50 mM Tris HCl, pH 8.0, and thermolysin buffer contained 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) at 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) at 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) at 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) at 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, a final 1% of formic acid was added to the solution. After proteolysis, 10 μL of the peptide solution generated by proteolysis was loaded onto a nano-liquid chromatography system (Ultimate 3000-RSLC) with the following settings: A: 95 / 05 / 0.1H2O / ACN / HCOOH v / v / v; B: 20 / 80 / 0.1H2O / ACN / HCOOH v / v / v, gradient 5-40% B in 35 minutes, injection volume 10 μL, pre-column 300-μm ID x 5-mm C18 PepMapTM, pre-column flow rate 20 μL / min, chromatographic column 75-μm ID x 15-cm C18 PepMapRSLC, column flow rate, 200 nL / min.

[0393] ELISA determination of cross-reactivity between human and cynomolgus monkey BTN2A1

[0394] The cynomolgus monkey BTN2A1 orthologous sequence (XM_015448906.1) was identified after a BLAST search using the human BTN2A1 amino acid sequence and its extracellular domain was cloned into the pFUSE-hIgG1FC2 vector (InvivoGen) using EcoRI / EcoRV restriction sites. TM The obtained pFUSE-hIgG1FC2-cynoBTN2A1 plasmid was transfected into Expi293F TMRecombinant cynoBTN2A1-Fc fusion protein was produced in cells. The cell culture supernatant collected on day 6 was used for purification by affinity purification column. The molecular weight and purity of the purified cynoBTN2A1-Fc protein were measured by SDS-PAGE and Western blot analysis. The cynoBTN2A1-Fc protein concentration was determined by Bradford assay with BSA as the standard. For ELISA, cynoBTN2A1-Fc protein or recombinant human BTN2A1-Fc (huBTN2A1-Fc, GTP Technologies) was coated (5 μg / mL in 1X PBS) at 4°C overnight. After washing three times in PBS, the plate was saturated with BSA 2% v / v in PBS for 1 hour at room temperature, and the saturation buffer was discarded. Reference mAbs 101G5 and 107G3 or control human IgG1 were diluted in 2% PBS BSA in a 10-fold serial dilution series from 1 μM to 1 pM, and 100 μL of each dilution was added to each well and incubated on a shaker for 90 minutes at room temperature. All wells were washed three times in PBS, and then goat anti-mouse IgG HRP (Jackson ImmunoResearch, diluted 1:10,000 in PBS BSA 2%) was added and incubated for 1 hour at room temperature. Then, all wells were washed three times in PBS and 1-step ABTS solution (ThermoFisher) was added for binding visualization, as assessed by absorbance at 405 nm in a Spark spectrometer (Tecan). All samples were evaluated in duplicate.

[0395] result:

[0396] Identification of the reference antibody anti-BTN2A1 107G3

[0397] The reference anti-BTN2A1 107G3 antibody was identified as follows: mice were immunized with the BTN2A1-Fc antigen, and splenocytes from mice expressing the highest titer of BTN2A1-specific serum were collected and fused with a myeloma cell line to obtain hybridomas. The hybridoma culture supernatants that showed the highest affinity for BTN2A1 were selected for the first round of screening based on their ability to modulate IFN-γ secretion by Vγ9 / Vδ2-T cells. The clones selected from the first round of screening were subcloned and tested for their ability to induce IFN-γ secretion and degranulation of Vγ9 / Vδ2-T cells and IFN-γ secretion, in particular, their ability to induce degranulation of Vγ9Vδ2-T cells ( Figure 1 c and 2), and led to the identification of the reference mAb 107G3. The VH and VL regions of these subclones were sequenced (see Table 2 below).

[0398] Anti-BTN2A1 107G3 antibody induces Vγ9Vδ2-T cell degranulation against different cancer cell targets

[0399] Purified Vγ9 / Vδ2 T cells were expanded from PBMCs of healthy donors and co-cultured with different cancer cell lines including Daudi (Burkitt lymphoma), Jurkat (acute T-cell leukemia), L-IPC (pancreatic cancer), and MDA-MB-134 (breast cancer) as target cells in the presence or absence of anti-BTN2A1 107G3 hybridoma culture supernatant. Figure 2 As shown in Table 3, addition of anti-BTN2A1 107G3 hybridoma supernatant induced cytolytic function of Vγ9 / Vδ2 T cells, as measured by the percentage of CD107+ degranulated cells, compared to co-culture with target cells alone or in the presence of control hybridoma culture medium. As expected, PMA / ionomycin treatment of Vγ9 / Vδ2 T cells maximally induced their cytolytic function independent of target cells.

[0400] The percentage of CD107+ cells induced by anti-BTN2A1 107G3 hybridoma supernatant ranged from 71.1±7.4% in Daudi cells to 17.1±2.9% in MDA-MB-134 cells, compared to 24.9±4.7% and 4.9±0.4%, respectively, in cocultures with control hybridoma medium. In cocultures of Vγ9 / Vδ2 T cells targeting all tested cancer cell lines, anti-BTN2A1 107G3-induced Vγ9 / Vδ2 T cell degranulation was increased 2-8-fold in the presence of control hybridoma medium compared to the same cocultures.

[0401] Purified anti-BTN2A1 mAb 107G3 induced Vγ9 / Vδ2 T cell degranulation in Daudi cells co-cultured with increasing concentrations (0-18 μg / ml) of anti-BTN2A1 107G3 mAb. 50 was 0.77 μg / mL (95% IC 0.32-13.22 μg / mL), as shown by the percentage of CD107+ cells ( Figure 2 b).

[0402] Table 3

[0403]

[0404]

[0405] Anti-BTN2A1 107G3 antibody recognizes BTN2A1 but not BTN3

[0406] To confirm that the anti-BTN2A1 mAb 107G3 is specific for only the BTN2A1 isoform, HEK-293T BTN2 KO cells with CRISPR-Cas9-mediated inactivation of both BTN2 isoforms were transiently transfected with a plasmid encoding BTN2A1 as a CFP-fusion protein. Figure 3 As shown in a, purified anti-BTN2A1 mAb 107G3 staining was detected only in HEK-293T BTN2KO cells transfected with a BTN2A1 encoding plasmid but not in HEK-293T BTN2 KO cells alone.

[0407] Anti-BTN3 mAb 103.2, which recognizes all BTN3 isoforms, readily detects BTN3 expression in HEK-293T BTN2 KO cells. Therefore, anti-BTN2A1 107G3 is specific for BTN2A1 isoforms and does not cross-react with BTN3.

[0408] Affinity of anti-BTN2A1 107G3 mAb for BTN2A1 in cells

[0409] To measure the affinity of anti-BTN2A1 107G3 mAb to its target, HEK-293T BTN2 KO cells transfected with BTN2A1 encoding plasmid were stained with increasing concentrations (5 ng / mL-75 μg / mL) of purified anti-BTN2A1 107G3 mAb or control mIgG1 ( Figure 3 b) Nonlinear regression analysis of mean fluorescence intensity data revealed that the EC of anti-BTN2A1 107G3 mAb 50 The active ingredient in the drug was 0.32 μg / mL (95% IC 0.21-0.46 μg / mL).

[0410] Generation, affinity measurement, and BTN2A isoform specificity of chimeric anti-BTN2A mAbs:

[0411] Twenty-three monoclonal antibodies were transiently produced in HEK-293T cells, achieving varying 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), had very low yields in HEK-293T cells, ranging from 6-8 mg / L. Amino acid sequence analysis revealed an N-glycosylation site in its Fab portion, in the CDR1 of its VH. The other two antibodies, anti-BTN2A mAb9 and mAb11, also exhibited N-glycosylation sites within their Fab regions (in the CDR1_VH of mAb9 and the CDR1_VL of mAb11). The purity levels of six antibodies were low (<95% in monomers), but only the purity levels of anti-BTN2A mAb1 and mAb3 were <90% (86% and 75%, respectively). All the final purified anti-BTN2A mAbs showed very low endotoxin levels (within the range of 0.1 EU / mg). Only mAb3 had an endotoxin level (0.73 EU / mg) higher than the other mAbs, but still within the acceptable standard range (<1 EU / mg). The affinity constants (K) of 23 anti-BTN2A chimeric mAbs were determined using biotinylated recombinant Fc fusion soluble proteins using Octet technology. D 、k on and k off Table 4 summarizes the K values ​​of each anti-BTN2A mAb. D For mAb6 and mAb9, no dissociation was observed during the measurement (k off <10 -7 s -1 ), so K cannot be calculated D , which can be explained by the avidity effect of these antibodies slowing their dissociation from the target. Eight anti-BTN2A mAbs were found to bind only to the BTN2A1 isoform (mAb2, mAb3, mAb4, mAb5, mAb6, mAb8, mAb9, and mAb10), eight anti-BTN2A mAbs were found to bind only to the BTN2A2 isoform (mAb16, mAb17, mAb18, mAb19, mAb20, mAb21, mAb22, and mAb23), and seven mAbs were found to bind to both isoforms (mAb1, mAb7, mAb11, mAb12, mAb13, mAb14, and mAb15).

[0412] Table 4: Summary of chimeric anti-BTN2A mAb production and affinity.

[0413]

[0414]

[0415] *N-glycosylation site present in VH or VL.

[0416] BTN2A1 and BTN2A2 plasma membrane expression on monocytes and NK cells:

[0417] We sought to determine whether anti-BTN2A mAbs could target non-Vγ9Vδ2 T cell compartments of peripheral blood, namely monocytes and NK cells. Therefore, we performed phenotypic analysis of monocytes and NK cells from peripheral blood using mAb5 and mAb17, which were found in our octet assay to bind only to BTN2A1 or BTN2A2, respectively. Figure 4 As shown, only anti-BTN2A1 mAb 5 stained the plasma membrane of monocytes and NK cells, with a stronger signal observed in monocytes. Thus, BTN2A1, but not BTN2A2, was detected on the plasma membrane of monocytes and NK cells, providing a rationale for screening mAbs that recognize BTN2A1, as they are able to modulate immune function in these immune cell compartments.

[0418] Screening of anti-BTN2A mAbs for their ability to modulate monocyte-to-macrophage polarization

[0419] In response to signals from their microenvironment, monocytes can polarize into either M1 or M2 macrophages. M1 macrophages have pro-inflammatory and anti-tumor properties, while M2 macrophages have anti-inflammatory properties and are associated with tumor development. Given that only the BTN2A1 isoform is found on the plasma membrane of monocytes, the ability of anti-BTN2A mAbs that recognize only BTN2A1 or both the BTN2A1 and BTN2A2 isoforms to interfere with monocyte polarization into M2 macrophages in vitro was evaluated in the presence of M-CSF. 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 mAb or its control IgG1 was assessed by flow cytometry (Table 4). As expected, M1 cells showed low CD14 expression and undetectable CD163 expression (Tables 5 and Figure 4 ), whereas M2 macrophages showed high expression of both markers. Interestingly, anti-BTN2A mAb1, which will be referred to as 101G5 from now on, induced the strongest reduction in CD14 and CD163 expression in the presence of M-CSF, thereby polarizing M-CSF-induced macrophages toward an M1-like phenotype (Tables 4 and Figure 4B). The second best inhibitor of M-CSF-induced M2 macrophage polarization was mAb2, 107G3 (Table 5 and Figure 4 B) This is in contrast to the phenotype of macrophages obtained in the presence of M-CSF and control IgG1, which resemble untreated M2 macrophages.

[0420] Table 5: Effect of anti-BTN2A mAbs on CD14 and CD163 expression after monocyte polarization into M2-macrophages.

[0421]

[0422]

[0423] Figure 5 The dose dependence of the M2 inhibitory effect of the reference 101G5 and 107G3 anti-BTN2A mAbs in terms of inhibition of CD14 and CD163 expression compared to isotype control is shown ( Figure 5 A and 5B) and increased expression of PDL1 and CD86 as characteristic of the M1 phenotype ( Figure 5 C and 5D). Cytokine secretion profiles are also distinguishing features between M2 and M1 macrophages. Therefore, after LPS stimulation of culture supernatants from M-CSF-induced macrophages (with or without reference anti-BTN2A mAb), IL-10 (anti-inflammatory, M2-associated) and TNFα (pro-inflammatory, M1-associated) secretion were assessed by ELISA. Figure 5 As shown in Figures E and 5F, the reference anti-BTN2A mAb inhibited IL-10 secretion and increased TNFα secretion in a dose-dependent manner compared to the isotype control. These observations confirm that 101G5 and 107G3 inhibit M-CSF-induced monocyte polarization to M2 macrophages by biasing them toward an M1-like phenotype in terms of phenotype and cytokine secretion. Furthermore, these effects of 101G5 and 107G3 were dose-dependent. The IC values ​​for each mAb were 0. 50 and EC 50 These are shown in Table 6. Notably, 101G5 obtained the lowest IC for all parameters except PD-L1 compared to 107G3. 50 and EC 50 .

[0424] Table 6: IC of reference anti-BTN2A mAbs for M2 vs. M1-associated phenotypes and cytokine secretion 50 and EC 50

[0425]

[0426] Other stimuli from the tumor microenvironment have 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 effects of 101G5 and 107G3 on the differentiation of so-called pro-tumor "M2+IL-4" macrophages generated from monocytes after stimulation with M-CSF and IL-4. After 5 days of culture under these conditions, 101G5 and 107G3 suppressed the expression of "M2+IL-4" related markers (CD14, CD163 and DC-SIGN, Figure 6 A, 6B and 6D) expression and IL-10 secretion ( Figure 6 F), while increasing the expression of M1-related markers (CD86, PDL1) and the secretion of TNFα ( Figure 6 C, 6E, and 6G). Thus, in a pro-tumor environment (M-CSF and IL-4), 101G4 and 107G3 inhibit "M2+IL-4" differentiation and enhance pro-inflammatory M1 macrophage differentiation.

[0427] Furthermore, the effects of 101G5 and 107G3 on cancer cell-induced monocyte M2 polarization were assessed by culturing sorted monocytes in the presence of PANC-1 (pancreatic adenocarcinoma cell line) conditioned culture supernatant. When 101G5 or 107G3 were added to this setting, M2 polarization was inhibited, as evidenced by the expression of M2-associated markers (CD14, CD163) and IL-10 secretion ( Figure 7 AC) and increased expression of M1-associated pro-inflammatory TNFα ( Figure 7 D) shown.

[0428] Reference effect of anti-BTN2A mAbs 101G5 and 107G3 on M2-macrophage reprogramming to M1

[0429] The potential of the reference 101G5 and 107G3 mAbs to revert M2 polarized macrophages to an M1 phenotype was evaluated. For this purpose, M2 macrophages previously polarized for 5 days in the presence of M-CSF were seeded into wells previously coated with 101G5 and 107G3 mAbs and cultured for an additional 2 or 4 days. M2 macrophages were treated with IFNγ as a positive control for M2->M1 reversion. Figure 8 As shown, 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 101G5 and 107G3 mAbs resulted in the expression of CD14 ( Figure 8 A) and CD163 ( Figure 8B) expression decreased, and CD86 expression increased ( Figure 8 C). After treatment with 101G5 or 107G3, moderate to no upregulation of PDL1 was observed ( Figure 8 D). In addition, treatment of M2 macrophages with 101G5 and 107G3 inhibited IL-10 secretion ( Figure 8 E) and enhance TNFα secretion ( Figure 8 F), indicates M1 phenotype.

[0430] Figure 8 GI showed inhibition of CD163 expression compared to isotype control ( Figure 8 G), Dose-dependent effects of 101G5 and 107G3 mAbs on the reprogramming of M2-macrophages to M1 macrophages, decreased IL-10 and increased TNFα secretion ( Figure 8 H and I). IC of each mAb for the relevant specific M1 / M2 markers. 50 and EC 50 As shown in Table 7, anti-BTN2A101G5 mAb showed the best activity on reprogramming M2-macrophages into M1 macrophages.

[0431] Table 7: IC of reference anti-BTN2A mAbs for M2 reversal 50 and EC 50

[0432]

[0433]

[0434] Reference anti-BTN2A mAbs 101G5 and 107G3 release M2-mediated inhibition of T cell proliferation

[0435] The ability of the reference 101G5 and 107G3 mAbs to affect the function of M2 macrophages, i.e., to inhibit M2-mediated T cell proliferation, was investigated. To this end, allogeneic preactivated 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 CD3+ T cell numbers, T cell proliferation (assessed by CTV dilution), and IFNγ production (P < 0.05) compared to M1 macrophages. Figure 9 A, C, G, E, and I). In contrast, M2 macrophages generated in the presence of 101G5 and 107G3 did not appear to suppress T cell proliferation, as shown by higher percentages and absolute numbers of CD3+ T cells compared to M2 macrophage cultures generated in the presence of control IgG1 ( Figure 9In addition, the percentage and number of IFNγ-producing T cells were also higher in co-cultures containing macrophages produced in the presence of 101G5 and 107G3 compared to control IgG1 ( Figure 9 D and 9F).

[0436] Thus, compared with M2 macrophages induced by M-CSF alone, macrophages generated in the presence of 101G5 or 107G3 in addition to M-CSF allowed for the proliferation of allogeneic CD3+ T cells and Th1 function (IFNγ production), similar to M1 macrophages.

[0437] Reference anti-BTN2A mAbs 101G5 and 107G3 trigger NK cell activation and cytotoxicity

[0438] Since BTN2A1 is found on the plasma membrane of NK cells, the potential ability of 101G5 and 107G3 to modulate NK cell activation was investigated. Purified NK cells from healthy donors were cultured for 5 days in the presence of 101G5 or 107G3, with or without further activation (IL-2 and IL-15 or IL-2 alone, respectively). Figure 10 As shown, 101G5 and 107G3 enhanced the expression of CD69 on the plasma membrane of NK cells under all tested conditions ( Figure 10 A). In addition, 101G5 and 107G3 also enhanced the expression of CD25 induced by IL-2 and IL-15 treatment of NK cells ( Figure 10 B). Since 101G5 and 107G3 are able to activate purified NK cells, we investigated whether these mAbs could also enhance NK cell cytotoxicity. Therefore, NK cell degranulation (% CD107+ cells) was assessed against the cancer cell lines HL-60 (myeloid leukemia), HT-29 (colon cancer), MDA-MB-231 (breast cancer), and A549 (lung adenocarcinoma) in the presence of 101G5 or 107G3, with or without IL-2 and IL-15 stimulation. As expected, only HL-60 cells triggered NK cell degranulation in the presence of control IgG1 ( Figure 10 C), which was enhanced by stimulation with IL-2 and IL-15 ( Figure 10D). In the presence of control IgG1 and IL2+IL-15, moderate NK cell degranulation for solid tumor cell lines HT-29, MDA-MB-231 and A549 was also observed. Table 8 summarizes the NK cell degranulation for these and other (Raji, HCT116, DU-145) cancer cell lines tested. Interestingly, reference mAb 101G5 and 107G3 enhanced NK cell degranulation for solid tumor cell lines MDA-MB-231 and A549, and in the absence of IL-2+IL-15 stimulation, the effect on HT-29 was smaller. Adding IL-2 and IL-15 enhanced the effect of 101G5 and 107G3 in MDA-MB-231, A549 and DU145 cells (Table 8). By adding reference 101G5 or 107G3 mAb, this enhancement was not observed in HL-60 and Raji blood cancer cell lines (Tables 8 and Figure 10 C and D). In addition, when pre-incubated with NK cells before co-culture, reference mAbs 101G5 and 107G3 were able to trigger NK cell degranulation against A549 cells without the need for further addition of mAb to the co-culture ( Figure 10 E). This indicates that the reference 107G3 and 101G5 mAbs induce direct effects on cytotoxicity against cancer cells by directly binding to NK cells. In addition, the dose-dependence of 101G5 and 107G3 on enhancing NK cell degranulation against the prostate adenocarcinoma DU-145 cell line was evaluated. In fact, 101G5 and 107G3 enhanced the degranulation of NK cells against DU-145 cells in a dose-dependent manner (EC for 101G5 and 107G3, respectively). 50(无刺激) = 0.14 and 0.54 nM; EC 50(IL-2+IL-15) = 0.08 and 0.2 nM).

[0439] Table 8: NK cell degranulation (% CD107+ cells) against different cancer cell lines in the absence of IL-2 and IL-15 stimulation

[0440]

[0441]

[0442] Finally, we tested the ability of 101G5 and 107G3 to enhance NK cell-mediated cancer cell killing by assessing the percentage of caspase 3 / 7 expressing cells after co-culture of purified NK cells with the leukemia cell line HL-60 or the lung adenocarcinoma cell line A459. Figure 11As shown, 101G5 and 107G3 enhanced NK cell-mediated killing of adenocarcinoma A549 cells (~2-fold), but had no effect on HL-60 leukemia cells. Taken together, these observations suggest that 101G5 and 107G3 preferentially enhance NK cell cytotoxicity against cancer cells from solid tumors.

[0443] Reference anti-BTN2A 101G5 and 107G3 mAbs recognize different epitopes of BTN2A1

[0444] Both 101G5 and 107G3 bind to BTN2A1 and share the ability to inhibit M2 macrophage polarization and enhance NK cell activation and cytotoxicity. Therefore, we investigated whether these mAbs recognize the same epitope region on the BTN2A protein. Therefore, an octet-based binning experiment was performed in which 101G5 and 107G3 competed for BTN2A1 binding using a “tandem” setup. Figure 12 As shown, 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 on BTN2A1.

[0445] Epitope mapping of reference mAbs 101G5 and 107G3

[0446] To characterize BTN2A1, we submitted samples to trypsin, chymotrypsin, Asp-N, elastase, and thermolysin hydrolysis, followed by nLC-LTQ-Orbitrap MS / MS analysis. After trypsin hydrolysis, 32 peptides were identified in the BTN2A1 sequence, covering 79.84% of the sequence; after chymotrypsin hydrolysis, 27 peptides were identified, covering 94.76% of the BTN2A1 sequence; after Asp-N protein hydrolysis, 2 peptides were identified, covering 12.50% of the BTN2A1 sequence; after elastase hydrolysis, 33 peptides were identified, covering 89.11% of the BTN2A1 sequence; and after thermolysin hydrolysis, 29 peptides were identified, covering 78.23% of the BTN2A1 sequence. Based on the results obtained, overlapping localization of trypsin, chymotrypsin, Asp-N, elastase, and thermolysin peptides was designed ( Figure 13Peptides derived from proteolysis with 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 a deuterated cross-linker followed by multienzyme cleavage. Following proteolysis of the BTN2A1 / 107G3 protein complex by trypsin, chymotrypsin, ASP-N, elastase, and thermolysin, nLC-orbitrap MS / MS analysis detected 17 cross-linked peptides between BTN2A1 and antibody 107G3.

[0447] Table 9: Sequences and positions of cross-links between BTN2A1 / 107G3

[0448]

[0449]

[0450]

[0451] Therefore, our analysis showed that the interaction between BTN2A1 and 107G3 mAb involves the following amino acids on BTN2A1: 65, 68, 69, 72, 78, 84, 85, 95, 97, 100. These results were Figure 14 A and Figure 15 Instructions.

[0452] After trypsin, chymotrypsin, ASP-N, elastase, and thermolysin proteolysis of the protein complex BTN2A1 / 101G5, nLC-orbitrap MS / MS analysis detected 14 cross-linked peptides between BTN2A1 and antibody 101G5.

[0453] Table 10: Sequences and positions of cross-links between BTN2A1 / 101G5

[0454]

[0455]

[0456] Therefore, our analysis showed that the interaction between BTN2A1 and 101G5 involves the following amino acids on BTN2A1: 212, 213, 218, 220, 224, 229. These results are shown in Figure 14 B and Figure 16 shown.

[0457] Reference anti-BTN2A 101G5 and 107G3 mAbs cross-react with cynomolgus monkey BTN2A1 orthologs

[0458] BTN2A1 orthologs are present in most non-human primates, including cynomolgus monkeys (Macaca fascicularis). To determine the cross-reactivity of reference mAbs 101G5 and 107G3 with the cynomolgus monkey BTN2A1 ortholog (cynoBTN2A1; NCBI ref. XM_015448906.1, 93.31% identical to human BTN2A1), we generated a recombinant Fc fusion protein containing the cynoBTN2A1 extracellular domain (cynoBTN2A1-Fc), and we performed ELISA assays to evaluate the binding of the reference mAbs to this protein. We also performed ELISA using recombinant human BTN2A1-Fc protein to compare the affinity of the reference mAbs between human and cynomolgus monkey BTN2A1 orthologs. As Figure 13 As shown, both 101G5 and 107G3 were able to bind to the extracellular domain of cynoBTN2A1, EC 50 were 0.60 and 0.57 nM, respectively, which were consistent with the corresponding EC values ​​obtained for huBTN2A1. 50 (0.82 and 0.56 nM, respectively) are comparable.

[0459] Table 11: Brief description of useful amino acid and nucleotide sequences for practice

[0460]

[0461] The present invention:

[0462]

[0463]

[0464]

[0465] References:

[0466] Throughout this application, various references describe prior art related to the present invention. The disclosures of these references are hereby incorporated by reference into this disclosure.

Claims

1. An anti-butyrophilin-2A (BTN2A) antibody comprising: - a heavy chain variable region CDR1 consisting of SEQ ID NO: 3, a heavy chain variable region CDR2 consisting of SEQ ID NO: 4, a heavy chain variable region CDR3 consisting of SEQ ID NO: 5, a light chain variable region CDR1 consisting of SEQ ID NO: 6, a light chain variable region CDR2 consisting of SEQ ID NO: 7, and a light chain variable region CDR3 consisting of SEQ ID NO: 8, or - A heavy chain variable region CDR1 consisting of SEQ ID NO:21, a heavy chain variable region CDR2 consisting of SEQ ID NO:22, a heavy chain variable region CDR3 consisting of SEQ ID NO:23, a light chain variable region CDR1 consisting of SEQ ID NO:24, a light chain variable region CDR2 consisting of SEQ ID NO:25, and a light chain variable region CDR3 consisting of SEQ ID NO:

26.

2. The antibody according to claim 1, It is characterized in that Has at least one of the following features: i. It inhibits the polarization of monocytes to M2 macrophages, ii. It induces the reversal of M2 macrophages to anti-tumor M1 macrophages, iii. It directly triggers NK cell activation, iv. It enhances NK cell-mediated cytotoxicity.

3. The antibody of claim 1 or 2, which binds to an epitope comprising the residues located at positions 65, 68, 69, 72, 78, 84, 85, 95, 97 and 100 of SEQ ID NO:

17.

4. The antibody of claim 1 or 2, which binds to an epitope comprising the residues located at positions 212, 213, 218, 220, 224 and 229 of SEQ ID NO:

17.

5. The antibody according to any one of claims 1 to 4, which does not cross-react with human BTN3A isoforms, and / or which cross-reacts with the cynomolgus monkey BTN2A1 ortholog.

6. The antibody according to any one of the preceding claims, comprising: - a heavy chain variable region consisting of a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region consisting of a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, or - a heavy chain variable region consisting of a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 19 and a light chain variable region consisting of a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

20.

7. The antibody according to any one of claims 1 to 6, wherein the antibody further has at least one of the following functions: i. It activates the secretion of cytolytic molecules of Vγ9Vδ2T cells, ii. It activates the cytolytic function of Vγ9Vδ2 T cells, and / or iii. It activates the proliferation of Vγ9Vδ2 T cells.

8. The antibody of claim 7, which competes for binding to BTN2A with reference mouse antibody 107G3, wherein the reference mouse antibody mAb 107G3 comprises (i) a heavy chain variable region having an amino acid sequence consisting of SEQ ID NO: 1 and (ii) a light chain variable region having an amino acid sequence consisting of SEQ ID NO:

2.

9. The antibody according to any one of claims 7 or 8, comprising: - a heavy chain variable region CDR1 consisting of SEQ ID NO: 3, a heavy chain variable region CDR2 consisting of SEQ ID NO: 4, a heavy chain variable region CDR3 consisting of SEQ ID NO: 5, a light chain variable region CDR1 consisting of SEQ ID NO: 6, a light chain variable region CDR2 consisting of SEQ ID NO: 7, and a light chain variable region CDR3 consisting of SEQ ID NO: 8, or - a heavy chain variable region consisting of a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region consisting of a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:

2.

10. The antibody according to any one of claims 6 to 9, which is specific for human BTN2A1 isoform.

11. The antibody according to any one of the preceding claims, which is a human antibody, a chimeric antibody or a humanized antibody.

12. A nucleic acid molecule encoding the heavy chain and / or light chain of the antibody according to any one of claims 1 to 11.

13. A host cell comprising the nucleic acid molecule according to claim 12.

14. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 11 and at least one pharmaceutically acceptable carrier.

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