RU2821703C2 - Btn2-specific antibodies and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is an antibody specific to BTN2A1. Also disclosed is the use of said antibody for enhancing macrophage-mediated immunity in a patient suffering cancer or an infectious disease. Invention also relates to a nucleic acid coding said antibody and a host cell containing such a nucleic acid.

EFFECT: invention can find application in therapy for treating cancer and infectious diseases.

21 cl, 56 dwg, 10 tbl

Description

Field of technology to which the invention relates

This invention relates to activating antibodies to BTN2A1, which bind to BTN2A1, induce a shift in the macrophage population towards the anti-tumor M1 phenotype and directly activate NK cells, as well as cytotoxicity directed against cancer cells. Alternatively, these antibodies are capable of activating Vγ9/Vδ2 T cells, or they have all of these activities. Such antibodies are particularly useful for treating cancer.

State of the art

There are different phenotypes of macrophages, ranging from classically activated cells with the M1 phenotype to differently activated cells with the M2 phenotype. M1 macrophages rapidly differentiate from monocytes after migration of the latter as a result of activation by agents of bacterial origin, such as lipopolysaccharides (LPS), as well as signaling agents produced in connection with infection, such as interferon gamma (IFNγ). M1 macrophages have high pro-inflammatory, phagocytic and bactericidal abilities. They release important proinflammatory cytokines, such as tumor necrosis factor alpha (TNFα) and interleukins 1, 6 and 12 (IL-1, IL-6, IL-12), as well as reactive oxygen species (ROS). In contrast, M2 macrophages appear later in the healing process, when granulation tissue has already formed; they counteract the inflammatory response, thereby promoting the initiation of healing. These anti-inflammatory cells mobilize fibroblasts and activate their differentiation to form myofibroblasts, which release proangiogenic factors to recruit endothelial progenitor cells and form new vessels; this process is mediated by the secretion of key anti-inflammatory cytokines IL-4, IL-10 and IL-13, while the production of ROS, nitric oxide (NO) and TNFα is also reduced.

The tumor microenvironment (TME) significantly influences macrophage differentiation toward the M2 phenotype, especially in cancers that recur after anticancer treatment. This shift not only promotes angiogenesis but also suppresses immune defenses in the tumor microenvironment. Thus, it has long been known that in most solid cancers, increasing infiltration of tumor-associated macrophages (TAMs) is associated with poor prognosis, suggesting the potential value of these cells as diagnostic and prognostic markers in cancer.

For these reasons, “reprogramming” and selectively killing M2 macrophages has been proposed as a promising cancer treatment strategy (Zhu Y et al. Cancer Res 2014).

In addition, cells of the innate immune system, namely natural killer or NK cells, perform key functions in cancer-related immunosurveillance mechanisms. NK cells are capable of eliminating various abnormal or stressed cells without prior sensitization, and they preferentially destroy stem cells or malignant stem cells. Having formed a conjugate with a target cell, the NK cell releases cytolytic granules already present in it, containing perforin, and granzymes, which cause lysis of the target cell.

A number of studies based on the above have been carried out, in which the adoptive transfer of N-cells was successfully carried out to affect various tumors, in particular in malignant lesions of the circulatory system. However, in cancer, various mechanisms operate that delay, distort, or even stop antitumor immunological reactions, which is why the body cannot control tumor growth. The antitumor effects of NK cells also face numerous limitations. In particular, the tumor microenvironment remains the main obstacle to the effective action of NK cells, especially NK cells transferred into the body during adoptive cancer therapy. For example, tumor-infiltrating immune cells, including dendritic cells (DCs), macrophages with suppressor or tolerogenic activity, and regulatory T cells (Treg cells), as well as tumor-associated fibroblasts that are embedded in the extracellular matrix, may interfere with NK cell activation by releasing immunosuppressive cytokines or affecting the expression of receptor proteins. For example, in the tumor microenvironment, the main cytokine that suppresses NK cells is transforming growth factor beta (TGF-β), secreted mainly by M2 macrophages, which limits the number of NK cells and prevents tumor metastasis.

From a therapeutic point of view, it would be extremely valuable to have a means of stimulating antitumor activity in both ways: i) suppressing the immunosuppressive effect of the tumor microenvironment and ii) directly initiating NK cell activation and NK cell-mediated cytotoxicity.

Butyrophilins are a family of transmembrane proteins that include butyrophilins (BTN), butyrophilin-like proteins (BTNL), and proteins involved in the selection and maintenance of intraepithelial T cells (SKINT) (Arnett H.A., Viney J.L., Nat. Rev. Immunol. , 2014). In these proteins, the extracellular part contains domains similar to the variable and constant regions of immunoglobulins (IgV-L and IgC-L domains), which are homologous to the corresponding domains of costimulatory proteins/protein complexes of type B7 (Arnett H.A., Viney J.L., 2014); thus, butyrophilins are considered members of the extensive B7 or Ig superfamily.

The butyrophilin (BTN) gene family in humans includes 13 genes, forming 8 distinct groups (Abeler-Dorner et al. Trends Immunol., 2012; Afrache et al. Immunogenetics, 2012). In humans, seven BTN genes form a cluster in the region of chromosome 6 where major histocompatibility complex class I (MHC I) proteins are encoded; these genes are divided into three subfamilies, representing phylogenetically related groups: BTN1, BTN2 and BTN3. The BTN1 subfamily includes only one prototype gene, BTN1A1, represented by one copy, and the BTN2 and BTN3 subfamilies have three genes each - BTN2A1, BTN2A2 and BTN2A3 (the latter is a pseudogene), BTN3A1, BTN3A2 and BTN3A3, respectively. Several gene polymorphisms have been described for the BTN family of genes and are associated with various diseases, including hypertension, chronic renal failure, inclusion body myositis, type I and II diabetes, and hepatitis C virus (HCV) infection (Chen et al. Int. J. Clin. Exp. Pathol, 2015; Am. J. Hypertens., 2014; Genet., 2013; Pacheco et al. Orphanet J. Rare Dis., 2016). Single nucleotide polymorphic variants have been described for BTNL2, BTN2A1, BTN3A2 and BTN3A3, and deleterious copy number variation of the BTNL3 and BTNL8 genes is known (Aigner et al. BMC Genet., 2013).

The first butyrophilin identified, BTN1A1, is required for the formation, secretion and stabilization of milk fat droplets (Ogg et al. Proc. Natl. Acad. Sci., 2004). The B7 and MHC class I and II protein genes are thought to be derived from 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 ). The BTN2A1 and BTN2A2 protein isoforms have an extracellular IgV and IgC domain. transmembrane domain and specific intracellular domain of B30.2, similar to BTN3A1 and BTN3A3, but not to BTN3A2. In mice, the gene encoding the BTN2A2 protein is present in a single copy and is orthologous to the human BTN2A2 gene. Using the recombinant human BTN2A1-Fc protein, it was determined that a specific glycoform of BTN2A1 binds to the DC-SIGN lectin present on the surface of dendritic cells (DCs). The binding of BTN2A1 to DC-SIGN depends on the glycosylation profile, namely the high mannose content of the protein when expressed in tumor cells (Malcherek et al. J. Immunol., 2007).

Increasing evidence suggests that butyrophilins play various roles in the immune system. RNA sequencing from 53 human tissue samples as part of the Tissue-Specific Gene Expression and Epigenomics Data Resource Project (Genotype-Tissue Expression Project; GTEx Consortium, 2013) showed that the BTN2A1 transcript is present in all normal tissues. Comparison of these data and information from The Cancer Genome Atlas (TCGA) database using the Gene Expression Profiling Interactive Analysis platform (Tang, Z. et al. Nucleic Acids Res., 2017) indicates changes in BTN2A1 expression , determined by the presence of the transcript, in a number of cancers, including squamous cell carcinoma of the cervix, endocervical adenocarcinoma, small cell lung carcinoma, ovarian carcinoma, pancreatic cancer and endometrial carcinoma.

Antibodies recognizing both isoforms of BTN2A were previously reported (WO2019057933), but these antibodies suppressed the production of IFN-γ and/or TNF-α by activated Vγ9/Vδ2 T cells, and/or the cytolytic function of activated Vγ9/Vδ2 T cells, and/or proliferation of activated Vγ9/Vδ2 T cells.

Vγ9/Vδ2 T cells are important effector elements of immune defense. They directly carry out the lysis of cells infected with pathogenic agents or abnormal cells. In addition, Vγ9/Vδ2-T cells regulate the immune response by inducing dendritic cell maturation as well as isotype switching and immunoglobulin formation. This important part of the immune system is tightly regulated by cell surface receptor proteins, chemokines and cytokines. Vγ9/Vδ2-T cells are activated by non-peptide phosphorylated metabolites of the isoprenoid biosynthetic pathway called phosphoantigens (PAg).

Development of antibodies targeting BTN2A1 and activation of immune system cells, preferentially in more than one cellular compartment, such as macrophages, NK cells and/or γδT cells, particularly Vγ9/Vδ2 T cells, may provide a significant contribution to treatment cancer and infectious diseases.

Disclosure of the Invention

The present invention provides for the first time antibodies that bind to the BTN2A protein (mainly the BTN2A1 isoform, e.g. the human BTN2A1 polypeptide) that have at least one of the following properties:

i. suppress the differentiation of monocytes into macrophages with the M2 phenotype;

ii. cause a reversion of the M2 phenotype towards the M1 phenotype with antitumor activity;

iii. directly trigger the activation of NK cells;

iv. enhance NK cell-mediated cytotoxicity.

In particular, the antibody of this invention has at least one of properties i) and ii) and at least one of properties iii) and iv), more specifically, the antibody of this invention has properties i) to iv).

Such antibodies in accordance with the invention are capable of

- favorably influence the antitumor properties of the tumor microenvironment, causing a change in the phenotype of macrophages and their functional activity towards the pro-inflammatory M1 phenotype, which leads to the release of pro-inflammatory cytokines, and/or

- directly trigger the activation of NK cells and thereby enhance their cytolytic activity.

Therefore, such antibodies in accordance with the present invention represent a powerful tool that can be used in various approaches in the treatment of cancer.

In certain embodiments of the present invention, the anti-BTN2A antibodies of the invention compete for binding to BTN2A with any of the following agents:

- with the mouse reference antibody mAb 101G5, comprising (i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO: 20, or

- with the reference mouse antibody mAb 107G3, comprising (i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO: 2.

In some embodiments of the present invention, the anti-BTN2A antibodies of the invention bind to an epitope comprising amino acid residues located:

- in positions 65, 68, 69, 72, 78; 84, 85, 95, 97, 100 of SEQ ID NO 17 or

- at positions 212, 213, 218, 220, 224, 229 of SEQ ID NO 17.

In certain embodiments of the present invention, the provided anti-BTN2A antibodies comprise:

- CDR1 region of the heavy chain variable region containing the sequence SEQ ID NO:3; the CDR2 region of the heavy chain variable region containing the sequence of SEQ ID NO:4; the CDR3 region of the heavy chain variable region containing the sequence of SEQ ID NO:5; the CDR1 region of the light chain variable region containing the sequence SEQ ID NO:6; the CDR2 region of the light chain variable region containing the sequence SEQ ID NO:7; and the CDR3 region of the light chain variable region containing the sequence of SEQ ID NO:8; or

- CDR1 region of the heavy chain variable region containing the sequence SEQ ID NO:21; the heavy chain variable region CDR2 region containing the sequence SEQ ID NO:22; the CDR3 region of the heavy chain variable region containing the sequence of SEQ ID NO:23; the CDR1 region of the light chain variable region containing the sequence of SEQ ID NO:24; the CDR2 region of the light chain variable region containing the sequence of SEQ ID NO:25; and the CDR3 region of the light chain variable region containing the sequence of SEQ ID NO:26.

In certain embodiments of the present invention, the provided anti-BTN2A antibodies comprise:

- a heavy chain variable region containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a light chain variable region containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2, or

- a heavy chain variable region containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:19, and a light chain variable region containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:20.

In a certain embodiment of the present invention, the provided antibodies also have at least one of the following properties:

- activate the secretion of cytolytic agents by Vγ9Vδ2-T cells;

- activate the cytolytic function of Vγ9Vδ2-T cells; and/or

- activate the proliferation of Vγ9Vδ2-T cells.

In particular, anti-BTN2A antibodies of such an embodiment of the present invention may compete for binding to BTN2A, typically the murine reference antibody mAb 107G3 comprising (i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO:1, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO:2.

More specifically, such anti-BTN2A antibodies may comprise:

- CDR1 region of the heavy chain variable region containing the sequence SEQ ID NO:3; the CDR2 region of the heavy chain variable region containing the sequence of SEQ ID NO:4; the CDR3 region of the heavy chain variable region containing the sequence of SEQ ID NO:5; the CDR1 region of the light chain variable region containing the sequence SEQ ID NO:6; the CDR2 region of the light chain variable region containing the sequence SEQ ID NO:7; and the CDR3 region of the light chain variable region containing the sequence of SEQ ID NO:8; or

- a heavy chain variable region containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1, and a light chain variable region containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2.

In some embodiments of the present invention, the antibodies provided are specific for BTN2A1.

In certain embodiments of the present invention, the anti-BTN2A antibodies provided are human, chimeric, or humanized.

Also included within the scope of the present invention are nucleic acid molecules (polynucleotides) encoding the heavy and/or light chain of the anti-BTN2A antibodies described above.

The present invention also relates to host cells containing such polynucleotides, particularly for use in the production of any of the anti-BTN2A antibodies described above.

Another aspect of the present invention relates to the above-described anti-BTN2A antibodies for therapeutic use, particularly in the treatment of cancer and infectious diseases.

In general, the cancers disclosed herein include circulatory and solid cancers, including cervical carcinomas, eg, squamous cell carcinoma and endocervical adenocarcinoma, ovarian carcinoma; skin cancers, including squamous cell carcinoma and melanoma; lung cancer, including small cell lung carcinoma; prostate cancer, colon cancer, pancreatic cancer, endometrial carcinomas, more specifically squamous cell carcinoma and endocervical carcinoma, squamous cell carcinoma, ovarian carcinoma, small cell lung carcinoma, prostate cancer, colon cancer, pancreatic cancer and endometrial carcinomas.

The present invention also provides a pharmaceutical composition comprising the anti-BTN2A antibodies described above and at least a pharmaceutically acceptable carrier.

The present invention also provides a method of activating an immune response in a subject, comprising administering to the subject an effective amount of the anti-BTN2A antibodies described herein.

Carrying out the invention

Definitions

As used herein, the designation “BTN2”, as used in the art, refers to human BTN2 polypeptides, including BTN2A1 having the amino acid sequence of SEQ ID NO:17, or BTN2A2 having the amino acid sequence of SEQ ID NO:18.

SEQ ID NO:17 - amino acid sequence of the precursor of BTN2A protein isoform 1 (Homo sapiens):

MESAAALHFSRPASLLLLLLSLCALVSAQFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVII RDKSVRHM VLLIPQNGFWTLEMHKGQYRAVSSPDRILPLKESLCRVGVFLDYEAGDVSFYНМRDRSHIYTCPRSAFSVPVRPFFRLGCEDSPIFICPALTGANGVTVPEEGLTLHRVGTHQSL

SEQ ID NO:18 - amino acid sequence of the precursor of the BTN2A protein isoform 2 (Homo sapiens):

MEPAAALHFSLPASLLLLLLLLLLSLCALVSAQFTVVGPANPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRITFVSKDINRGSVALVIHNVTAQENGIYRCYFQEGRSYDEAILRLVVAGLGSKPLIEIKAQEDGSIWLECISGGWYPEPLTVWRDPYGEVVPALKEVSIADADGLFMVT TAVIIRDKYVRNVSCSVNNTLLGQEKETVIFIPESFMPSASPWMVALAVILTASPWMVSMTVILAVFIIFMAVSICCIKKLQREKKILSGEKKVEQEEKEIAQQLQEELRWRRTFLHAADVVLDPDTAHPELFLSEDRRSVRRGPYRQRVPDNPERFDSQPCVLGWESFASGKHYWEVEVENNVMVWTVGVCRHSVERK GEVLLIPQNGFWTLEMFGNQYRALSSPERILPLKESLCRVGVFLDYEAGDVSFYNM RDRSHIYTCPRSAFTVPVRPFFRLGSDDSPIFICPALTGASGVMVPEEGLKLHRVGTHQSL

In the present description, the terms “antibody” or “immunoglobulin” mean the same thing and are used interchangeably.

As used herein, the term “antibody” refers to immunoglobulin molecules and their immunologically active moieties, that is, molecules containing an antigen-binding site that binds to the antigen immunospecifically. In this sense, the term "antibody" covers not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments.

As used herein, the term "antibody" also includes bispecific or multispecific molecules. The antibody molecule may be chemically modified (derivatized) or attached to another functional molecule, such as another protein or peptide (such as another antibody or a receptor ligand), such that a bispecific molecule is formed that binds to at least two different binding sites or target molecules. An antibody molecule can be derivatized or coupled to more than one functional molecule so that a multispecific molecule is formed that binds to more than two different binding sites and/or target molecules; in the present description, such multispecific molecules are also included in the term "bispecific molecules". To produce a bispecific molecule, an antibody of the present invention is operably linked (e.g., chemically bonded, genetically fused, formed a non-covalently linked complex, or otherwise) to one or more other binding molecules, e.g., another antibody, an antibody fragment, peptide or mimetic, resulting in a bispecific molecule. Moreover, in the case of an embodiment of the present invention in which the bispecific molecule is multispecific, the molecule also includes a third specific binding site in addition to the specific binding sites of the first and second target epitopes. In one embodiment of the present invention, the bispecific molecules disclosed herein contain as a specific binding site at least one antibody or antibody fragment, including, for example, Fab, Fab', F(ab')2, Fv, unitel or single chain Fv fragments (scFv). The antibody of this invention may also be a light or heavy chain dimer, or some minimal fragment, such as an Fv or single chain construct, as described in US Pat. No. 4,946,778 (Ladner et al).

Other antibodies may also be used in the bispecific molecules described herein: murine, chimeric and humanized monoclonal (mAb).

The bispecific molecules of the present invention are prepared by linking specific binding sites in one way or another using methods known in the art. For example, sections are connected one after another. If the specific binding sites are proteins or peptides, covalent coupling is accomplished using a variety of coupling agents or cross-links. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-( 2-pyridyldithio)propionate (SPDP) and 4-(N-maleimidomethyl)cyclohexanoic acid sulfosuccinimide ester (sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). Other methods include those described in Brennan et al., 1985; Glennie et al., 1987; Paulus, 1985. Another approach is that both nucleotide sequences encoding two different specific binding sites are inserted into one vector, introduced into host cells, so that they are expressed together and combined. This approach is particularly valuable when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') ₂ or ligand x Fab fusion protein. The bispecific molecule of the present invention may be a single chain polypeptide containing one antibody chain and a binding region; or a single-chain bispecific molecule contains two binding sites. The binding of a bispecific molecule to its targets for which it is specific can be verified, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), flow cytometry by fluorescence-activated cell sorting (FACS), biological assay (e.g. inhibition of cell growth or apoptosis) or Western blotting. All these analytical methods, as a rule, detect the presence of the desired protein-antibody complexes using reagents specific to the desired complex, labeled in one way or another (for example, labeled antibodies).

Natural antibody molecules consist of two light and two heavy chains, connected in pairs by disulfide bonds. In humans, two types of light chain are known - lambda (λ) and kappa (κ) and five main types of heavy chain (isotypes) - IgM, IgD, IgG, IgA, IgE - which determine the functional activity of the antibody. In each chain, the amino acid sequence forms several domains. The light chain has two domains - variable (VL) and constant (CL). The heavy chain has four domains - variable (VH) and three constant (CH1, CH2 and CH3, which are collectively designated CH). The variable regions of the light and heavy chains determine the specificity of an antibody, that is, its ability to recognize and bind to a specific antigen. The constant regions of light and heavy chains also perform important biological functions, such as association of chains into an antibody molecule, secretion of antibodies from the cell, ability to cross the placenta, fixation of complement, and binding to Fc receptors (FcR).

The Fv fragment of an antibody is part of the Fab fragment of an immunoglobulin molecule at the N-termini of the polypeptide chains; Fv consists of variable regions of one light and one heavy chain. Antibody specificity is created by structural complementarity between the binding site of the antibody and the antigenic determinant. The antigen-binding regions of the antibody molecule are formed mainly by amino acid residues that are part of the complementarity determining regions, also called hypervariable regions (denoted CDRs). Sometimes amino acid residues from non-hypervariable or framework regions (denoted FR) can participate in the formation of the antigen-binding region and influence the overall domain structure and thereby the binding region. The amino acid sequences of the CDRs together determine the antigen binding affinity and specificity of the natural immunoglobulin Fv region. In each chain of the antibody molecule - both light and heavy - there are three CDR regions, designated in the light chains L-CDR1, L-CDR2, L-CDR3 and in the heavy chains - H-CDR1, H-CDR2, H- CDR3. Thus, the antigen-binding region of an antibody typically includes six CDRs, three from each of the light and heavy chain variable regions. The amino acid sequences of the framework regions are located between the CDRs. Accordingly, the variable regions of the light and heavy chains, as a rule, contain four framework regions and three hypervariable regions: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In the numbering of amino acid residues in the variable domains of immunoglobulins, it is customary to follow the system proposed by Kabat et al.., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (this source is given briefly below: Kabat et al.) This numbering system is used in the present description. The number of an amino acid residue according to the Cabot system does not always coincide with its position in the linear amino acid sequence presented in the present description under the designation SEQ ID NO. In the actual linear amino acid sequence there may be fewer or more amino acids. residues than in strict Cabot numbering; accordingly, a component of the basic structure of the variable domain - a framework region or a hypervariable region - can be shortened or lengthened due to insertion. The correct Cabot numbering for a given antibody is established by aligning the amino acid sequences by homology with the “standard” one. sequence numbered according to Cabot. The heavy chain variable domain CDRs occupy positions of amino acid residues 31-35 (H-CDR1), 50-65 (H-CDR2) and 95-102 (H-CDR3) in Cabot numbering. In the light chain, the CDR regions occupy positions of amino acid residues 24-34 (L-CDR1), 50-56 (L-CDR2) and 89-97 (L-CDR3) in Cabot numbering.

In certain embodiments of the present invention, the antibody of the invention is an antibody fragment, more specifically, any protein comprising the antigen-binding domain of an antibody described herein. Antibody fragments include, but are not limited to, Fv, Fab, F(ab') ₂ , Fab', dsFv, scFv, sc(Fv) ₂ and scFv dimer (diabody).

The term “isolated antibody” as used herein refers to a preparation of that antibody that is substantially free of antibodies of other antigen specificities (e.g., an isolated antibody that specifically binds to BTN2A1 is substantially free of antibodies that specifically bind to antigens other than BTN2A) . An isolated antibody that specifically binds to BTN2 may, however, be cross-reactive to other antigens, for example to proteins related to BTN2 but of a different species origin. Also, the isolated antibody is substantially free of other cellular material and/or other chemicals.

The term “antibody affinity” refers to the degree of affinity (binding strength) of the antigen binding site (paratope) of a given antibody to an antigenic determinant (epitope) of an antigen, for example BTN2A1 as used herein. Affinity is assessed by the value of the dissociation constant of the antigen-antibody complex (Kd).

The term "dissociation equilibrium constant" (denoted K _D ) in the present description refers to the equilibrium constant measured by the ratio k _off /k _on (where k _off is the rate constant of dissociation of the antigen-antibody complex, k _on is the rate constant of its formation) and expressed in units of molar concentration (M).

The K _D value reflects the antibody concentration (the amount of antibody required in a particular experiment); the smaller it is, i.e. the lower the concentration of the antibody, the higher the affinity of the antibody. K _D values for antibodies are determined by standard methods known in the art. Preferred methods for determining the K _D of monoclonal antibodies can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988); Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, 1992, 1993; Muller, Meth Enzymol 1983, the contents of which are incorporated herein by reference. K _D of antibodies is determined by surface plasmon resonance or using a biosensor system, for example Biacore ^® (for details on determining affinity, see also Rich RL, Day YS, Morton TA, Myszka DG High-resolution and high-throughput protocols for measuring drugs/ human serum albumin interactions using BIACORE®. Anal Biochem. 2001) or the Octet® system based on biomolecule layer interferometry (BLI) technology. The BLI method comes down to the analysis of interference patterns of white light reflected from two surfaces - a layer with immobilized proteins on the surface of the biosensor and an internal control layer. The binding between the ligand immobilized on the surface of the biosensor and the analyte in solution leads to a change in the optical thickness of the layer of molecules on the surface of the biosensor and, accordingly, to a shift in the interference pattern. The wavelength shift (δλ in nanometers) is proportional to the change in the optical thickness of the layer of molecules on the surface of the biosensor; δλ is measured over a certain period of time, and a graph of its values versus time is plotted, which gives an association/dissociation curve. These changes due to the interaction of the ligand with the analyte are recorded in real time, allowing monitoring of binding specificity, association/dissociation rates and analyte concentration (see Abdiche et al. 200 and also the Results section). Affinity measurements are usually carried out at 25°C.

The term "association constant", denoted by k_assoc, or k_a,or k_on, as used herein, refers to the rate of association (complex formation) when a particular antibody interacts with a particular antigen, and the term “dissociation constant,” denoted k_dis, or k_d, or k_off, as used herein, refers to the rate of dissociation (breakdown of the complex) upon interaction of a particular antibody with a particular antigen.

The term “monoclonal antibody” or “monoclonal antibody composition” as used herein refers to a preparation of antibodies of a single molecular composition. A monoclonal antibody composition has one specificity and one binding affinity for a particular epitope.

The term “specificity” as used herein refers to the ability of an antibody to detectably bind to an epitope present on an antigen molecule, for example, in the BTN2A protein isoforms (including BTN2A1 and BTN2A2) of the present invention. Generally, as used herein, the term refers to an antibody or protein that binds to human BTN2A, namely BTN2A1, wherein the binding is characterized by a K _D of 10 nM or less, 5 nM, 1 nM or less, 100 pM or less, or 10 pM or less. Typically, the etv value is from ^10-3 pM to 10 nM, namely from 0.1 pM to 10 nM, namely from 0.1 pM to 5 nM or from 1 pM to 10 nM, namely from 1 pM to 5 nM or from 10 pM to 5 nM. Typically, the antibody of the present invention is specific for BTN2A1 or both BTN2A1 and BTN2A2, and is characterized by the K _D specified above. In some embodiments of the present invention, specificity is determined by expressing BTN2A1 in cultured cells (eg, b HEK-293T cells) and by staining transfected cells in the presence of increasing concentrations (eg, 5 ng/ml to 75 μg/ml) of purified monoclonal antibodies to BTN2A, as described herein, with a negative control, for example, an isotype control. By applying nonlinear regression analysis to the obtained data on average fluorescence intensity, EC ₅₀ is determined - the concentration of the specified monoclonal antibody at which the fluorescence intensity is 50% of the maximum value. Typically, the BTN2A1-specific antibody of the present invention binds to this protein with an EC ₅₀ value of less than 50 μg/ml, namely less than 40 μg/ml (see Examples section), namely from 0.1 μg /ml to 50 µg/ml or from 0.5 µg/ml to 20 µg/ml.

The expressions “an antibody that recognizes an antigen” and “an antibody that is specific for an antigen” are used interchangeably herein with the phrase “an antibody that specifically binds an antigen.”

The term "selective binding" generally means that the antibody binds more strongly to the target it is specific for, such as an epitope, than other targets. An antibody binds more strongly to the first target than to the second if its affinity for the first target is higher than for the second. Typically, an antibody binds more strongly to the first target than to the second if the dissociation equilibrium constant (K _D ) or EC ₅₀ discussed above when binding to the first target is less than the K _D or EC ₅₀ when binding to the second target. Most preferably, the agent does not bind to the second target to any significant extent at all. In some embodiments of the present invention, the antibodies of the invention selectively bind to BTN2A, primarily BTN2A1. Binding selectivity can also be expressed in terms of affinity: for example, the ratio of affinities for an antigen for which an antibody is specific and for molecular agents for which it is not specific (for example, an antibody is specific for a BTN2A polypeptide, namely BTN2A1, and a molecular agent for which it is not specific) for which it is not specific is the BTN3 isoform), is about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or more.

The selectivity of antibodies in the above sense can be tested by analyzing the cross-reactivity of the antibody against closely related proteins (eg, BTN3 protein isoforms) compared with the “true” protein target of the antibody (eg, BTN2A isoforms). If, when detecting the reactivity of a given antibody with its “true” target, a significant signal is observed, but with other proteins at the same antibody concentration and for the same period of time - no, then this antibody is considered selective (see the results described in the “Examples” section) when discussing Fig. 3). An antibody is considered to be cross-reactive with a given antigen if its binding exhibits a K _D of 10 nM or less, 1 nM or less, or 100 pM or less. An antibody is considered not to be cross-reactive with a given antigen if its binding exhibits a K _D of 100 nM or greater, 1 μM or greater, or 10 μM or greater. In some embodiments of the present invention, the binding of antibodies to an antigen to which they are not cross-reactive is not detected by standard analytical methods applicable to these proteins (see Table 3 or Fig. 3A).

In certain embodiments of the present invention, the anti-BTN2A1 antibodies disclosed herein exhibit cross-reactivity with the cynoBTN2A1 protein orthologous to the BTN2A1 protein [cynoBTN2A1; registration number NCBI ref XP_015304392.1] having the amino acid sequence SEQ ID NO: 35 given below:

MQRQFSKASRPCLPWVLMEPAAALHFSLPASLILLLLLLRLCALVSAQFTVVGPTDPILAMVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGRTTFVSKDISRGSVALIIHNVTAQENGTYRCYFQEGRSYDEAILHLMVAGLGSKPLVEMRGHEDGGIRLECISRGWYPKPLTVWRDPYGRVVP ALKEVFPPDTDGLFMVTTAVIIRDKSMRНМSCSISDTLLGQKKESVIFIPESFMPSVSPCVVALPIIVVFLMIIIAVCIYWINRLQKETKILSGEKESERK TREIAVKELKKERVQKEKELQVKEQLQEELRWRRTVLHAVDVVLDPDTAHPDLLLSEDRRSVRRCPLGHLGESVPDNPERFNSEPCVLGRESFASGKHYWE VEVENVIEWTVGVCRDSVERKEEVLLRPRNGFWTLEMCKGQYRALSSPKRILPLKESLCRVGVFLDYEAGDVSFYНМRDRSHIYTCPRLAFSVPVRPFFRIGSDDSPIFICPALTGASGITVPEEGLILHRVGTNQSLMPVGTRCYGHGMRPTGFIRMREERGIHRTTREEREPDMQNFDLGAHWSNNLPSARSREFLNSDLVPDHSLESPVTP GLANKTGEPQAEVTCLCFSLPSSELRAFPSTATNHNHKATALGSDLHIEVKGYEDGGIHLECRSTGWYPQPQIQWSNTKGQHIPAVKAPVVADGVGLYAVAASVIMRGSSGEGVSCIIRNSLLGLEKTASISITDPFFRNAQPWIAALAGTLPISLLLLAGASYFLWRQQKEKIALSRETEREREMKEMGYAATKQEISL RGGEKSLAYHGTHISYLAAPERWEMAVFPNSGLPRCLLTLILLQLPKLDSAPFDVIGPPEPILAVVGEDAELPCRLSPNASAEHLELRWFRKKVSPAVLVHR DGREQEAEQMPEYRGRATLVQDGIAEGRVALRIRGVRVSDDGEYTCFFREDGSYEEALVHLKVAALGSDPHISMQVQENGEIWLECTSVGWYPEPQVQWRTSK GEKFPSTSESRNPDEEGLFTVAASVIIRDTSVKNVSCYIQNLLLGQEKEVEIFIPG.

In some embodiments of the present invention, binding of an antibody disclosed herein to the ectodomain of cynoBTN2A1 exhibits an EC ₅₀ comparable (+/- 10%) to the EC ₅₀ obtained for the human protein (huBTN2A1).

The term "identity" refers to the similarity of two amino acid sequences of polypeptides or nucleotide sequences of nucleic acid molecules. If the same monomers (amino acid residues or nucleotides, respectively) are located at a given position in both compared sequences, then the compared molecules at this position are identical. The degree of identity (as a percentage) of two sequences is the number of positions at which monomers match in these sequences, divided by the total number of positions in the sequence and multiplied by 100. Typically, when comparing two sequences, they are aligned to reveal maximum similarity. The degree of identity is calculated by alignment using special computer programs, such as PileUp (Genetics Computer Group, Program Manual for the GCG Package, version 7, Madison, WI, USA) and sequence comparison algorithms, such as BLAST, FASTA or CLUSTALW.

Functional variants of the reference molecules (eg, monoclonal antibody 107G3 mAb) of this invention have functional properties substantially the same as or superior to the functional properties of the reference molecules. The expression "substantially the same" as used herein means that the variant has a functional capacity of the reference molecule that is at least about 50%, 60%, 70%, 80%, 90%, 95% or 100% of that reference molecule. molecules.

In one aspect, the present invention provides an antibody specific for the BTN2A1 protein, as described above. Typically, the binding of such an antibody to human BTN2A1 is characterized by a K _D of 10 nM or less, as described above.

In some embodiments of the present invention, the antibodies provided do not cross-react with BTN3 isoforms.

An antibody specific for BTN2A, namely BTN2A1, according to this invention is generally characterized in that it has at least one of the following properties:

i. suppresses the differentiation of monocytes into macrophages with the M2 phenotype;

ii. causes a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype;

iii. directly triggers NK cell activation;

iv. enhances NK cell-mediated cytotoxicity.

The antibody of this invention preferably has at least one of properties i and ii and at least one of properties iii and iv, most preferably it has properties i - iv.

Antibodies to BTN2A having the preferred properties described above can be detected among antibodies to BTN2A using analytical methods such as those described in the Examples section. These methods and their practical applications are briefly described below.

To determine the formation of the M1 and M2 phenotypes, M2 macrophages are prepared in a manner generally accepted in the art, namely, differentiation is carried out in the presence of antibodies to BTN2A, more specifically to BTN2A1, described above, or under negative control conditions, for example, in the presence of an isotype control. To control the inhibition of differentiation of monocytes into M2 macrophages, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ are added to monocytes during M-CSF-induced differentiation. In contrast, M1 macrophages formed in the presence of GM-CSF can usually serve as a phenotypic control. After differentiation, the resulting population of macrophages is analyzed to detect the expression of plasma membrane markers associated with the M1 and M2 phenotypes using flow cytometry.

Markers of the M1 phenotype that can be readily detected in accordance with the present invention include, but are not limited to those listed here, PDL1, CD86, CD40, CD80 and/or SOCS3. Usually PDL1 and/or CD86 are determined. M2 phenotype markers detectable according to the present invention include, but are not limited to those listed herein, CD14, CD163, CD206 and CD20. Usually CD14 and/or CD163 are determined.

Typically, the anti-BTN2A antibodies of this invention cause suppression of M2 phenotype macrophage differentiation when:

- there is a significant increase in the level of at least one marker of the M1 phenotype when M2 macrophages are formed in the presence of antibodies to BTN2A of the present invention, compared with the isotype control; and/or

- there is a significant reduction in the level of at least one marker of the M2 phenotype when M2 macrophages are formed in the presence of antibodies to BTN2A of the present invention, compared to a negative control, for example an isotype control

It is also possible to establish a cytokine secretion profile in the cell culture supernatant (including, in particular, the anti-inflammatory cytokine IL-10 associated with M2 and the proinflammatory cytokine TNFα associated with M1), which is a defining functional indicator for the separation of macrophages into M1 and M2 phenotypes; cytokines are detected using commercially available kits (eg ELISA). Other cytokines that are also readily detectable to define the M1 and M2 phenotypes of macrophages include IL-1, IL-6, IL-12, and IL-23, which are associated with the M1 phenotype, and TGFβ and IL-10, which are associated with the M2 phenotype.

Thus, in some embodiments of the present invention, differentiation of monocytes into macrophages with the M2 phenotype can be considered to result from the action of the anti-BTN2A antibodies of the present invention when:

- there is a significant reduction in the secretion of at least one cytokine associated with the M2 phenotype when M2 macrophages are cultured in the presence of antibodies to BTN2A according to this invention, compared to a negative control, for example, an isotype control; and/or.

- there is a significant increase in the secretion of at least one cytokine associated with the M1 phenotype when M2 macrophages are cultured in the presence of antibodies to BTN2A according to this invention, compared to a negative control, for example, an isotype control.

In general, the anti-BTN2A antibodies of the present invention inhibit the differentiation of monocytes into macrophages with the M2 phenotype in a dose-dependent manner, as assessed by attenuated expression of markers associated with the M2 phenotype (e.g., CD14 and/or CD163) and/or reduction in cytokine secretion associated with the M2 phenotype (eg IL-10). For such antibodies, the half-maximal inhibition concentration (IC ₅₀ ) of the secretion of said at least one cytokine associated with the M2 macrophage phenotype or the expression of the at least one marker associated with the M2 macrophage phenotype can be determined from a dose-response curve as described in "Examples" section. In certain specific embodiments of the present invention, the anti-BTN2A antibodies of the invention are characterized by:

- IC₅₀For expression of a marker associated with the M2 macrophage phenotype (typically CD14 and/or CD163) ranging from 0.05 μg/ml, more specifically from 0.1 μg/ml to 100 μg/ml, more specifically up to 50 μg/ml; and/or

- IC ₅₀ for the secretion of a cytokine associated with the M2 phenotype (usually IL-10) ranging from 0.01 μg/ml, more specifically from 0.05 μg/ml to 100 μg/ml, more specifically up to 50 μg/ml, most specifically up to 20 μg/ml.

Additionally, or alternatively, the anti-BTN2A antibodies of the present invention, in a dose-dependent manner, shift monocyte differentiation toward the formation of macrophages with the M1 phenotype rather than promoting the predominance of the M2 phenotype; this was determined by increased expression of markers associated with the M1 phenotype (eg, CD86 and/or PDL1) and/or decreased secretion of cytokines associated with the M2 phenotype (eg, IL-10). The half-maximal effective concentration (EC ₅₀ ) of such antibodies with respect to the secretion of said at least one cytokine associated with the M1 phenotype or the expression of said at least one marker associated with the M1 phenotype is determined from a dose-response curve as described in the Examples section " In certain specific embodiments of the present invention, anti-BTN2A antibodies exhibit the following properties:

- EC ₅₀ for the expression of markers associated with the M1 phenotype (e.g. CD86 and/or PDL1) is from 0.01 μg/ml, specifically from 0.1 μg/ml to 100 μg/ml, specifically up to 50 μg/ml , most preferably from 1 to 50 μg/ml; and/or

- The EC ₅₀ for the secretion of cytokines associated with the M1 phenotype (e.g. TNFα) is from 0.01 μg/ml, specifically from 0.05 μg/ml to 100 μg/ml, specifically from 50 μg/ml, most preferably from 1 to 10 µg/ml.

For the M2 phenotype reversion assay, macrophages with the M2 phenotype, which normally differentiate from monocytes in the presence of M-CSF, are cultured with or without lipopolysaccharide (LPS) in the presence of anti-BTN2A antibodies (most preferably anti-BTN2A1, as discussed above) or a negative control. for example, with isotype control. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFNγ are added to M2 macrophage culture as a positive control for reversing the M2 phenotype. M1 macrophages generated in the presence of GM-CSF can generally be used as a phenotypic control. After phenotype reversal, those macrophages that exhibit such reversion without LPS are analyzed by flow cytometry to determine the expression of markers associated with the M1 and M2 phenotypes, as discussed above. Quantitative determination of cytokine secretion is carried out in the same way as described above.

Typically, reversion of the M2 to M1 macrophage phenotype is caused by the anti-BTN2A antibodies of this invention when:

- when culturing M2 macrophages in the presence of these antibodies to BTN2A, a significant increase in the level of markers associated with the M1 phenotype is observed compared to isotype controls; and/or

- when M2 macrophages are cultured in the presence of these antibodies to BTN2A, a significant decrease in the level of markers associated with the M2 phenotype is observed compared to a negative control, for example, an isotype control.

To determine NK cell activation, these cells (usually taken from healthy donors) are cultured with anti-BTN2A antibodies as described herein, or with isotype control antibodies as a negative control, with or without IL-2 and/or IL-15. After at least 48 hours of cultivation, specifically after at least 4 days, the extracellular phenotype of NK cells is determined by markers of their activation, for example, CD69 and/or CD25. NK cell activation is generally considered to be caused by the anti-BTN2A antibodies of this invention when, in the presence of said anti-BTN2A antibodies (with or without further activation by IL-2 and/or IL-15), a significant increase in the level of activation markers is observed NK cells, eg CD69 and/or CD25, compared to a negative control, eg isotype control. The results presented herein clearly demonstrate that the antibodies of this invention initiate direct activation of NK cells.

Increased cytotoxicity involving NK cells is also determined by NK cell degranulation in vitro. This functional assay involves co-culturing the NK cells described above (previously activated with antibodies to BTN2A or treated with a negative control as described above) with cells of a cancer lineage, such as leukemia (myelogenous leukemia) or carcinoma (for example, colon or lung carcinoma, or breast or adenocacinoma) in the presence or absence of IL-2 and/or IL-15. NK cell degranulation is typically determined by flow cytometry as the relative number (percentage) of NK cells having CD107 (in the presence or absence of IL-2 and/or IL-15).

Generally, NK cell cytotoxicity is considered to be caused by anti-BTN2A antibodies when NK cell degranulation [eg, the relative number (percentage) of NK cells having CD107] is significantly increased following activation of NK cells by said anti-BTN2A antibodies compared with a negative control, such as an isotype control. Also typically, the anti-BTN2A antibodies of the present invention cause N-cell degranulation in a dose-dependent manner (specifically against cancer cells of various lineages, as described above). The semi-effective concentration (EC ₅₀ ) of such antibodies for degranulation of NK cells is determined from the dose-response curve, as described in detail in the Examples section.

In the functional assay methods described above, reference antibodies 101G5 and/or 107G3 can be used as a positive control.

In some embodiments of this invention, the anti-BTN2A antibodies described herein inhibit the differentiation of monocytes into M2 macrophages, cause reversion of the M2 macrophage phenotype to the antitumor M1 phenotype, directly initiate NK cell activation, and/or enhance NK cell-mediated cytotoxicity to a level substantially equal to or greater than that of the reference antibodies mAb 101G5 or mAb 107G3, as detailed below. The formulation “inhibits the differentiation of monocytes into macrophages with the M2 phenotype, causes a reversion of the M2 macrophage phenotype to the antitumor M1 phenotype, directly initiates NK cell activation and/or enhances NK cell-mediated cytotoxicity to a level substantially equal to or exceeding that of the reference antibody.” , as used herein means that when a given anti-BTN2A1 antibody is tested, there is a change in that functional activity of less than 20%, specifically less than 15%, less than 10%, and generally less than 5%, compared to any of reference antibodies mAb 107G3 or 101G5.

Also included within the scope of this invention are antibodies that

1) specific for BTN2A, especially BTN2A1, as described above; in particular, antibodies having at least one of the following characteristics:

- bind to human BTN2A1, typically expressed in a cell line, for example HEK293T, transfected with a plasmid encoding human BTN2A1, as described in the Examples section; more specifically, this binding is characterized by an EC ₅₀ of less than 50 μg/ml, more preferably 40 μg/ml; or the binding of said antibodies to BTN2A1 is characterized by a K _D = 10 nm or less and/or the ratio of its affinity to that of nonspecific binding is about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1, or more;

- do not show cross-reactivity with BTN3;

And

2) additionally or alternatively exhibit one or more of the following functional properties:

- activate the formation of cytolytic agents (namely, IFN-γ) by Vγ9Vδ2-T cells; and/or

- activate the cytolytic function of Vγ9Vδ2-T cells; and/or

- activate the proliferation of Vγ9Vδ2-T cells.

It has been observed that proliferation, cytolytic function and production of cytolytic agents (namely, IFN-γ) by Vγ9/Vδ2-T cells are carried out by activated Vγ9/Vδ2-T cells, which are typically activated by the antibodies described herein.

Antibodies to BTN2A1 according to this invention, having such valuable properties, can be detected among antibodies to BTN2A1 using the cellular analysis methods described in the Examples section, in particular the ELISA method using the determination of IFN-γ secretion by Vγ9/Vδ2-T cells and/or or detecting degranulation using CD107 in different cancer cell lines, such as Daudi, Jurkat, L-IPC or MDA-MB-134.

The cytolytic agents of this invention are typically the cytokines IFNγ or TNFα.

As used herein, the phrase “activation of production of cytolytic agents” (typically IFNγ and/or TNFα) means that when comparing the production of at least IFNγ or TNFα by activated Vγ9/Vδ2-T cells and control activated Vγ9/Vδ2-T cells (with IgG1 or hybrid culture medium as a control), a significant increase in this indicator is observed, and these Vγ9/Vδ2 T cells are activated by co-cultivation with target cells (cells of the Daudi, Jurkat, L-IPC or MDA-MB-134 lines ) or phosphoantigens (pAg). In general, activation of IFNγ or TNFα production by activated Vγ9/Vδ2-T cells can be quantified by cellular assays by intracellular labeling with anti-IFNγ or anti-TNFα antibodies as determined by flow cytometry, or by enzyme-linked immunosorbent assay (ELISA) at various concentrations of IFNγ or TNF released by Vγ9/Vδ2-T cells into their culture medium. Such studies are described in more detail below in the “Examples” section (chapter “Materials and methods”).

As used herein, the phrase “activation of cytolytic function of activated Vγ9/Vδ2-T cells” means that when comparing the cytolytic function of activated human Vγ9/Vδ2-T cells and control activated human Vγ9/Vδ2-T cells (with IgG1 or hybridoma culture medium as a control), in the former this function is much stronger, and these human Vγ9/Vδ2 T cells are activated by co-cultivation with target cells (cell lines Daudi, Jurkat, L-IPC or MDA-MB-134) or phosphoantigens (pAg ). As a rule, activation of the cytolytic function of activated Vγ9/Vδ2-T cells is determined by data on the activation of induction of degranulation of Vγ9/Vδ2-T cells by cells of standard lines; in this case, CD107a and CD107b are used together as degranulation markers to identify degranulated Vγ9/Vδ2-T cells. As a positive control when determining activation. Vγ9/Vδ2-T cells are typically treated with phorbol 12-myristate 13-acetate (PMA) with ionomycin. This study is described in more detail below in the “Examples” section.

As used herein, the phrase “increased proliferation of activated Vγ9/Vδ2-T cells” means that when comparing the proliferation of activated Vγ9/Vδ2-T cells and control IgG1-activated Vγ9/Vδ2-T cells, the former exhibit significantly greater proliferation than wherein said Vγ9/Vδ2 T cells are activated by co-cultivation with target cells (cell lines Daudi, Jurkat, L-IPC or MDA-MB-134) or phosphoantigens (pAg). Typically, the proliferation of activated Vγ9/Vδ2-T cells is determined by assessing the functional state of the cells using carboxyfluorescein succinimidyl ester (CFSE) or Cell Trace Violet dye and by flow cytometry of purified Vγ9/Vδ2-T cells from peripheral blood, or by monitoring population proliferation Vγ9/Vδ2-T cells among peripheral blood mononuclear cells with and without a stimulating agent.

In some embodiments of the present invention, anti-BTN2A1 antibodies activate the cytolytic function of activated Vγ9/Vδ2-T cells to a level substantially equal to or greater than the reference mAb 107G3 antibody as described above. The phrase “activation of the cytolytic function of activated Vγ9/Vδ2-T cells to a level substantially equal to or exceeding that of the reference antibodies” as used herein means that when testing antibodies to BTN2A1 in comparison with the reference antibodies mAb 107G3, the difference is less than 15%, specifically less than 10% and typically less than 5% of the cytolytic function of activated Vγ9/Vδ2-T cells.

In some embodiments of the present invention, antibodies to BTN2A1 activate the production of cytolytic agents (i.e., at least IFNγ or TNFα) by activated Vγ9/Vδ2-T cells to a level substantially equal to or greater than that of the reference antibody mAb 107G3, as described above. The phrase “activating the production of at least IFNγ or TNFα by activated Vγ9/Vδ2-T cells to a level substantially equal to or exceeding that of the reference antibodies” as used herein means that when testing antibodies to BTN2A1 in comparison with the reference antibodies mAb 107G3 the difference is less than 15%, specifically less than 10% and generally less than 5% of the production of cytolytic agents by activated Vγ9/Vδ2-T cells.

Reference antibodies mAbs 101G5, 107G3 and their variants

The antibodies described in this application include the reference monoclonal antibodies mAb 101G5, the molecules of which contain the corresponding heavy (VH) and light (VL) chain variable regions represented by SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and mAb 107G3, whose molecules contain the corresponding VH and VL represented by SEQ ID NO:1 and SEQ ID NO:2, respectively.

Other antibodies described in this application include sequences that are at least 90%, namely 95, 96, 97, 98, 99 or 100% identical to the VH and VL regions represented by SEQ ID NO: 1 and 2 or SEQ ID 19 and 20, respectively.

In certain embodiments of the present invention, the anti-BTN2A antibodies described herein are typically humanized anti-BTN2A1 antibodies and contain hypervariable regions (CDRs) in the heavy chain variable region: CDR1 comprising the sequence of SEQ ID NO:3, CDR2 comprising the sequence SEQ ID NO:4 and CDR3 including the sequence SEQ ID NO:5; in the light chain variable region: CDR1 including the sequence SEQ ID NO:6, CDR2 including the sequence SEQ ID NO:7 and CDR3 including the sequence SEQ ID NO:8. In specific embodiments of the antibodies described herein, these six CDRs are 100% identical to the six CDRs of the reference antibody mAb 107G3 represented by SEQ ID NO:3-8.

In other specific embodiments of the present invention, the anti-BTN2A1 antibodies described herein are generally humanized anti-BTN2A1 antibodies, and contain in the heavy chain variable region a CDR1 region comprising the sequence SEQ ID NO:21, a CDR2 region comprising the sequence SEQ ID NO: :22 and CDR3 comprising the sequence SEQ ID NO:23; in the light chain variable region: CDR1 comprising the sequence SEQ ID NO:24, CDR2 comprising the sequence SEQ ID NO:25 and CDR3 comprising the sequence SEQ ID NO:26.

In specific embodiments of the antibodies described herein, these six CDRs are 100% identical to the six CDRs of the reference antibody mAb 101G5 represented by SEQ ID NOs:21-26. Other antibodies described herein include proteins containing regions of CDRs whose amino acid sequences are the result of a mutation - deletion, insertion or substitution of an amino acid residue - but by at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identical to the CDR regions of the reference antibody mAb 101G5. In general, in the antibodies of this invention, 1, 2, 3 or 4 amino acid residues in one or more CDRs may be changed (by deletion, insertion or substitution) compared to the amino acid sequences of the corresponding CDRs in the reference antibody mAb 107G3, represented by SEQ ID NO:21-26.

In other specific embodiments of the antibodies described herein, the six CDRs are 100% identical to the six CDRs of the reference antibody mAb 107G3 represented by SEQ ID NO:3-8. Other antibodies described herein include proteins containing regions of CDRs whose amino acid sequences are the result of a mutation - deletion, insertion or substitution of an amino acid residue - but by at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100% identical to the CDRs of the reference antibody mAb 107G3. In general, in the antibodies of this invention, 1, 2, 3 or 4 amino acid residues in one or more CDRs may be changed (by deletion, insertion or substitution) compared to the amino acid sequences of the corresponding CDRs in the reference antibody mAb 107G3, represented by SEQ ID NO:3-8.

In some embodiments of the present invention, the antibodies provided are mutant variants of mAb 101G5 or mAb 107G3, in which the six CDRs are 100% identical to the corresponding six CDRs of the reference antibodies mAb 101G5 or 107G3, wherein said mutant antibody variants include mutant amino acid sequences resulting from the deletion , insertions or substitutions of no more than 1, 2, 3, 4 or 5 amino acid residues in the framework regions FR1, FR2, FR3 and FR4 compared to the corresponding regions in the corresponding reference antibody.

Functional variants of antibodies

As evidenced by the experimental data presented in the “Examples” section, the reference antibody mAb 107G3 binds to human BTN2A1, interacting with amino acid residues located at positions 65, 68, 69, 72, 78; 84, 85, 95, 97, 100 amino acid sequence of BTN2A1. Thus, within the scope of the present invention are monoclonal antibodies that bind to a conformational epitope containing amino acid residues located at positions 60 to 100 of SEQ ID NO:17, most preferably to a conformational epitope containing amino acid residues located in positions 65, 68, 69, 72, 78; 84, 85, 95, 97, 100 of SEQ ID NO:17, and which have one or more of the functional properties of the reference antibody mAb 107G3 described above.

As experimental data also shows. given in the Examples section, the reference antibody mAb 101G5 binds to BTN2A1 by interacting with amino acid residues located at positions 212, 213, 218, 220, 224, 229 of the BTN2A1 amino acid sequence. Thus, within the scope of the present invention are monoclonal antibodies that bind to a conformational epitope containing amino acid residues located at positions 210 to 230 of SEQ ID NO:17, most preferably to a conformational epitope containing amino acid residues located at positions 212, 213, 218, 220, 224, 229 of SEQ ID NO:17, and which have one or more of the above-described functional properties of the reference antibody mAb 101G5.

In other embodiments of the present invention, the functional variants of the antibodies described herein have full-length amino acid sequences of the heavy and light chains, or heavy and light chain variable regions, or six CDRs regions that are homologous or, more preferably, identical to the corresponding amino acid sequences of any of the reference monoclonal antibodies 101G5 or 107G3 described above, and such functional variant antibodies retain the desired functional properties of the specified reference antibody.

Functional variants of the reference antibody mAb 101G5 or mAb 107G3, specifically functional variants VL, VH or CDRs, according to this invention provide the antibodies described in this application with at least a significant proportion (at least about 50%, 60%, 70% , 80%, 90%, 95% or 100%) affinity (usually defined as _KD , measured by surface plasmon resonance (SPR) or Octet® system interferometry in a layer of biological molecules, which is usually carried out at a temperature of 25 °C) and/or binding selectivity of the parent antibody (e.g., mAb 101G5 or 107G3), and in some cases, such monoclonal antibodies of this invention are found to have greater affinity, selectivity, and/or specificity than the parent antibody (e.g., mAb 101G5 or 107G3 ).

The desired functional properties of the reference antibody mAb 101G5, or 107G3, or a variant of the specified reference antibody described herein, are selected from the group consisting of the following properties:

i. specificity for BTN2A1, in particular for human BTN2A1 expressed in a cell line, for example HEK-293T BTN2 KO, transfected with a plasmid encoding BTN2A1, as described in the Examples section, characterized by preferably an EC ₅₀ of less than 50 μg/ml, more preferably less than 40 µg/ml or K _D measured by SPR at a temperature typically 25°C, or by flow microsphere fluorometry (Luminex platform) as described in the Examples section, or using the Octet® system (Abdiche et al. 2008), equal to 10 nm or less, and the ratio of affinities for binding to BTN2A1 compared to nonspecific binding of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or more and/ or

ii. suppression of differentiation of monocytes into macrophages with an M2 phenotype, usually defined as described in the Examples section, and/or

iii. inducing a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype, usually defined as described in the Examples section, and/or

iv. directly triggering NK cell activation, typically defined as described in the Examples section, and/or

v. enhancement of NK cell cytotoxicity, usually defined as described in the Examples section.

In some more specific embodiments of the present invention, the desired functional properties of the reference antibody mAb 107G3 or a variant of the specified reference antibody described herein are also selected from the group consisting of the following properties:

vi. activation of the production of cytolytic agents (eg, IFNγ or TNFα) by Vγ9/Vδ2-T lymphocytes, usually determined as described in the Examples section, and/or,

vii. activation of the cytolytic function of Vγ9/Vδ2-T cells, usually determined as described in the Examples section, and/or,

viii. activation of Vγ9/Vδ2-T cell proliferation, usually defined as described in the Examples section.

In general, the functional properties according to points (ii) to (v) above of a functional variant of mAb 101G5 or 107G3 are substantially the same as or superior to those of the reference antibody mAb 101G5 or 107G. The expression "substantially the same" as used herein implies that the functional variant has at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% of the property. , 98%, 99% or 100% of the corresponding functional property of the reference antibody mAb 107G3.

Generally, the functional properties of (vi) to (viii) above of the functional variant of the reference antibody mAb 107G3 are substantially the same as or superior to those of the reference antibody mAb 107G3. The expression "substantially the same" as used herein implies that the functional variant has at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% of the property. , 98%, 99% or 100% of the corresponding functional property of the reference antibody mAb 107G3.

For example, this invention relates to functional variants of the reference antibody mAb 101G5 containing the amino acid sequences of the variable heavy chain region (V _H ) and the variable region of the light chain (V _L ), in which the sequences of six CDRs, namely HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2, LCDR3 are at least 60, 70, 90, 95 or 100% identical to the corresponding CDRs of the reference antibody mAb 101G5 represented by SEQ ID NO:21-26, wherein said functional variant specifically binds to BTN2A and has at least one of the functional properties i) - iv):

i. suppresses the differentiation of monocytes into macrophages with the M2 phenotype;

ii. causes a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype;

iii. directly triggers NK cell activation;

iv. enhances cytotoxicity with the participation of NK cells.

Preferably, such an antibody has at least one of properties i) and ii) and at least one of properties iii) and iv), most preferably it has properties i)-iv).

This invention also relates to functional variants of the reference antibody mAb 107G3 containing the amino acid sequences of the variable heavy chain region (V _H ) and the variable region of the light chain (V _L ), in which the sequences of six CDRs, namely HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are at least 60, 70, 90, 95 or 100% identical to the corresponding CDRs of the reference antibody mAb 107G3 represented by SEQ ID NO: 3-8, wherein said functional variant specifically binds to BTN2A1 and has at least one of the following functional properties:

i. suppresses the differentiation of monocytes into macrophages with the M2 phenotype;

ii. causes a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype;

iii. directly triggers NK cell activation;

iv. enhances cytotoxicity involving NK cells

v. activates the production of IFNγ or TNFα by Vγ9Vδ2-T cells;

vi. activates the cytolytic function of Vγ9Vδ2-T cells; and/or

v. activates the proliferation of Vγ9Vδ2-T cells.

Preferably, said functional variant has at least one of the functional activities i) - iv) (preferably at least activities i and/or ii, and iii and/or iv) and at least one of the functional activities v) - vii).

This invention also provides functional variants of the reference antibody mAb 101G5 comprising variable heavy chain and variable light chain amino acid sequences that are at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding light and heavy chain variable regions of said reference antibody mAb 101G5 represented by SEQ ID NO: 19 and 20, respectively, wherein said functional variant specifically binds to BTN2 and has at least one of the following functional properties :

i. suppresses the differentiation of monocytes into macrophages with the M2 phenotype;

ii. causes a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype;

iii. directly triggers NK cell activation;

iv. enhances cytotoxicity with the participation of NK cells

Preferably, such an antibody has at least one of properties i) and ii) and at least one of properties iii) and iv), most preferably it has properties i)-iv).

This invention also provides functional variants of the reference antibody mAb 107G3 comprising variable heavy chain and variable light chain amino acid sequences that are at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding light and heavy chain variable regions of said reference antibody mAb 107G3 represented by SEQ ID NO: 1 and 2, respectively, wherein said functional variant specifically binds to BTN2A and has at least one of the following functional properties :

i. suppresses the differentiation of monocytes into macrophages with the M2 phenotype;

ii. causes a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype;

iii. directly triggers NK cell activation;

iv. enhances cytotoxicity involving NK cells

v. activates the formation of cytolytic agents (IFNγ or TNFα) by Vγ9Vδ2-T cells;

vi. activates the cytolytic function of Vγ9Vδ2-T cells; and/or

vii. activates the proliferation of Vγ9Vδ2-T cells.

In some embodiments of the present invention, said functional antibody variants exhibit at least one of functional activities i) - iv) (mainly at least activities i and/or ii, and iii and/or iv) and at least one of the functional activities v) - vii).

In some embodiments of the present invention, said functional antibody variants exhibit at least one of functional activities i - iv or one or more functional activities v - vii.

In general, the functional properties of (i) to (iv) above of a functional variant of mAb 101G5 or 107G3 are substantially the same as or superior to the corresponding functional properties of the corresponding reference antibody mAb 101G5 or 107G3. The expression “substantially the same” as used herein means that the functional embodiment has at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% of this property. , 99% or 100% of the corresponding functional property of the reference antibody mAb 107G3.

In various embodiments of the present invention, the antibody of the invention may exhibit one or more of the desired functional properties discussed above.

The antibody of this invention may be, for example, human, humanized or chimeric. Typically, the antibody or protein of this invention is a humanized antibody, more preferably a humanized silent antibody.

As used herein, the term “silent antibody” refers to antibodies exhibiting low or no antibody-dependent cell-mediated cytotoxicity (ADCC), as determined by an appropriate in vitro method for lysis of target cells.

In one embodiment of the present invention, the term “low or no ADCC activity” means that the silent antibody exhibits less than 50% ADCC activity; for example, less than 10% of the ADCC activity observed in a corresponding wild-type (non-silenced) antibody, such as wild-type human IgG1. As a rule, when determining ADCC activity in vitro ADCC in a silent antibody in comparison with a control Fab fragment of an immunoglobulin, this activity is not detected.

Silent effector function is achieved by mutating the Fc region of the antibody; this is described in the art in Strohl 2009 (LALA &N297A); Baudino 2008, D265A (Baudino et al., J. Immunol. 2008, Strohl, CO Biotechnology 20 2009). For example, silent antibodies of the IgG1 isotype carry mutations that reduce ADCC activity at positions 234, 235 and/or 331 of the amino acid sequence of the IgG1 Fc region (EU numbering of amino acid residues). Another silent antibody of the IgG1 isotype contains the N297A mutation, which affects the glycosylation of the antibody molecule.

The amino acid sequences of the CDRs in antibody variants may differ from those of the original antibody as a result of substitutions of mostly conservative amino acid residues; there may be at least 10 such substitutions, for example at least 9, 8, 7, 6, 5, 4, 3, 2 or 1. In the context of this invention, conservative substitutions are those substitutions when a different amino acid residue replaces the original one. from the same group of amino acids, divided according to the specificity of the side chains, namely:

Aliphatic - I, L, V, M

Aromatic - F, H, W, Y

Hydrophobic - A, C, F, G, H, I, L, M, R, T, V, W, Y

Negatively charged - D, E

Polar - C, D, E, H, K, N, Q, R, S, T

Positively charged - H, K, R

Small - A, C, D, G, N, P, S, T, V

Very small - A, G, S

Providing rotation of the polypeptide chain - A, C, D, E, G, H, K, N, Q, R, S, P, T

"Flexible" - Q, T, K, S, G, P, D, E, R

Substitutions in the following subgroups are more conservative: valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, asparagine/glutamine. Conservatism in terms of hydrophobicity/hydrophilicity and weight/size properties is also generally observed in the CDRs of the antibody variants compared to any of the monoclonal antibodies 1-6. The importance of the hydrophobicity index of a protein molecule for its interactions with other molecules and biological function is well known in the art. The relative hydropathic nature of amino acids is believed to influence the secondary structure of a protein molecule, which in turn determines its interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and others. Each amino acid is assigned a so-called hydrophobicity index (hydropathicity), based on its hydrophobicity/hydrophilicity and electrical charge, namely: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine(+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); arginine (-4.5). Also or additionally, the consistency of the similarity of amino acid residues is determined by the numerical similarity value in the BLAST group computer programs (for example, in BLAST 2.2.8, available from the US National Center for Biotechnology Information, using the standard BLOSUM62 parameters: gap opening = l l, gap extension = l ). Antibody variants suitable for use in the present invention are typically at least about 80% identical to the original sequence. In accordance with the invention, the term “a first amino acid sequence is at least 70% identical to a second amino acid sequence” means that the first sequence is 70% identical to the second; 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%. The term “a first amino acid sequence is at least 50% identical to a second amino acid sequence” means that the first sequence is 50% identical to the second; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100%.

In some embodiments of the present invention, the antibodies provided are chimeric, typically mouse-human chimeric. The term "chimeric antibody" refers to a monoclonal antibody whose molecule contains the VH and VL domains of an antibody of non-human origin and the CH and CL domains of a human antibody. The source of the non-human antibody can be any animal, such as a mouse, rat, hamster, rabbit, or other. In particular, the specified mouse-human chimeric antibody contains the VH and VL domains of any of the reference antibodies mAb 101G5 or mAb 107G3.

In some embodiments of the present invention, the antibodies provided are humanized. In certain embodiments of the present invention, the antibody of the invention is humanized, comprising the six CDRs of any of the reference antibodies mAb 101G5 or mAb 107G3. As used herein, the term “humanized antibody” refers to antibodies in which framework regions (FRs) have been modified to contain amino acid sequences from a donor immunoglobulin of a different species (e.g., human) than the parent immunoglobulin (e.g., murine CDRs). .

In some embodiments of the present invention, the antibody of interest is selected from the group consisting of Fab, F(ab') ₂ , Fab' and scFv antibody fragments. As used herein, the designation “Fab” means an antibody fragment with a molecular weight of about 50,000, capable of binding antigen; Fab contains approximately half of the N-terminal part of the heavy chain and the entire light chain, connected by a disulfide bond - such a fragment is obtained by treating immunoglobulin with the proteolytic enzyme papain. The designation “F(ab') ₂ ” refers to an antibody fragment with a molecular weight of about 100,000, capable of binding antigen and containing, in addition to those parts of the immunoglobulin molecule that are present in Fab, also a hinge region - such a fragment is obtained by cleavage of the immunoglobulin molecule by another protease, pepsin. The designation “Fab'” refers to a 50,000 molecular weight antibody fragment capable of binding antigen and resulting from cleavage of the disulfide bond at the hinge region of the F(ab') ₂ fragment. The designation "scFv" refers to a single chain polypeptide that is a covalently linked VH:VL heterodimer that is formed by expression of a fusion gene comprising nucleotide sequences encoding VH, VL and a peptide linker. The human scFv fragment according to this invention includes CDR regions that retain the original conformation, which is achieved, as a rule, by recombinant DNA methods.

Functional variants of antibodies with mutant amino acid sequences are obtained by mutagenesis (e.g., site-directed or by polymerase chain reaction) of the encoding polynucleotides, followed by testing for retention of the desired function (e.g., those functions discussed above) in the mutant antibodies using functional analytical methods described in this application.

Cross-competing antibodies

with reference antibodies mAb101G5 or mAb107G3

Additional antibodies having properties similar to the preferred properties of the reference antibody mAb 101G5 or mAb 107G3 described herein are identified by their ability to cross-compete (i.e., competitively inhibit binding to a given antigen in a statistically significant manner) with said reference antibody mAb 107G3 for binding with BTN2A1, which is detected by standard analytical methods.

First, the antibodies to be tested are determined for binding affinity to BTN2A1, for example, using a library of recombinant human antibodies and using a phage display technique or by immunizing transgenic mice that express human variable region antibodies with BTN2A1 antigens, as described in the Examples section (see . "Materials and methods").

In another embodiment of the present invention, antibodies are provided that bind to the same epitope as at least the reference antibody mAb 107G3 or mAb 101G5. As evidenced by the results presented in the Examples section, the reference antibodies mAb 101G5 and mAb 107G3 do not bind to the same BTN2A1 epitope.

The ability of the test antibody to cross-compete with the antibodies of the present invention to human BTN2A1, that is, to inhibit their binding to this protein, demonstrates that the test antibody competes with the antibodies of the present invention for binding to human BTN2A1; It is contemplated, without limitation, that such an antibody may bind to the same or a similar (eg, structurally related or spatially separated) epitope of human BTN2A1 as the antibody for which competition is observed.

To screen anti-BTN2A1 antibodies for their ability to cross-compete with the reference antibody mAb 107G3 and/or to screen anti-BTN2A1 antibodies for their ability to bind to the same epitope as the reference antibody, the following test is used, for example. BTN2KO cells transfected with the human BTN2A1 gene (typically HEK293T cells, as described in the Examples section) are treated with the reference antibody mAb 107G3 at a saturating concentration (eg, 10 μg/ml). Different doses of the BTN2A1 mAbs tested are then examined to determine the competing potential of the mAbs relative to the reference antibody mAb 107G3. Those monoclonal antibodies that compete with the reference antibody will not recognize BTN2A1 in the presence of that reference antibody. The data obtained are expressed as the average fluorescence intensity. Alternatively, competition is determined by epitope-specific sorting using a biosensor system, as described in the Examples section. Typically, for epitope-specific antibody sorting, recombinant human BTN2A1 is immobilized on a biosensor and applied to a reference antibody and then a competing antibody.

Next, the selected antibodies are tested for the preferred properties of the reference antibody mAb 101G5 or mAb 107G3, particularly as described previously.

In one embodiment of the present invention, an isolated antibody is provided that competes with the reference antibody mAb 101G5 or mAb 107G3 for binding to BTN2A1, wherein said antibody

i. has specificity for BTN2A1, in particular binding to human BTN2A1 expressed in cells, for example, the HEK-293T BTN2 KO line, transfected with polynucleotides encoding human BTN2A1, as described in the Examples section; More specifically, this binding is characterized by an EC ₅₀ of less than 50 μg/ml, more specifically less than 40 μg/ml or K _D measured by surface plasmon resonance (SPR) or microsphere flow fluorometry (Luminex platform), as described in section "Examples" or using the Octet® system (Abdiche et al. 2008) of 10 nm or less, and BTN2A1 binding affinity versus nonspecific binding ratios of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or more (see also above for details) and/or

ii. suppresses the differentiation of monocytes into macrophages with the M2 phenotype;

iii. causes a reversion of the M2 macrophage phenotype towards the antitumor M1 phenotype;

iv. directly triggers NK cell activation;

v. enhances cytotoxicity with the participation of NK cells.

Or additionally the specified antibody:

vi. activates the formation of cytolytic agents (IFNγ or TNFα) by Vγ9Vδ2-T cells, determined, as a rule, as illustrated in the “Examples” section; and/or

vii. activates the cytolytic function of Vγ9Vδ2-T cells, determined, as a rule, as illustrated in the “Examples” section; and/or

vii. activates the proliferation of Vγ9Vδ2-T cells, determined, as a rule, as illustrated in the “Examples” section.

In such embodiments of the present invention, the antibodies preferably do not cross-react with BTN3.

Typically, an antibody that competes with the reference antibody mAb 101G5 or 107G3 for binding to BTN2A1 will have functional properties in (iii) to (v) above that are substantially the same as or superior to those of the reference antibody mAb 101G5 or 107G3 their. The expression "substantially the same" as used herein means that the functional variant has at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% of a particular property. 98%, 99% or 100% of the corresponding functional property of the reference antibody mAb 101G5 or 107G3.

Typically, an antibody of the invention that competes with the reference antibody mAb 101G5 or 107G3 for binding to BTN2A1 retains at least a significant portion (at least 50%, 60%, 70%, 80%, 90%, 95 % or 100%) of affinity and/or selectivity compared to the reference antibody (e.g., mAb 107G3), and in some cases it has greater affinity, selectivity and/or specificity than the reference antibody (e.g., mAb 107G3).

In some embodiments of the present invention, the cross-blocking antibodies or antibodies that compete with the reference antibody mAb 101G5 or 107G3 for binding to BTN2A1 are chimeric, humanized, or human recombinant antibodies.

Preparation of transfectomes producing monoclonal antibodies

Antibodies of this invention can be produced by any method known in the art, for example, but not limited to those listed here, any chemical, biological, genetic or enzymatic method, using any one or combination thereof. As a rule, a person skilled in the art, knowing the amino acid sequence of the desired polypeptide, can easily obtain these antibodies using standard methods for preparing polypeptides. For example, they can be synthesized by well-known solid-phase synthesis methods, typically using commercially available peptide synthesis equipment (eg, those from Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions. Alternatively, the antibodies of this invention are produced by recombinant DNA methods well known in the art. For example, antibodies are produced as expression products by inserting nucleotide sequences encoding the antibody into an expression vector and introducing such vector into a suitable eukaryotic or prokaryotic host organism in which the desired antibodies will be expressed, after which they are isolated by known methods.

Accordingly, the next object of the present invention is a nucleic acid (polynucleotide) encoding the antibody described in this application. In particular, such a nucleic acid of the present invention encodes the heavy and light chains of an antibody according to the invention. More specifically, such a nucleic acid contains nucleotide sequences encoding VH or VL that are at least 70%, 80%, 90%, 95% or 100% identical to the corresponding nucleotide sequences encoding a heavy (VH) or mild variable region (VL) chains of the reference antibody mAb 107G3.

Typically, these polynucleotides are DNA or RNA molecules that can be included in any suitable vector, for example, a plasmid, cosmid, episome, artificial chromosome, phage or viral vector. As used herein, the terms “vector,” “cloning vector,” and “expression vector” mean a vehicle in which DNA or RNA nucleotide sequences (e.g., a foreign gene) are introduced into a host organism that is transformed by the vector, thereby causing expression (e.g. , transcription and translation) of the introduced nucleotide sequence. Thus, the next object of this invention relates to a vector containing the polynucleotide described in this application. Such vectors may contain regulatory elements, for example, promoters, enhancers, transcription terminators, etc., to initiate and regulate the expression of the specified antibody after introduction of the vector into the host organism. Examples of promoters and enhancers that are used in animal cell expression vectors include the SV40 simian virus early gene promoter and enhancer, the Moloney murine leukemia virus promoter and enhancer, the immunoglobulin heavy chain gene promoter and enhancer, etc. In animal cells, any expression vector can be used as long as a gene encoding the constant region of a human antibody can be inserted into it, and this gene will be expressed. Examples of vectors suitable for use according to this invention include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, etc. Other examples of plasmids include replicating plasmids containing an origin of replication, or integrative plasmids such as, for example, pUC, pcDNA , pBR, etc. Other examples of viral vectors include adenoviral, retroviral, herpesvirus, and adeno-associated virus (AAV) vectors. Such recombinant vectors are prepared by methods known in the art, for example, by transfection of packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virion packaging cells include PA317, PsiCRIP, GPenv ⁺ , 293, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95/14785, WO 96/22378 and WO 94/ 19478, and also in US patents No. 5,882,877; 6,013,516; 4,861,719 and 5,278,056.

A further aspect of the present invention relates to host cells transfected, infected or transformed with a polynucleotide and/or vector as described above. As used herein, the term “transformation” means the introduction into a host organism/cell of a foreign (i.e. not originating from the organism or cells) gene, DNA sequence or RNA sequence, resulting in expression of the introduced gene or nucleotide sequence and the desired substance is formed, usually a protein or enzyme encoded by the introduced gene or nucleotide sequence. A host cell/organism that has received an introduced gene or nucleotide sequence and produces the product it encodes is called transformed.

The polynucleotides described in this application can be used to produce antibodies of this invention in an expression system suitable for this purpose. The term "expression system" as used herein means a host cell and a compatible vector under suitable conditions, for example, conditions that allow expression of a protein encoded by foreign DNA introduced into the cell as part of the vector. Commonly used expression systems include Escherichia coli host cells and plasmid vectors, insect cells and baculovirus vectors, mammalian cells and vectors suitable therefor. Other examples of host cells include, but are not limited to, prokaryotic cells (eg, bacteria) and eukaryotic cells (eg, yeast, mammals, insects, plants, etc.). Specific examples of host cells include E. coli, Kluyveromyces or Saccharomyces, mammalian cell lines HEK-293, Vero, CHO, 3T3, COS, etc., as well as primary or established cultures of mammalian cells (for example, derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples of host cells also include mouse SP2/0-Ag14 cell (ATCC CRL1581), P3X63-Ag8.653 (ATCC CRL1580), CHO cells with a defective dihydrofolate reductase gene (hereinafter referred to as DHFR) (Urlaub G. et al; 1980), rat cells YB2/3HL.P2.G11.16Ag.20 (ATCC CRL1662; hereinafter these cells are designated YB2/0), etc.

This invention also provides a method for producing recombinant host cells in which the antibodies described herein are expressed, said method comprising the following steps: (i) introducing in vitro or ex vivo the recombinant polynucleotide or vector described above into competent cells; owners; (ii) culturing in vitro or ex vivo the resulting recombinant host cells and (iii) optionally selecting cells that express and/or secrete said antibody. Such recombinant host cells are used to produce the antibodies of the present invention.

Antibodies of the present invention are suitably isolated from the culture medium by conventional immunoglobulin purification methods, for example by protein A-Sepharose or hydroxyapatite column chromatography, gel electrophoresis, dialysis or affinity chromatography.

In some embodiments of the present invention, human chimeric antibodies are prepared as follows: nucleotide sequences encoding the VL and VH domains are prepared as previously described; constructing a vector for expressing human chimeric antibodies by inserting these sequences into an expression vector for animal cells containing genes encoding the CH and CL regions of a human antibody, and expressing the coding sequences by introducing the vector into animal cells. The CH domain of a human chimeric antibody can be any region of the human immunoglobulin molecule, the most suitable being IgG of any subclass, namely IgG1, IgG2, IgG3 and IgG4. Also, the CL domain of a human chimeric antibody can be any region of the human immunoglobulin molecule; for example, polypeptides of the κ and λ classes can be used. The production of chimeric antibodies involves conventional recombinant DNA and transfection techniques well known in the art (see Morrison S. L. et al. (1984) and US Pat. Nos. 5,202,238 and 5,204,244).

The humanized antibodies of this invention are prepared as follows: obtain the nucleotide sequences encoding the CDRs as described previously; constructing a vector for the expression of humanized antibodies by inserting these sequences into an expression vector containing genes encoding (i) a heavy chain constant region and heavy chain variable framework regions identical to those of a human antibody, and (ii) a light chain constant region and variable heavy chain framework regions light chain regions identical to those of the human antibody, and express these genes by introducing an expression vector into cells of a suitable cell line. An expression vector for a humanized antibody can be a vector in which the gene encoding the heavy chain of the antibody and the gene encoding the light chain of the antibody are part of different vector particles, or a vector that contains both of these genes (tandem). For reasons of ease of construction and ease of introduction of the expression vector into host cells, as well as the proper balance between the levels of expression of the H and L chains in the cell, a tandem-type expression vector is preferred for a humanized antibody. Examples of tandem expression vectors for a humanized antibody include pKANTEX93 (WO 97/10354), pEE18, and the like.

Methods for humanizing antibodies based on recombinant DNA technology and transfection are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger M. S., et al. 1985). Antibodies are humanized by various methods known in the art, such as grafting of CDRs (EP 239,400; application WO91/09967; US Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), masking, or modification surface amino acid residues of the variable region (EP 592 106 and 519 596; Padlan E. A. (1991); Studnicka G. M. et al. (1994); Roguska M. A. et al. (1994)), and the combination of light and heavy chain variable domains (US patent No. .5 565 332). The general strategy for using recombinant DNA technology to produce such antibodies is also known (see European Patent Application No. 125 023 and WO 96/02576).

The Fab fragment of this invention is obtained by treating an antibody that specifically interacts with AMH with the proteolytic enzyme papain. Fab can also be obtained by inserting the DNA encoding the Fab of a given antibody into a vector for a prokaryotic or eukaryotic expression system and introducing it, respectively, into a prokaryotic or eukaryotic organism in which the Fab will be expressed.

The F(ab') ₂ fragment of this invention is obtained by treating an antibody that specifically interacts with AMH with the proteolytic enzyme pepsin. F(ab') ₂ can also be prepared by joining the Fab' fragments described below with a thioether or disulfide bond.

The Fab' fragment of this invention is obtained by treating F(ab') ₂ , which specifically binds AMH, with the reducing agent dithiothreitol. Fab' can also be obtained by inserting the DNA encoding the Fab' of a given antibody into a prokaryotic or eukaryotic expression vector and introducing it into a suitable prokaryotic or eukaryotic organism in which the Fab' will be expressed.

To obtain the scFv fragment of this invention, complementary DNA (cDNA) encoding the VH and VL domains is prepared as described above; construct DNA encoding scFv; insert this DNA into a prokaryotic or eukaryotic expression vector and introduce it into a suitable prokaryotic or eukaryotic organism in which the scFv will be expressed.

To obtain a humanized scFv fragment, the well-known method of “grafting” complementarity determining regions (CDRs) is used: CDRs are taken from a donor scFv fragment and combined with scFv framework regions of known spatial structure (see, for example, applications W098/45322; WO 87 /02671; US patents No. 5 859 205; EP No. 0173494;

Engineered antibodies of the present invention also include those in which changes are made to the amino acid residues of the heavy and/or light chain variable region framework regions, for example, to improve the properties of the antibodies. Typically, such changes in the framework regions are made in order to reduce the immunogenicity of the antibody. For example, one approach here is to “backward” mutate one or more amino acid residues of the framework regions to the same as the corresponding germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain amino acid residues in its framework regions that are different from the corresponding amino acid residues in the germline sequence from which the antibody originates. Such amino acid residues can be identified by comparing the amino acid sequences of the framework regions of a given antibody and the corresponding sequences of the germ line from which the antibody is derived. In order to return the framework sequences to the original structure of the germline sequence, the somatic mutation that has occurred must be “reversed” by returning to this position the amino acid residue that was there in the germline sequence, which is achieved by, for example, site-directed mutagenesis or using polymerase chain reaction. Such "reverse mutant" antibodies are also within the scope of this invention. Another modification of the framework regions involves mutating one or more amino acid residues in these regions, or even in one or more CDRs, to eliminate T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, called deimmunization, is described in detail in published US patent application No. 20030153043 (Carr et al.).

Construction of the Fc fragment

The antibodies described herein are characterized by one or more functional or structural properties in the aspects discussed above or a combination of selected functional and structural properties.

The antibodies described herein can be of any isotype. The choice of isotype is generally determined by what effector functions of the antibody are required, such as turning off ADCC activity. The isotypes are, for example, the following: IgGl, IgG2, IgG3 and IgG4. The light chain constant region can be either κ or λ. If necessary, the class of the antibody can be changed by known methods. Typically, antibody class switching methods are used to change the subclass within the IgG class, for example, IgG2 instead of IgG1. Thus, the effector functions of the antibodies of the present invention can be altered by switching the isotype and producing, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies for various therapeutic purposes. In some embodiments of the present invention, the antibody provided is full-length. In some embodiments of the present invention, the provided full-length antibody is of the IgG1 subclass. In some embodiments of the present invention, the provided full-length antibody is of the IgG4 subclass. In some embodiments of the present invention, the BTN2A-specific IgG4 antibody is a stabilized IgG4 antibody. Examples of stabilized IgG4 antibodies suitable for use according to the present invention are those having an arginine residue at position 409 [the numbering of amino acid residues is indicated in the EU numbering system consistent with the Cabot system (Kabat E.A. et al. 1991)] of the amino acid sequence of the constant region the heavy chain of human IgG4 is replaced by a lysine, threonine, methionine or leucine residue, typically lysine (described in application WO2006033386) and/or in which the hinge region contains the sequence Cys-Pro-Pro-Cys. Other suitable stabilized IgG4 antibodies are described in WO2008145142.

In some embodiments of the present invention, the antibodies of the invention do not contain the Fc region required for antibody-dependent cell-mediated cytotoxicity (ADCC). The terms "Fc domain", "Fc region" and "Fc region" refer to the C-terminal portion of the heavy chain of an antibody, for example positions 230 to 450 of the human heavy chain type γ amino acid sequence or the corresponding sequence in heavy chains other types (for example, α, δ, ε and μ in the case of human antibodies), or their natural allotype. Unless otherwise indicated, the generally accepted Kabot numbering of immunoglobulin amino acid sequences is used herein (see Kabat et al. Sequences of Protein of Immunological Interest, 5th edition, US Public Health Service, National Institutes of Health, Bethesda, MD, USA , 1991). In some embodiments of the present invention, the antibodies of the invention do not contain an Fc domain capable of significantly binding to the FcgRIIIA (CD16) polypeptide. In some embodiments of the present invention, the antibodies of the present invention have an incomplete Fc domain (eg, lack a CH2 and/or CH3 domain) or contain an Fc domain of the IgG2 or IgG4 isotype. In some embodiments of the present invention, the antibodies of the invention comprise a Fab, Fab', Fab'-SH, F(ab')2, Fv, a diabody, a single chain antibody fragment, or any of these structures, or contain a multispecific antibody comprising a plurality of different antibody structures, or are such a multispecific antibody. In some embodiments of the present invention, the antibody of the invention is not associated with a toxic agent. In some embodiments of the present invention, one or more amino acid residues are replaced with other amino acid residues, resulting in the antibody having impaired binding to the C2q protein and/or reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in detail in US Patent No. 6,194,551.

Another modification of antibodies disclosed in this application is the addition of polyethylene glycol molecules (PEGylation). This is done, for example, to prolong the biological half-life of the antibody (eg in serum). To attach polyethylene glycol to an antibody or antibody fragment, a reaction is carried out with a reactive ester or aldehyde derivative of polyethylene glycol under conditions in which one or more polyethylene glycol groups are attached to the antibody molecule or antibody fragment. The addition of polyethylene glycol can be accomplished by an acylation or alkylation reaction with reactive PEG (or similar reactive water-soluble polymer) molecules. As used herein, the term "polyethylene glycol" includes any form of PEG that has been used to derivatize other proteins, such as mono(CI-CIO)alkoxy- or aryloxypolyethylene glycol or polyethylene glycol-maleimide. In some embodiments of the present invention, the antibody to which the PEG is attached is not glycosylated. Methods for attaching polyethylene glycol to proteins are well known in the art and are applicable to the antibodies of this invention. See, for example, EP no. 0154 316 (Nishimura et al.) and 0 401 384 (Ishikawa et al.).

Another modification of antibodies contemplated herein are conjugates or fusion proteins comprising at least the antigen-binding portion of an antibody of the present invention and a serum protein, such as human serum albumin or a fragment thereof, thereby increasing the half-life of the entire molecule.

In some embodiments of the present invention, multispecific antibodies are also provided. Examples of multispecific antibody formats of the present invention include, but are not limited to, (i) a heteroconjugate of two chemically cross-linked antibodies, one specific for BTN2A and the other specific for a different antigen; (ii) one antibody containing two different antigen-binding sites; (iii) a single-chain antibody containing two different antigen-binding sites, for example two scFv fragments connected one after another by an additional peptide linker; (iv) antibodies with dual variable domains (DVD-Ig), in which each light chain and each heavy chain contains two variable domains connected one after another by a short peptide linker (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg 2010); (v) a chemically linked bispecific fragment (Fab') ₂ ; (vi) tandem dimeric antibody (TandAb) - two fused single-chain diabodies that together form a tetravalent bispecific antibody, each of which contains two binding sites for each of the target antigens; (vii) flexibody - a combination of scFv fragments with a diabody, forming a multivalent molecule; (viii) a so-called “dock and lock” molecule based on the dimerization and docking domain of protein kinase A, which, when applied to Fabs, results in the formation of a bispecific trivalent binding protein consisting of two identical Fab fragments linked to another Fab fragment ; (5) a so-called scorpion molecule containing, for example, two scFvs fused to both ends of a human Fab; (x) diabody is a non-covalent dimer of scFv. Another example of a bispecific antibody format is IgG-like molecules with complementary CH3 domains, which promote the formation of heterodimers. Such antibodies are produced by known methods, for example, using Triomab/Quadroma technologies (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatic control strategy (Amgen), LUZ-Y (Genentech), method replacement of Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genm ab A/S) chains. In some embodiments of the present invention, bispecific antibodies are produced by controlled exchange of Fab arms, typically using DuoBody technology. Methods for producing bispecific antibodies in vitro through controlled exchange of Fab arms are described in WO 2008119353 and WO 2011131746 (both Genmab A/S). In one typical method, described in WO 2008119353, a bispecific antibody is formed by exchanging "Fab arms" or "half molecules" (a heavy chain and an associated light chain are exchanged) between two monospecific antibodies that both contain IgG4-like CH3 regions , when incubated under reducing conditions. The resulting product is a bispecific antibody containing two Fab arms, which may have different amino acid sequences. According to another exemplary method described in WO 2011131746, bispecific antibodies of the present invention are produced by a method comprising the following steps, wherein at least one of the first and second antibodies is an antibody of the present invention: a) preparing a first antibody containing an Fc region immunoglobulin, this Fc region containing the first CH3 region; b) producing a second antibody containing an Fc region of an immunoglobulin, wherein the Fc region contains a second CH3 region; wherein the amino acid sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions between the first and second CH3 regions; c) incubating said first and second antibodies together under reducing conditions; and d) producing a bispecific antibody, wherein the first antibody is an antibody of the present invention and the second antibody has a different binding specificity, or vice versa. Reducing conditions are created, for example, by adding a reducing reagent, for example selected from 2-mercaptoethylamine, dithiothreitol and tris-(2-carboxyethyl)phosphine. Step d) may further include returning to non-reducing or less reducing conditions, for example by removing the reducing agent, for example by desalting. Typically, the amino acid sequences of the first and second CH3 regions are different and have only a few relatively conservative, asymmetric mutations, such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions between the first and second CH3 regions. More details about these interactions and how they can be accomplished are disclosed in WO 2011131746, the contents of which are incorporated herein by reference in their entirety. The following are typical embodiments of combinations of such asymmetric mutations, where, if necessary, one or both Fc regions are of the IgG1 isotype.

Thus, the present invention provides bispecific or multispecific antibodies (proteins) containing the anti-BTN2A antibody described herein. Accordingly, within the scope of this invention are bispecific antibodies comprising at least one first BTN2A-specific binding site, for example, one antigen-binding site of an antibody described herein, and a second specific binding site to a second target epitope. For example, a bispecific antibody of the present invention may include a single antigen binding region comprising at least:

- heavy chain variable region CDR1 region containing the sequence SEQ ID NO:3, heavy chain variable region CDR2 region containing the sequence SEQ ID NO:4, heavy chain variable region CDR3 region containing the sequence SEQ ID NO:5, variable region CDR1 region light chain comprising the sequence of SEQ ID NO:6, a light chain variable region CDR2 region containing the sequence SEQ ID NO:7 and a light chain variable region CDR3 region containing the sequence SEQ ID NO:8, or

- heavy chain variable region CDR1 region containing the sequence SEQ ID NO:21, heavy chain variable region CDR2 region containing the sequence SEQ ID NO:22, heavy chain variable region CDR3 region containing the sequence SEQ ID NO:23, variable region CDR1 region light chain comprising the sequence of SEQ ID NO:24, a light chain variable region CDR2 region containing the sequence SEQ ID NO:25 and a light chain variable region CDR3 region containing the sequence SEQ ID NO:26.

In one embodiment of the present invention, the proposed bispecific protein contains a second specific binding site for BTN3. More specifically, this bispecific protein also includes the antigen-binding site of activating antibodies to BTN3A, which specifically bind to BTN3A and activate the cytolytic function of Vγ9/Vδ2 T lymphocytes.

Moreover, in an embodiment of the present invention in which the bispecific protein is multispecific, it also includes a third specific binding site in addition to those that bind the first and second epitopes of the target.

In one embodiment of the present invention, the bispecific proteins described herein contain as a specific binding site at least one antibody or antibody fragment, including, for example, Fab, Fab', F(ab') ₂ , Fv, unitel and single-chain Fv. The at least one antibody may be a light or heavy chain dimer, or some minimal fragment, such as an Fv or single chain construct, as described in US Pat. No. 4,946,778 (Ladner et al.).

Other antibodies that can be used in the bispecific proteins described herein include murine, chimeric and humanized monoclonal antibodies.

Pharmaceutical compositions

In another aspect of the present invention, there is provided a composition, for example a pharmaceutical composition, comprising at least one antibody described herein combined with a pharmaceutically acceptable carrier. Such compositions may include a single antibody or a combination of antibodies (eg, two or more different antibodies) described above. The pharmaceutical compositions disclosed herein may also be administered to an individual as part of combination therapy, that is, in combination with other therapeutic agents.

For example, an antibody of this invention is typically combined with at least one antiviral or anti-inflammatory agent or other antiproliferative agent. Examples of therapeutic agents that can be used in combination therapy are described in detail below in the section on the use of antibodies of this invention.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coating agents, antibacterial and antifungal agents, osmotic pressure agents, absorption retarding agents, and other agents. The carrier of this invention should be suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal or intraepidermal administration (eg, by injection or infusion). In one embodiment of the present invention, the carrier is suitable for subcutaneous administration.

Depending on the route of administration into the body, the active substance, that is, the antibody of this invention, can be coated with a material that protects it from the action of acids and other natural factors that can inactivate it. The dosage form of the pharmaceutical composition according to this invention, the route of its administration into the body, the dosage and the schedule of administration depend, of course, on the specifics of the condition to be treated, the severity of the disease, the age, body weight and gender of the patient and other factors.

The pharmaceutical compositions of this invention have a composition suitable for the desired route of administration into the body - topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraorbital, etc.

Applications and methods of this invention

Antibodies of this invention can be used for therapeutic purposes and for diagnostic purposes in vitro and in vivo. For example, these proteins can be administered to cell cultures, for example, in vitro, ex vivo or in vivo, or to a subject, for example in vivo, for the treatment, prevention or diagnosis of various disorders.

The antibodies of this invention are anti-BTN2A1 antibodies capable of inhibiting the differentiation of monocytes into macrophages with a pro-tumor M2 phenotype, cytokine secretion and/or T-cell inhibitory properties.

Alternatively or preferably - in addition to the above - the antibodies of this invention directly bind to BTN2A (mainly BTN2A1) on the plasma membrane of NK cells and trigger their activation and cytotoxicity against cancer cells.

In some embodiments of the present invention, the antibodies also activate the proliferation and cytolytic function of Vγ9/Vδ T cells and their production of cytokines.

Thus, the antibodies of this invention can be used to cope with the mechanisms of immunosuppression observed in cancer and chronic infections

In some embodiments of the present invention, the antibodies of the invention can be used to reduce the immunosuppressive effects of the tumor environment.

The anti-BTN2A antibodies described in this application are also capable of enhancing the cytotoxic effect of NK cells and/or Th1 lymphocytes, affecting both the tumor microenvironment (promoting the differentiation of monocytes into macrophages with the M1 phenotype and suppressing differentiation according to the M2 phenotype), and directly on NK cells.

In some embodiments of the present invention, the antibodies provided (eg, antibody 107G3 and its variants described herein) also activate the proliferation and cytolytic function of Vγ9/Vδ T cells and their production of cytokines. Such antibodies can act simultaneously in three compartments of the immune defense: NK cells, macrophages and γδ T cells, thereby representing a powerful tool for the treatment of cancer, namely solid tumors.

Preclinical studies have convincingly shown that NK cells are capable of eliminating myeloid leukemia cells. However, for example, in chronic myeloid leukemia, as the disease progresses, the number of NK cells becomes smaller, they respond less well to stimulation and exhibit reduced cytolytic activity. In acute myeloid leukemia, higher cytolytic activity of NK cells both at diagnosis and in remission is a sign of better treatment outcome in the long term (Carlsten M, Järås M. Natural Killer Cells in Myeloid Malignancies: Immune Surveillance, NK Cell Dysfunction, and Pharmacological Opportunities to Bolster the Endogenous NK Cells. Front Immunol. Therefore, in some embodiments of the present invention, antibodies exhibiting NK cell-priming properties can be used in combination with NK cell-targeting therapies, such as adoptive cell therapy, which involves transferring NK cells to a patient to restore their function, and/ or triggering or enhancing their cytotoxicity. In particular, the antibodies of this invention can be used in the treatment of solid tumors that are generally resistant to the killing action of NK cells. As used herein, the terms “cancer/cancerous disease,” “hyperproliferative/hyperproliferative,” and “neoplasia/neoplastic” refer to cells that have the ability to autonomously reproduce, that is, an abnormal condition characterized by the rapid proliferation (multiplication and spread) of cells. A hyperproliferative and neoplastic disease/condition can be classified as pathological, that is, characterized by or being a disease state, or can be classified as non-pathological, that is, deviating from the normal, but not associated with a disease state. These terms include all types of malignant growth or oncogenic processes, formation of metastases or malignantly transformed cells, tissues or organs, without regard to histopathological type or stage of invasiveness.

The terms “cancer” or “neoplasm” include malignant neoplasms/tumors of various organ systems, such as lesions of the lungs, breast, thyroid gland, lymphoid organs, gastrointestinal tract and genitourinary tract, as well as adenocarcinomas, which include malignancies such as most colon cancer, squamous cell carcinoma of the lung, skin or vagina, renal cell carcinoma, prostate cancer and/or testicular tumors, non-small cell lung carcinoma, small cell lung carcinoma, endometrial carcinoma, ovarian carcinoma, endocervical adenocarcinoma, pancreatic cancer, small intestinal cancer, esophageal cancer, and in a broader sense, any cancer that can be treated by applying in vivo stimulation of γδ-T cell activation and/or proliferation to patients with these cancers.

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

Examples of cancers that do not involve the circulatory system 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, malignant tumors of bone cells, cervical cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, testicular cancer and skin cancer.

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

Accordingly, the present invention provides a method of treating one or more of the disorders set forth above in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the anti-BTN2A antibodies described herein.

Antibodies for the uses described above are administered alone, as a single active ingredient, or in combination with auxiliary ingredients or with other therapeutic agents, for example, cytokines, antiviral, anti-inflammatory or cytotoxic, antiproliferative, chemotherapeutic or antineoplastic agents, cell therapy agents (eg , γδ-T-cell or N-cell compositions), for example for the treatment or prevention of the diseases mentioned above.

For example, antibodies for the above applications can be used in combination with cell therapy, in particular using γδ-T cells, NK cells, or with chemotherapy, antineoplastic or immunotherapeutic agents.

As used herein, the term “cell therapy” refers to a therapeutic approach comprising administering to a subject in need thereof in vivo at least a therapeutically effective amount of a cellular composition. The introduced cells can be allogeneic or autologous to the patient. The term “γδ T cell therapy” refers to a cell therapy in which the cell composition includes γδ T cells, particularly Vγ9/Vδ2 T cells, as the active principle (e.g., adoptive transfer of γδ T cells or γδ-T cells, which express the chimeric antigen receptor). The term “NK cell therapy” refers to a cell therapy in which the cell composition includes NK cells as an active component, for example, adoptive transfer of NK cells or NK cells expressing chimeric antigen receptor (CAR-NKs).

The term "cell therapy agent" refers to a composition of cells that is administered to a specified patient for therapeutic purposes. Said cell therapy agent comprises a therapeutically effective dose of cells and, if necessary, additional excipients, excipients or other pharmaceutically acceptable carriers.

Suitable antineoplastic agents include, but are not limited to those listed herein, alkylating agents (eg, cyclophosphamide, mechlorethamine, chlorambucil, melphalan, nitrosourea, temozolomide), anthracyclines (eg, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin), taxanes (eg, pacitaxel , docetaxel), epothilones, topoisomerase II inhibitors (for example, etoposide, teniposide or tafluposide), nucleotide analogues and precursors (for example, azacitidine, azathioprine, capecitabine, cytarabine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrecesate or thioguanine), peptide antibiotics ( e.g., carboplatin, cisplatin and oxaliplatin), retinoids (e.g., tretinoin, alitretinoin, bexarotene), vinca alkaloids and their derivatives (e.g., vinblastine, vincristine, vindesine, vinorelbine), targeted therapies, e.g., kinase inhibitors (e.g., ibrutinib, idelalisib , erlotinib, gefitinib, imatinib, vemurafenib, vismodegib), proteasome inhibitors (for example, bortezomib, carfilzomib), histone deacetylase inhibitors (for example, vorinostat or romidepsin).

Examples of immunotherapeutic agents include, but are not limited to, phosphoantigens (eg, zoledronic acid or other bisphosphonates), programmed cell death protein (PD-1) antibodies, PD-L1 antibodies, CTLA-4 antibodies and cytokines [for example, 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 their derivatives or recombinant forms, or any cytokines capable of inducing lymphocyte activity (eg proliferation, or cytokine production, or metabolic changes). The term “derivative” is used to refer to any modification of a cytokine, such as the addition of polyethylene glycol (PEG), mutations, such as deletions, substitutions or insertions of amino acid residues, or association with potentiating agents [for example, IL15/IL15Ra complexes fused to the IgG1 Fc, where IL-15 has an additional mutation (asn72asp), which further increases biological activity, so that this complex becomes a superagonist of IL-2 and IL-15Rβγ (Rhode P.R. et al, Cancer Immunol. Res. 2016) (Barroso-Sousa R. et al , Curr. Oncol. Rep. 2018).

The term "IL-2" has its common meaning and as used herein refers to human interleukin-2. IL-2 mainly regulates the activity of lymphocytes by binding to its receptor on their surface.

The term "IL-15" has its common meaning and as used herein refers to human interleukin-15. Like IL-2, IL-15 signals lymphocytes by binding to 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 NK cells.

The term "IL-21" has its common meaning and as used herein refers to human interleukin-21. IL-21 is associated with pleiotropic properties, including, but not limited to, increased cytotoxicity of NK cells and (CD8 ⁺ ) T cells, effects on plasma cell differentiation and suppression of T _reg cells.

The term "IL-33" has its common meaning and as used herein refers to human interleukin-33. IL-33 is thought to serve as an endogenous alarm signal that is released when tissue is stressed or damaged; it belongs to the interleukin-1 family and binds to the ST2 receptor. IL-33 is known to effectively stimulate T _H 1 immune cells, NK cells, invariant lymphocytes bearing both NK cell markers and T cell differentiation antigens (iNKT cells), and (CD8) T cells.

The designation “PD-1” has its common meaning and as used herein refers to programmed cell death receptor protein 1. It also belongs to the type I transmembrane protein of the CD28-B7 family of receptor signaling proteins, which includes the co-stimulatory coreceptor CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), inducible costimulatory protein (ICOS), and an inhibitory receptor protein called B/ T-lymphocyte attenuator (BTLA) (Greenwald RJ et al., 2005, Riley JL et al. 2005).

The terms “anti-PD-1 antibody” or “anti-PD-L1 antibody” have their common meaning and refer to antibodies that have binding affinity for PD-1 or its ligand PD-L1, respectively, and are PD-1 antagonists, i.e. inhibitory a signal transduction cascade involving PD-1 and inhibiting the binding of PD-1 to its ligands PD-L1 and PD-L2. Such anti-PD-1 or anti-PD-L1 antibodies preferentially inactivate PD-1 with greater affinity and potency than other subtypes and isoforms of the CD28-B7 family of signaling receptor proteins (CD28; CTLA-4 ; ICOS; BTLA). Analytical methods for determining PD-1 antagonism are well known to those skilled in the art; they are described, for example, in Greenwald et al., 2005; Riley et al., 2005.

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

In accordance with the above, in another aspect of the present invention there is provided:

a method described above, comprising co-administration, for example, simultaneous or sequential, of a therapeutically effective amount of an anti-BTN2A1 antibody of this invention and at least one other drug, wherein said other drug is an antiviral or anti-inflammatory agent, or an immunotherapeutic agent, or a cytokine or cell therapy agent (eg, γδ T cells), for example, as above.

In one embodiment of the present invention, the antibodies of the invention are used to detect the level of soluble BTN2A1 or the number of cells in which BTN2A1 is expressed. This is achieved, for example, by contacting a sample (e.g., an in vitro sample) and a control sample with antibodies to BTN2A1 under conditions that allow such antibody to form a complex with BTN2A1 (in a form exposed on the cell surface or in a soluble form, e.g. in a sample blood). Any complexes formed by the indicated antibody and BTN2A1 are detected and the sample is then compared with the control. For example, standard methods well known in the art, such as ELISA and flow cytometry, can be used with the compositions of this invention.

One aspect of the invention also provides methods for detecting the presence of BTN2A1 (eg, human BTN2A1 antigen) in a sample or quantifying BTN2A1, comprising contacting the sample and control sample with an antibody or protein of the invention, or an antigen-binding site thereof, that bind specifically to BTN2A1, under conditions that ensure the formation of a complex of the antibody or its part with BTN2A1. The formation of the complex is then detected and the presence of BTN2A1 in the sample is determined from the difference between the data obtained for the sample and the control.

Also included within the scope of this invention are kits consisting of compositions (eg, humanized antibodies, conjugated antibodies, and multispecific proteins) of this invention and instructions for their use. The kit of this invention may also contain at least one additional reagent, or one or more additional antibodies, or one or more additional proteins. Typically the kit includes a label indicating the intended use of the kit's contents. The term “label” includes information attached to the kit on paper or other media; the label may be on or inside the packaging, or otherwise accompany the kit. The kit of this invention may also contain means for determining whether a given patient is in a group that will benefit from treatment with anti-BTN2A1 antibodies as described above.

Another therapeutic approach is based on the use of humanized antibodies described herein as agents that selectively activate NK cells isolated from a human sample.

Thus, this invention relates to a method of treating a subject in need thereof, comprising:

(a) isolating blood cells including NK cells, such as peripheral blood mononuclear cells (PBMCs), from a blood sample of the subject;

(b) expansion of NK cells in vitro in the presence of antibodies to BTN2A1, as described in this application, and, if necessary, other cells - tumor or auxiliary;

(c) collecting expanded NK cells;

(d) if necessary, preparing a preparation of expanded NK cells and administering a therapeutically effective amount of said NK cells to the subject.

This invention also relates to the use of humanized antibodies described herein as agents that selectively activate chimeric antigen receptors (CAR) of NK cells. CAR NK cell proteins and their use in adoptive NK cell immunotherapy for cancer are described, for example, in Rezvani, K. et al. “Adoptive cell therapy using engineered natural killer cells” (Bone Marrow Transplant 2019).

The present invention also provides anti-BTN2A1 antibodies disclosed herein for use in vivo as potentiating agents in NK cell therapy in a subject in need thereof, typically a cancer patient.

As used herein, the term “NK cell therapy” refers to a treatment comprising administering to a subject in need thereof at least an effective amount of NK cells. The N cells administered may be allogeneic or autologous to the subject. In specific embodiments of the present invention, the NK cells for such treatment are genetically modified by deletion or knockout, or insertion or knockout of certain genes. In specific embodiments of the present invention, said NK cells include NK cells that express the chimeric antigen receptor. These NK cells are then expanded and/or purified ex vivo. Alternatively, these NK cells are included in a cellular composition containing other blood cells and, for example, other cells of the immune system. References to papers that discuss γδ T-cell therapy can be found in Rezvani, K. Bone Marrow Transplant 2019.

The present invention also relates to a method for treating cancer patients, for example those suffering from malignant diseases of the circulatory system, in particular leukemias, for example acute myeloid leukemia, and having tumor cells, for example malignant blood cells, the method comprising:

(i) administering to said subject an effective amount of the anti-BTN2A1 antibodies described herein, and

(ii) administering to said subject an effective amount of the NK cell composition;

wherein said effective amount of anti-BTN2A1 antibodies may promote anti-tumor cytolysis mediated by said NK cell composition directed against said tumor cells. This invention also provides a method of treating a subject in need thereof, the method comprising co-administration (simultaneous or sequential) of NK cells, for example, CAR-bearing NK cells, and humanized antibodies described herein.

In alternative or additional embodiments of the present invention, the therapeutic approach also relies on the use of humanized antibodies described herein as agents that selectively cause the expansion and/or activation of Vγ9/Vδ2-T cells isolated from a human sample.

The present invention also relates to a method of treating a subject in need thereof, comprising:

(a) isolating blood cells including Vγ9/Vδ2 T cells, for example, isolating PBMC from a blood sample of a subject;

(b) in vitro expansion of Vγ9/Vδ2-T cells in the presence of antibodies to BTN2A1 described in this application, and, if necessary, other cells - tumor or auxiliary;

(c) collection of grown Vγ9/Vδ2-T cells,

(d) if necessary, preparing a preparation of expanded Vγ9/Vδ2-T cells and administering to the subject a therapeutically effective amount of said Vγ9/Vδ2-T cells.

This invention also relates to the use of humanized antibodies disclosed herein as agents that selectively cause the expansion of Vγ9/Vδ2-T lymphocytes bearing a chimeric antigen receptor (CAR). (CAR)γδ T cells and their use in adoptive T cell immunotherapy for cancer are described, for example, in Mirzaei et al, Cancer Lett. 2016.

The antibodies of this invention can also be used to produce an artificial T cell receptor [also known as chimeric T cell receptor or chimeric antigen receptor (CAR). For example, antibody variable regions can be used to create Fabs or scFvs that can be linked via a spacer to the transmembrane domain and signaling endodomain of a T cell receptor (TCR), and then displayed on the surface of T cells. Such CARs are used in therapy with adoptive transfer of immunocompetent cells, for example, for the treatment of proliferative disorders.

The present invention also provides anti-BTN2A1 antibodies for use in vivo as potentiating agents in γδ T cell therapy in a subject in need thereof, typically a cancer patient.

As used herein, the term “γδ T cell therapy” refers to a treatment comprising administering to a subject in need thereof at least an effective amount of γδ T cells. The γδ cells administered may be allogeneic or autologous to the subject. In specific embodiments of the present invention, the γδ T cells for such treatment are genetically modified by deletion or knockout, or insertion or knockout of certain genes. In specific embodiments of the present invention, said γδ T cells include γδ T cells that express the chimeric antigen receptor. These γδ T cells can then be expanded and/or purified ex vivo. Alternatively, these γδ T cells can be included in a cellular composition containing other blood cells and, for example, other cells of the immune system. References to works that discuss γδ-T-cell therapy can be found in the works of Pauza C. D. et al, Front. Immunol. 2018; J. Saudemont A. et al, Frontiers Immunol. 2018.

Without being limited by theory, the proposed mechanism of action of the anti-BTN2A1 antibodies of the present invention is that their binding to BTN2A1 exposed on the surface of a tumor cell initiates a conformational change that signals a counter-receptor on Vγ9Vδ2-T cells.

This invention also relates to a method of treating a patient with cancer, for example a malignant disease of the circulatory system, in particular leukemia, for example acute myeloid leukemia, who has tumor cells, for example malignant blood cells, the method comprising:

(i) administering to said subject an effective amount of the anti-BTN2A1 antibodies disclosed herein, and

(ii) administering to said subject an effective amount of the γδ-T cell composition;

wherein said effective amount of anti-BTN2A1 antibodies promotes anti-tumor cytolysis mediated by said γδ-T cell composition directed against said tumor cells. The present invention also provides a method of treating a subject in need thereof, the method comprising co-administration (simultaneous or sequential) of CAR-bearing T cells, for example, (CAR)γδ T cells, and humanized antibodies described herein.

The invention is further illustrated by figures and examples. However, these examples and figures should not be construed as limiting the scope of the invention in any way.

Captions for figures

Figure 1. Identification of mAb 107G3 to BTN2A1 mAb. A. Sequence of steps for screening monoclonal antibodies to BTN2A1 from immunization of mice to protein sequencing. B. The bar graph shows the number of clones with the affinity ranges indicated below (K _D ) as determined by flow microsphere fluorometry (Luminex platform) during primary screening. C. Stacked line graph shows the number of clones classified as neutral (gray), antagonist (white), or agonist (black) based on their ability to influence IFN-γ production by Vγ9/Vδ2-T cells during primary (1st cycle) and secondary (2nd cycle) screening.

Figure 2. Anti-BTN2A1 mAb 107G3 enhances the cytolytic function of Vγ9/Vδ2-T cells. Vγ9/Vδ2-T cells from PBMCs from three healthy donors were expanded (see Materials and Methods) and co-cultured at 37°C with target cells at an effector:target (E:T) ratio of 1:1 in the presence of antibodies to CD107a/b and an inhibitor of protein transport in the Golgi apparatus, in the presence or absence of these antibodies. After 4 hours, cells were harvested, fixed, and analyzed by flow cytometry. In fig. A shows data with various cell lines targeted, including Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), L-IPC (pancreatic adenocarcinoma), and MDA-MB-134 (breast carcinoma), in the presence or in absence of supernatant containing anti-BTN2A1 mAb 107G3 or control hybridoma culture medium. The bar graph shows the relative number (percentage) of CD107 ⁺ cells corresponding to Vγ9/Vδ2-T cell degranulation. In fig. B shows data for Daudi cells as target cells in the presence of the indicated concentrations of purified anti-BTN2A1 mAb 107G3 or the corresponding mouse IgG1 as isotype control. The graph is a dose-response curve from which the EC ₅₀ is calculated.

Figure 3. Anti-BTN2A1 mAb 107G3 recognizes BTN2A1 but not BTN3. HEK-293T BTN2 KO cells were transiently transfected with a plasmid encoding the BTN2A1-CFP fusion protein. A. Histograms show overlap between the indicated cells and transfected cells stained for binding to purified anti-BTN2A1 mAb 107G3 (top, black line), anti-BTN3 mAb 103.2 (bottom, black line), or control mIgG1 or IgG2a (dashed lines). For transfected cells, staining is shown after setting the CFP ⁺ cell discrimination window. B. Graph of dose-response curve for binding of purified mAb 107G3 to BTN2A1 on HEK-293T BTN2 KO cells transfected with a plasmid encoding BTN2A1-CFP. Staining was analyzed after setting the CFP ⁺ cell discrimination window.

Figure 4. Expression of BTN2A in NK cells and monocytes and the effect of reference monoclonal antibodies 101G5 and 107G3 to BTN2A on the differentiation of monocytes into macrophages with the M2 phenotype. (A) Histograms represent expression of BTN2A1 and BTN2A2 (white) versus isotype control (gray) on NK cells and monocytes from unstimulated PBMCs from healthy donors by flow cytometry. (B) 2D flow cytometry plots of CD14/CD163 after 5 days of in vitro differentiation for M1/M2 macrophages or macrophages induced by antibodies 101G5 and 107G3 in the presence of M-CSF.

Figure 5: Reference monoclonal antibodies 101G5 and 107G3 to BTN2A inhibit differentiation into M2 macrophages in a dose-dependent manner. Macrophages M1; M2; M2 differentiated by GM-CSF and IFNγ and macrophages induced by M-CSF in the presence of various concentrations of 101G5 or 107G3 antibodies (or their isotype control) acquired a phenotype within 5 days and were stimulated or not stimulated for another 2 days LPS. The expression of CD14 (A), CD163 (B), PDL1 (C) and CD86 (D) in unstimulated (A-C) or LPS-stimulated cells (D) was analyzed by flow cytometry. Results are presented as mean fluorescence intensity (MFI) subtracted from isotype control values. In the supernatant from stimulated macrophages. LPS, the amount of IL-10 (E) and TNFα (F) was determined by ELISA. Results are expressed in pg/ml.

Figure 6: Reference monoclonal antibodies 101G5 and 107G3 to BTN2A inhibit the differentiation of monocytes into M2+IL-4 macrophages. M1 macrophages were obtained; M2; M2+IL4; M2 differentiated by GM-CSF and IFNγ; and macrophages induced with M-CSF+IL-4 and antibody 101G5 or 107G3 mAbs (or their isotype control) at a concentration of 10 μg/ml for 5 days and for another 2 days they were stimulated or not stimulated with LPS. The expression of CD14 (A), CD163 (B), PDL1 (C), DC-SIGN (D), and CD86 (E) was analyzed by flow cytometry on unstimulated cells (A-D) and on LPS-stimulated cells (E). Results are presented as mean fluorescence intensity (MFI) subtracted from isotype control values. In the supernatant from macrophages stimulated with LPS, the amount of IL-10 (E) and TNFα (F) was determined by ELISA. Results are expressed in pg/ml.

Figure 7: Reference monoclonal antibodies 101G5 and 107G3 to BTN2A inhibit cancer cell-induced differentiation of monocytes into M2 macrophages. M1 macrophages were obtained; M2; M2, induced by medium conditioned by PANC-1 cells; M2 differentiated by GM-CSF and IFNγ; and macrophages induced with medium conditioned by PANC-1 cells and antibody 101G5 or 107G3 (or their isotype control) at a concentration of 10 μg/ml, for 5 days and for another 2 days they were stimulated or not stimulated with LPS. The expression of CD14 (A), CD163 (B) on unstimulated cells was analyzed by flow cytometry. Results are presented as mean fluorescence intensity (MFI) subtracted from isotype control values. In the supernatant from macrophages stimulated with LPS, the amount of IL-10 (C) and TNFα (D) was determined by ELISA. Results are expressed in pg/ml.

Figure 8: Reference monoclonal antibodies 101G5 and 107G3 to BTN2A change the phenotype of M2 macrophages towards a pro-inflammatory M1 phenotype and cytokine secretion.

Macrophages M1 and M2 were obtained from monocytes for 5 days. After this period, antibody 101G5 or 107G3 (or isotype control) at a concentration of 10 μg/ml or IFNγ was added to M2 macrophages and left for 2 days, after which they were stimulated or not stimulated with 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 (A–C) or on cells stimulated with LPS (D). Results are presented as mean fluorescence intensity (MFI) subtracted from isotype control values. In the supernatant from macrophages stimulated with LPS, the amount of IL-10 (E) and TNFα (F) was determined by ELISA. Results are expressed in pg/ml.

Figure 9. Reference monoclonal antibodies 101G5 and 107G3 to BTN2A abolish M2 macrophage-mediated suppression of T cell proliferation and IFNγ secretion. Differentiated M1, M2 macrophages or macrophages induced in the presence of antibodies 101G5 and 107G3 (or their isotype control) were co-cultured with allogeneic (CD3 ⁺ )T cells labeled with CTV that had been activated with OKT3 antibodies for 5 days. After coculture, cells were stimulated for 5 hours with PMA/ionomycin and the protein transport inhibitor GolgiStop; the number of (CD3 ⁺ )T cells (A and B), intracellular IFNγ production (CF) and proliferation (CellTrace Violet, CTV ^dim cells) (GJ) were then determined by flow cytometry. Proliferation was determined by CTV dye dilution (the CTV signal on Day 0 was taken as background). Results are presented as absolute counts of (CD3 ⁺ )T cells (B, E, F, I and J) using CountBright absolute counting beads for calibration, or as relative counts (percentage) of (CD3 ⁺ )T cells (C , D, G and H).

Figure 10. Effect of reference monoclonal antibodies 101G5 and 107G3 to BTN2A on the activation and cytotoxicity of purified NK cells. (A-B) Purified NK cells were cultured with reference monoclonal antibodies 101G5 and 107G3 to BTN2A (or isotype control) for 5 days under stimulation with IL-2 or IL-2/IL-15. NK cell activation was determined by the expression of CD69 (A) and CD25 (B), expressed as mean fluorescence intensity (MFI) in unstimulated and IL-2/IL-15 stimulated NK cells in the presence of the indicated antibodies or isotype control. (C-D) Purified NK cells were preincubated overnight with reference anti-BTN2A monoclonal antibodies 101G5 and 107G3 (or isotype control) with or without IL-2/IL-15 stimulation and then cocultured with human tumor cells for 4 hours Degranulation of NK cells with tumor cells of each line in the presence of the indicated antibodies or isotype control was determined by flow cytometry; The measure of degranulation was the relative number (percentage) of cells with CD107αβ among unstimulated (C) and IL-2/IL-15 stimulated NK cells (D). (E) Degranulation of NK cells with A549 cells when anti-BTN2A reference antibodies 101G5 and 107G3 (or isotype control) were preincubated with NK cells or target cells prior to 4-h coculture, compared with when these antibodies were added to the co-culture without preincubation.

Figure 11. Reference monoclonal antibodies 101G5 and 107G3 to BTN2A enhance NK cell degranulation and adenocarcinoma cell death. (A) Purified NK cells were preincubated with reference anti-BTN2A monoclonal antibodies 101G5 and 107G3 (or isotype control) with or without IL-2/IL-15 stimulation and then cocultured with DU-145 cells for 4 hours. NK cell degranulation was determined by flow cytometry as the relative number (percentage) of CD107αβ ⁺ cells. For the indicated antibodies, the EC ₅₀ of enhancing NK cell degranulation was calculated using a 4-parameter logistic curve in the Prism program. (B) Purified NK cells were preincubated overnight with reference anti-BTN2A monoclonal antibodies 101G5 and 107G3 (either isotype control or IL-2/IL-15 stimulation) and then cocultured for 4 hours with HL- cells. 60 and A549. NK cell-mediated cancer cell death was determined by the relative abundance of caspase 3/7 cells in the presence of the indicated antibodies or isotype control.

Figure 12. Epitope-specific sorting of reference monoclonal antibodies 101G5 and 107G3 to BTN2A. Epitope-specific sorting experiments were performed using the Octet Red96 system based on biological layer interoferometry (BLI). Antibodies 107G3 and 101G5 were tested by changing their order in the experiment with the rhBTN2A1-His protein. A. Antibody 107G3 acts as a saturating antibody, antibody 101G5 acts as a competitor; B: Antibody 101G5 is saturating, antibody 107G3 is competing; C: Measurement results in arbitrary binding units and self-blocking antibody pairs.

Figure 13. Peptides produced by cleavage of BTN2A1 by trypsin, chymotrypsin, endopeptidase ASP-N, edastase and thermolysin. The identified peptides cover 96.37% of the amino acid sequence of BTN2A1.

Figure 14. Interaction of reference monoclonal antibodies 101G5 and 107G3 with human BTN2A. 1. A. 107G3/BTN2A1. B. 101G5/BTN2A1.

Figure 15. BTN2A1/107G3 interaction. Structure of BTN2A,1 according to the PDB (identifier 4F9P); epitopes are highlighted in gray. The amino acid residues shown in blue occupy positions 65-78 (RWFRSQFSPAVFVY) and 84-100 (RTEEQMEEYRGRTTFVS) of the BTN2A1 amino acid sequence. A, B, C, D, E: Ribbon/surface representation of the molecule - front view (A), back view (B), side view 1 (C), side view 2 (D), top view (E). F, G, H, I, J: strip view - front view (F); rear view (G), side view 1 (H), side view 2 (I), top view (J).

Figure 16. BTN2A1/101G5 interaction. Structure of BTN2A1, according to the PDB (identifier 4F9P); epitopes are highlighted in gray. The amino acid residues shown in blue occupy positions 212-229 (KSVRHM SCSINNTLLGQK) of the BTN2A1 amino acid sequence. A, B, C, D, E: Ribbon/surface representation of the molecule - front view (A), back view (B), side view 1 (C), side view 2 (D), top view (E)F, G , H, I, J: strip view - front view (F); rear view (G), side view 1 (H), side view 2 (I), top view (J).

Figure 17. Determination of cross-reactivity of reference monoclonal antibodies 101G5 and 107G3 to BTN2A1 in relation to the cynomolgus monkey orthologous protein. The binding of antibodies 107G3 and 101G5 to recombinant human BTN2A1-Fc fusion protein or recombinant cynomolgus BTN2A1-Fc fusion protein coated on an ELISA plate was determined by ELISA. The graphs are dose-response curves from which EC ₅₀ is calculated using nonlinear regression analysis and using a variable slope.

EXAMPLES

Materials and methods

Cell culture, selection of monocytes and NK cells

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Eurobio) from ethylene diamine tetraacetic acid (EDTA) buffy coat blood from healthy donors provided by a local blood bank [Etablissement Français du Sang (EFS), Marseille, France]. Fresh PBMC were cultured at 37°C in an atmosphere containing 5% CO ₂ in RPMI-1640 medium [developed at the Institute. Roswell Park Memorial Institute, Buffalo, NY, USA and manufactured by Lonza) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

NK cells were obtained from fresh PBMCs by negative selection using the EasySep kit^TM Human NK Cell Enrichment Kit (StemCell Technologies) following the manufacturer's instructions. Human (CD14⁺)-monocytes were selected by magnetic separation using the CD14 kit⁺ Microbead kit (Miltenyi), following the manufacturer's instructions. Monocytes were cultured at a cell density of 10⁶/ml in RPMI medium containing 1% L-glutamine, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate, 10 mM [4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES)], 0.1 mM nonessential amino acids, 10% FBS (all reagents listed here are from Thermofisher), for 5 days at 37°C. Pancreatic adenocarcinoma cells (line PANC-1) were cultured in RPMI medium containing 10% FBS to a monolayer of 90%. After this, the culture medium was discarded, and the cells were washed twice with phosphate buffered saline (PBS x1). Then PANC-1 cells were cultured in RPMI medium containing 5% FBS for another 24 hours (30 ml in 175 cm flasks², to obtain a concentrated supernatant). The medium in which PANC-1 cells were cultured was collected, passed through a filter with a pore diameter of 0.2 μm) and maintained at -20°C until use. Other human cells and corresponding culture media are listed in Table 1 below.

Line
cells Textile Disease Cell type Wednesday HEK-293T Embryonic
bud N.A. Epithelial DMEM
Glutamax 10% FBS
1mM NaPyr DU-145 Prostate Carcinoma Epithelial RPMI
Glutamax MDA-MB-
231 Breast Adenocarcinoma Epithelial DMEM
Glutamax A-549 Lung Carcinoma Epithelial Wednesday F-
12K HT-29 Colon Colorectal
adenocarcinoma Epithelial DMEM
Glutamax HCT-116 Colon Colorectal
carcinoma Epithelial McCoy's 5a 10% FBS RAJI B lymphoblasts Burkitt's lymphoma B lymphocytes RPMI
Glutamax 10% FBS
1mM NaPyr HL60 Peripheral blood Acute promyelocytic leukemia Promyeloblasts

Table 1

NaPyr - sodium pyruvate

NA - not applicable

Human cell lines of the following were obtained from the American Type Culture Collection (ATCC): Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), MDA-MB-134 (ductal carcinoma of the breast) and HEK-293T (embryonic kidney). Human pancreatic adenocarcinoma cells [L-IPC line (PDAC087T)] were kindly provided by Dr/Juan IOVANNA. Daudi and Jurkat cells and PBMCs were cultured in RPMI 1640 medium containing 10% FCS, 1% sodium pyruvate, 1% L-glutamine (all reagents listed here are from Life Technologies). HEK-293T BTN2 KO cells were generated by inactivating the genes of all BTN2 isoforms using CRISPR-Cas9 genome editing technology (data not shown). MDA-MB-134, L-IPC, HEK-293, and HEK-293T BTN2 KO cells were cultured in DMEM (Life Technologies) containing 10% FCS. Hybridomas were cultured in a 1:1 mixture of DMEM and Ham's F12 medium (ThermoFisher Scientific) containing 4% FetalClone I (Hyclone), Chemically Defined Lipid Concentrate (1:250), 1% glutamine, 1 % sodium pyruvate and 100 µg/ml complex antibacterial drug PenStrep (all reagents listed here are from ThermoFisher Scientific). To collect the supernatant, hybridomas were cultured for 4-5 days without Fetalclone.

To determine the specificity of monoclonal antibodies to BTN2A1, HEK-293T BTN2 KO cells were independently transfected with plasmids pcDNA3-Zeo-BTN2A1-CFP encoding the BTN2A1 and BTN2A2 proteins fused to a blue fluorescent protein (CFP) tagged Nter using Lipofectamine 3000 (Thermofisher Scientific) according to manufacturer's instructions.

Identification of reference monoclonal antibody 107G3

to BTN2A1

To generate mouse anti-human BTN2A1 antibodies, 48 mice with six different MHC combinations were immunized with recombinant human BTN2A1-Fc fusion protein. After 21 days, blood was taken from the animals and the titer of polyclonal antibodies specific to BTN2A1 was determined in the blood serum using flow fluorometry on microspheres (Luminex platform). Individuals with the highest titers of antibodies specific to BTN2A1 were sacrificed. Splenic B lymphocytes were isolated by positive selection and hybridomas were obtained by performing polyethylene glycol-induced fusion of the isolated cells with myeloma cells.

Hybridomas were cloned by the limiting dilution method, and hybridoma culture supernatants were screened in two rounds for target specificity and ability to induce degranulation of Vγ9Vδ2-T lymphocytes (Figs. 1c and 2); As a result, the reference antibody mAb 107G3 was identified. The amino acid sequences of the variable regions of the light and heavy chains of these subclones were determined (see Table 10).

Proliferation of Vγ9Vδ2-T cells

An established culture of effector Vγ9/Vδ2-T cells was established by growing PBMC from healthy donors in the presence of zoledronic acid (Sigma, 1 μM) and recombinant human interleukin-2 (rhIL-2; Proleukin, 200 IU/ml); culture began on Day 0. Starting on Day 5, fresh rhIL-2 was added every other day; Cell density was maintained at 1.15 x 10 ⁶ /ml for 15 days. On the last day of this period, the purity of Vγ9/Vδ2 T cells was determined by flow cytometry. For functional analysis, only cultures with a purity greater than 80% were taken. Purified Vγ9/Vδ2-T cells were kept frozen until use.

Analysis using the Luminex platform

RhBTN2A1 protein (R&D) was attached to magnetic microspheres with COOH groups (Biorad) according to the manufacturer's instructions; The resulting microspheres were kept in storage buffer solution (Biorad) at -20°C until use. To titrate mouse blood sera, serial 4-fold dilutions were prepared in Luminex assay buffer (Nanotools), starting with a dilution of 1:50; 100 µl of microsphere suspension was mixed with 100 µl of serum dilution and incubated for 1 hour at room temperature, after which the microspheres were washed three times with wash buffer, incubated with biotinylated goat anti-mouse IgG-Fc in Luminex assay buffer and washed three times with the same buffer solution. Finally, the microspheres were incubated for 1 hour with 1 μg/mL streptavidin-phycoerythrin (PE) complex in Luminex assay buffer. then washed three times in Luminex Reader Buffer Solution (Nanotools). Microspheres were resuspended in Luminex reading buffer solution and measured on a Luminex 100/200 System multiplex flow analyzer. For identification, 30 μl of the supernatant was added to the wells of a 96-well plate and 90 μl of Luminex assay buffer was added. Then, 100 μl of the microsphere suspension was mixed with 100 μl of the supernatant dilution and incubated for 16 hours at room temperature, after which the protocol described above was followed. To identify the desired antibodies, those that had the highest affinity for binding to the target and the lowest affinity for contact with the control protein (Rank-Fc) were selected. To calculate affinity and Kd, hybridoma supernatants were serially diluted 1:4 in Luminex assay buffer starting at 40,000 pM and assayed as described above. The midpoint of the binding curve corresponds to Kd.

Flow cytometry

PBMCs, purified Vγ9Vδ2-T cells, or cell lines were incubated with specific monoclonal antibodies and analyzed on a BD LSRFortessa (BD Biosciences), CytoFlex LX, or CytoFlex S (Beckman Coulter) flow cytometer using FlowJo 10.5.3 software ( FlowJo). For degranulation of Vγ9Vδ2-T cells, the following antibodies were taken: to CD107a-FITC (BD Biosciences), to CD107b-FITC (BD Biosciences), to CD3-PeVio700 (Miltenyi), to PanTγδ-PE (Miltenyi), LIVE/DEAD Near IR (Thermofisher). For all immunostaining, purified monoclonal antibodies were used at a concentration of 10 μg/ml in the presence of FcR Block blocking reagent (Miltenyi), phycoerythrin-conjugated goat anti-mouse IgG 1:100 (Jackson Immunoresearch), and LIVE/DEAD Near IR antibody ( Thermofisher). Mouse anti-human CD277 antibodies (also referred to as BTN3A; clone 103.2 of the IgG2a isotype) have been described previously (WO2012/080351). To determine the specificity of anti-BTN2A1 antibodies, HEK-293T BTN2 KO cells (5x10 ⁴ /sample) were collected 24 hours after transfection and treated with the 107G3 anti-BTN2A1 monoclonal antibody described above at the indicated concentrations (5 ng/ml to 75 μg/ml). Mouse IgG1 antibodies (Miltenyi) were used for isotype control.

Functional analysis of Vγ9/Vδ2-T cells

Purified Vγ9/Vδ2-T cells from the blood of healthy donors were cultured overnight with rhIL-2 (200 IU/ml). They were then cocultured at 37°C with the indicated target cell lines [effector:target (E:T) ratio = 1:1] in the presence or absence of the following monoclonal antibodies (50 μl hybridoma supernatant or 10 μg/ml purified monoclonal antibodies, as indicated): to BTN2A1, mIgG1 (isotype control) or culture medium from hybridomas. Phorbol 12-myristate 13-acetate (PMA, 20 ng/ml) with ionomycin (1 μg/ml) was used as a positive control for Vγ9/Vδ2 T cell activation. In the first round of screening, hybridoma supernatants were collected after 4 hours and IFNγ content was determined as an indicator of Vγ9/Vδ2 T cell activation using the Human IFNγ ELISA kit (BD Biosciences). In the second round of analysis, to determine Vγ9/Vδ2-T cell degranulation, hybridoma supernatants were incubated for 4 hours in the presence of the protein transport inhibitor GolgiStop (BD Biosciences) and fluorescein isothiocyanate (FITC)-labeled soluble CD107(a&b). After 4 hours, cells were harvested, fixed in PBS with 2% paraformaldehyde, and analyzed on a CytoFlex LX flow cytometer (Beckman Coulter) using FlowJo 10.5.3 software (FlowJo).

Proliferation of Vγ9/Vδ2-T cells

Vγ9/Vδ2-T cells were isolated from PBMCs of healthy donors using a TCRγδ microsphere kit (Miltenyi Biotec). The purity of γδ-T cells according to flow cytometry was more than 80%. γδ-T cells were labeled with CellTrace violet dye for 20 min at 37°C. ^5x105 labeled cells were then cultured in a 96-well round-bottom plate with IL-2 (200 IU/ml), in the presence or absence of pAg gene product protein, and in the presence or absence of 107G3 anti-BTN2A1 antibody (10 μg /ml). After 5 days of cultivation, dye dilution was determined by flow cytometry on a CytoFlex LX cytometer (Beckman Coulter) using the FlowJo 10.5.3 program (FlowJo).

Statistical analysis

For Vγ9/Vδ2-T cell degranulation, results were expressed as mean ± standard error of the mean (SEM). For purified anti-BTN2A1 monoclonal antibodies on HEK-293T BTN2 KO cells transfected with the BTN2A1 gene, EC ₅₀ was determined from a log dose-response curve using nonlinear regression and variable slopes. GraphPad Prism 7.04 (GraphPad) was used for statistical analysis.

Identification of reference antibody mAb 101G5 to BTN2A

Having determined the amino acid sequences of the variable regions of the heavy and light chains, the authors obtained 23 monoclonal antibodies to BTN2A in a chimeric IgG1 format using mouse hybridomas, as described above. Briefly, we did the following. The nucleotide sequences encoding the VH and VL of the mouse monoclonal antibody to BTN2A were synthesized in vitro and amplified by polymerase chain reaction (PCR) using PrimeSTAR Max DNA polymerase (Takara). PCR products were cloned into heavy and light chain expression vectors (MI-mAbs) using the In-Fusion system (Clontech), and the plasmids were transformed into competent Escherichia coli Stellar cells (Clontech). Sequencing of the vectors (MWG Eurofins) was performed to confirm that the desired sequences for the anti-BTN2A monoclonal antibodies were obtained before preparing plasmids in large quantities for further transfection. Vectors encoding the desired heavy and light chains for each clone of antibodies to BTN2A were transiently transfected into HEK-293 cells (2.9x10 ⁶ cells/ml) at a H:L ratio of 1:1.2, and after 18 hours the culture medium was renewed. 7 days after transfection, culture supernatants were collected for purification of monoclonal antibodies. Affinity purification was carried out using Protein A Sepharose Fast Flow chromatographic medium; GE Healthcare), overnight at 44°C. Composition of the binding buffer solution: 0.5 M glycine, 3 M NaCl, pH 8.9. Eluted with 0.1 M citrate buffer solution (pH 3). Immediately after elution, samples were neutralized with 1 M Tris-HCl, pH 9 (10% v/v). Finally, chimeric monoclonal antibodies to BTN2A were dialyzed into PBS (1×) and passed through a 0.22 μm pore filter [Millex GV, hydrophilic PVDF; Millipore]. The concentration of chimeric monoclonal antibodies to BTN2A was determined using a Nanodrop 2000 spectrophotometer (ThermoScientific) taking into account the antibody extinction coefficient. The purity of the mAb monomer fraction was determined by ultra-performance liquid exclusion chromatography (UPLC-SEC) using an Acquity UPLC-HClass Bio system (Waters) with Acquity UPLC Protein-BEH-200A columns (1.7 μm; 4.6 x 50 mm; Waters). Antibody mass was determined using a Xevo G2-S Q-Tof mass spectrophotometer (Waters) using reverse phase columns (PLRP-S 4000 A, 5 μm, 50 x 2.1 mm (Agilent technologies). All samples were analyzed after deglycosylation of the peptide -N-glycosidase F (New England Biolabs) at 37°C. If an unexpected mass value was detected, the primary amino acid sequence was analyzed using bioinformatics methods to identify possible glycosylation sites within the Fab region by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (). SDS-PAGE) of purified antibodies revealed fragmentation and/or aggregation in the final dye-free Mini protean TGX gel 4-15 material (Biorad). Endotoxin levels were also determined using a bacterial endotoxin test kit using the Chromogenic LAL Limulus Amebocyte kinetic chromogenic method. Lysate kinetic assay (Charles River Endosafe) on a ClarioStar spectrophotometer (BMG Labtech).

Determination of macrophage differentiation according to M1 and M2 phenotypes in vitro

Macrophages were polarized, that is, they were differentiated according to the “polar” phenotypes M1 or M2, from sorted monocytes from the blood of healthy donors. To obtain M1 and M2 macrophages, sorted monocytes were cultured in the presence of GM-CSF or M-CSF (40 ng/ml; Miltenyi), respectively. After 5 days, the resulting macrophages were collected for phenotypic analysis or subjected to stimulation with lipopolysaccharide (200 ng/ml) for another 2 days in culture. In some experiments, IL-4 (20 ng/ml) was added to M2 macrophages on day 4, which resulted in the formation of “M2+IL-4” macrophages. In some experiments, M2 macrophages were obtained by culturing monocytes in the presence of PANC-1 cancer cell-conditioned culture medium (diluted to 30% v/v; on day 0 or day 3) without the addition of M-CSF. The resulting M2 macrophages are referred to herein as “Tum-ind-M2”. To test anti-BTN2A monoclonal antibodies for their ability to influence macrophage differentiation toward the M2 phenotype, M2 monocyte-derived macrophages were prepared as described above in the presence or absence of chimeric anti-BTN2A monoclonal antibodies or in the presence of an isotype control (human IgG1; Sigma) at the indicated concentrations. All antibodies were kept in PBS 1x overnight at room temperature to avoid drying out. To control the suppression of monocyte differentiation according to the M2 phenotype, GM-CSF (40 ng/ml) and IFNγ (100 ng/ml, BioTechne) were added to them during M-CSF-induced differentiation. M1 macrophages obtained in the presence of GM-CSF were used as phenotypic controls. After differentiation, formed macrophages and their culture supernatants were collected and the expression of M1 and M2 phenotype markers on the plasma membrane of cells was determined by flow cytometry. In addition, cytokine levels in the culture supernatants were determined using IL-10 and TNα ELISA kits (ThermoFisher Scientific) following the manufacturer's instructions.

Determination of M2 macrophage phenotype reversion in vitro

M2 macrophages were prepared from monocytes in the presence of M-CSF as described above, without reference monoclonal antibodies. M2 macrophages were collected and cultured for 2 days in the presence and absence of lipopolysaccharide, with the plate wells pre-incubated overnight with reference antibodies at a concentration of 10 μg/ml or with isotype control monoclonal antibodies (human IgG1; Sigma). As a control for phenotype reversion, GM-CSF (40 ng/ml) and IFNγ (100 ng/ml) were added to the M2 macrophage culture for 2 days. M1 macrophages formed in the presence of GM-CSF were used as a phenotypic control. After phenotype reversal experiments, macrophages were collected and incubated without added lipopolysaccharide for phenotypic determination by flow cytometry. Supernatants from cultures of reverted macrophages stimulated with lipopolysaccharide were collected for determination of cytokine levels by ELISA.

Determination of M2 macrophage-mediated suppression of proliferation

T cells and their production of IFNγ in vitro

M1 and M2 macrophages were prepared with the addition of reference antibodies or corresponding monoclonal antibodies for isotype control or without antibodies as described above. From PBMCs of healthy donors, (CD3 ⁺ ) T cells were sorted using a CD3 ⁺ Microbead kit (Miltenyi) according to the manufacturer's instructions and stored frozen until coculture. Activated (CD3 ⁺ ) T cells were prepared as follows. (CD3 ⁺ ) T cells were stained with CellTrace violet dye (ThermoFisher Scientific) at a concentration of 5 μM; then 10 ⁵ such cells were cultured on 96-well flat-bottomed plates, pre-coated with monoclonal antibodies to CD3 (clone OKT3, BD biosciences), in X-Vivo 10 medium containing 20 U/ml IL-2 (Miltenyi), lipopolysaccharide (200 ng /ml) and CountBright microspheres (absolute amount 5x10 ³ per well; ThermoFisher Scientific). For co-culture, 2x10 ⁴ allogeneic macrophages M1, M2, or macrophages formed in the presence of M-CSF and reference antibodies or isotype control antibodies were taken, added to activated CD3 ⁺ cells and cultured for 5 days, after which they were added to the coculture with PMA (20 ng/ml) and ionomycin (0.5 μg/ml) to enhance cytokine production, in the presence of the protein transport inhibitor GolgiStop for 5 hours. The phenotype was then analyzed by flow cytometry. A dilution of CellTrace violet dye was used as an indicator of (CD3 ⁺ ) T cell proliferation. Phenotype and proliferation data were expressed as a relative number (percentage) or an absolute number of cells in 1 ml (a ready-made calibration suspension of Absolute Counting Beads microspheres was used).

Effect of reference monoclonal antibodies to BTN2A1 on NK cells

Sorted NK cells from healthy donors were labeled and then cultured at 37°C in an atmosphere containing 5% CO ₂ in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin, IL-2 (50 IU/ml), in the presence or absence of IL-15 stimulation (10 ng/ml). On day 0, reference monoclonal antibodies to BTN2A or isotype control (10 μg/ml) were added to this cell culture. After 5 days, NK cells were extracellularly phenotyped based on the expression of activation markers. NK cell activation was assessed by induction of CD69 and CD25 expression [relative abundance (percentage) and mean fluorescence intensity (MFI)]. The discrimination window setup for NK cell flow cytometry is shown in Figure 4 . To determine NK cell cytotoxicity, sorted NK cells from three healthy donors were cultured at 37°C in an atmosphere containing 5% CO ₂ in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin, in the presence or absence of IL. -2 (50 IU/ml), IL-15 (10 ng/ml). Reference monoclonal antibodies to BTN2A or isotype control (10 μg/ml) were added to NK cells stimulated with IL-2/IL-15 or not stimulated and incubated overnight. The next day, NK cells were co-cultured with the indicated blood cells or carcinoma line cells in a 1:1 ratio; FITC-labeled anti-CD107a and anti-CD107b monoclonal antibodies (both BD Biosciences) were added to the coculture and incubated for 4 hours. NK cell degranulation was determined by flow cytometry as the relative percentage of cells carrying both indicated markers (CD107ab ⁺ ) in a sample of unstimulated NK cells and in a sample of NK cells stimulated with IL-2/IL-15. To calculate the EC ₅₀ of increased NK cell degranulation, anti-BTN2A monoclonal antibodies and isotype control were used at concentrations ranging from 0.005 nM to 300 nM. When determining cancer cell death involving NK cells, purified NK cells were preincubated overnight with reference monoclonal antibodies 101G5 and 107G3 or the corresponding control IgG1 at a concentration of 10 μg/ml at 37°C in an atmosphere containing 5% CO ₂ . in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin, with IL-2 (50 IU/ml) and IL-15 (10 ng/ml) serving as a positive control. The next day, NK cells were co-cultured with cancer cells of the indicated lines, labeled with CellTrace violet dye, in a 1:1 ratio for 4 days. Cancer cell death was determined as the relative percentage of those cells harboring caspase 3/7 ⁺ using the fluorogenic substrate for activated caspase-3/7 CellEvent ^TM Caspase-3/7 Green Detection reagent (Thermofisher Scientific).

Flow cytometry

PBMC/NK cells and monocytes/macrophages were incubated for 10 min with human Fc receptor blocker (Miltenyi) or human IgG1 (Sigma) to saturate Fc receptors, followed by staining. The following labeled monoclonal antibodies were used: 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 this antibody mixture for 30 min at 4°C. Dead cells were excluded using Live/Dead Near IR Dye (ThermoFisher Scientific) to define the area of live cells in the histogram of their distribution. For intracellular IFNγ staining, extracellularly stained cells were fixed, membrane permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience), and incubated with allophycocyanin (APC)-labeled anti-IFNγ antibodies (BD Biosciences). Data were obtained on a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo 10 software. For phenotyping of BTN2A1 and BTN2A2, purified monoclonal antibodies to BTN2A1 (mAb5) and to BTN2A2 (mAb17) were used at a concentration of 10 μg/ml and detected using phycoerythrin (PE)-labeled anti-IgG (H+L) antibodies (Jackson Immunoresearch). After single cell selection, NK cells were characterized as CD45 ⁺ CD14-CD3-CD56 ⁺ cells within the live cell region of the histogram. Monocytes were identified as CD45 ⁺ CD19 - CD3 - CD56 - CD14 ⁺ cells within the live cell region after single cell selection. Data were acquired on a Cytoflex LS (Beckman Coulter), iQue Screener (Intellicyt), or Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo 10 software. Results were expressed as mean fluorescence intensity (MFI) after subtracting values obtained from the corresponding control staining

BTN2A1 epitope affinity determination and epitope-specific sorting

using the Octet system

Having generated reference chimeric IgG1 antibodies to BTN2A, we determined their affinity for two different target isoforms (BTN2A1 and BTN2A2) and studied competitive binding to determine whether these monoclonal antibodies recognize the same BTN2A1 epitope. Affinity determination and epitope-specific sorting were performed using the Octet Red96 system (Fortebio/PALL), based on biological layer interferometry (BLI). For affinity determination, recombinant human (rh)BTN2A1-Fc (GTP) was biotinylated using the EZ-Link™ NHS-PEG4 Biotinylation Kit according to the manufacturer's instructions; biotinylated rhBTN2A2-Fc was provided by R&D Systems. In the case of determining the affinity for BTN2A1, biotinylated rhBTN2A1-Fc was applied to the biosensor with streptavidin (SA) (ForteBio) diluted in a ready-made Kinetic Buffer 1X buffer solution (ForteBio), with a target layering level of about 1 nm; The analyte was chimeric antibodies to BTN2A. For BTN2A2 affinity testing, chimeric anti-BTN2A antibodies were applied to FAB2G sensors (anti-human CH1; Fortebio) as described above; The analyte was biotinylated rhBTN2A2-Fc. In both cases, the analytes remained in solution, and their working concentration was achieved by dilution with a ready-made buffer solution Kinetic Buffer 10x (ForteBio). In the first run, the standard working concentration was 200-3.125 nM. If a second run was needed, the working concentration was adjusted to 80-1.25 nM. All runs (including layering, equilibration, association/immersion of the sensor into the analyte) were carried out at 30°C and stirring at 1000 rpm. The analysis used the Langmuir model for 1:1 or 2:1 binding (for BTN2A1 or BTN2A, respectively), calculated using Octet software, which provided better calculation of matches. For epitope-specific sorting, histidine-tagged BTN2A1 (rhBTN2A-His) was obtained from R&D Systems. Reference antibodies to BTN2A were tested in pairs with BTN2A1. The sorting was carried out “one after another,” that is, rhBTN2A-His was immobilized on a biosensor (anti-Penta-His “HIS1K”; ForteBio/PALL) and contacted with two competing antibodies sequentially. For kinetic analysis, rhBTN2A-His was applied to HIS1K (signal intensity 1 nm), followed by an association step at an antibody concentration of 10 μg/ml for 3 min, followed by a dissociation step for 3 min. Activity. rhBTN2A1-His was confirmed by the results of a kinetic assay performed in the same format as the binding assay (BTN2A1 as the ligand immobilized on the sensor and antibody as the analyte). All antibodies (saturating or competing) were taken at a concentration of 10 μg/ml, diluted with a ready-made buffer solution Kinetic Buffer 1x. For kinetic analysis, loading of rhBTN2A1-His onto HIS1K (signal intensity 1 nm) was followed by an association step for 3 min, followed by a dissociation step for 3 min. Assay steps: Baseline > Antigen Binding -> Baseline -> Saturating Antibody -> Baseline -> Competing Antibody -> Regeneration, then back-to-back stage. Epitope-specific sorting data were analyzed using Octet Data Analysis HT 11 software using panels of antibodies that bind different epitopes.

Mapping of epitopes recognized by reference monoclonal antibodies 107G3 and 101G5

The interaction between BTN2A1 and the reference monoclonal antibodies 107G3 and 101G5 was assessed by the difference in peptide mass fingerprinting data for BTN2A1 alone and with antibody 107G3 or 101G5. Prior to epitope mapping, rhBTN2A1-Fc protein (GTP Technologies) was analyzed by high-mass MALDI mass spectrometry to check integrity and level of aggregation using an Autoflex II mass spectrometer MALDI ToF (Bruker) equipped with a CovalX HM4 module (CovalX). As a result, it was confirmed that there were no non-covalent aggregates or multimers of BTN2A1 in the sample. In order to characterize BTN2A1 and determine its epitopes to which monoclonal antibodies 107G3 and 101G5 bind, the sample was treated with trypsin, chymotrypsin, endoprotease Asp-N, elastase and thermolysin and, after proteolysis, analysis was carried out in a mass spectrometric complex based on a mass spectrometer type ion trap and nanoflow high performance liquid chromatography nLC-LTQ-Orbitrap MS/MS using an nLC Ultimate 3000-RSLC system with an LTQ-Orbitrap mass spectrometer (Thermo Scientific). In cases of BTN2A1/107G3 and BTN2A1/101G5 binding, the protein complexes were incubated with deuterated pepper cross-linking agents before multienzyme digestion. After enrichment of cross-linked peptides, samples were analyzed and the resulting data were processed using XQuest 2.0 and Stavrox 3 programs. Reductive alkylation was performed to obtain samples as follows. BTN2A1 (4.04 µM) was mixed with disuccinimidyl suberate ((DSS d0/d12; 2 mg/ml in dimethylformamide (DMF)) and incubated for 180 min at room temperature. At the end of incubation, the reaction was stopped by adding 1 µl ammonium bicarbonate (final concentration 20 mM) and incubated for 1 hour at room temperature. Then the solution was dried with a SpeedVac vacuum concentrator, after which an aqueous suspension of 8 M urea (10 μl) was added. After mixing, 1 μl of dithiothreitol (DTT; 500 mM) was added. The resulting mixture was incubated for 1 hour at 37ºC. At the end of the incubation, 1 μl of iodoacetamide (1M) was added to the mixture and incubated for 1 hour at room temperature in the dark. Then, 100 μl of proteolysis buffer solution containing 50 mM ammonium bicarbonate (Ambic) was added. ), 5% acetonitrile; the buffer solution for proteolysis with chymotrypsin contained Tris-HCl 100 mM, CaCl ₂ 10 mM (pH 7.8); the buffer solution for proteolysis with endoprotease Asp-N was phosphate buffer solution 50 mM (pH 7.8); the elastase proteolysis buffer contained Tris-HCl 50 mM (pH 8.0) and the thermolysin proteolysis buffer contained Tris-HCl 50 mM, CaCl ₂ 0.5 mM (pH 9.0). For tryptic digestion, 100 μl of reduced/alkylated BTN2A1 was mixed with 1 μl of trypsin (Roche Diagnostic) at a ratio of 1/100. This mixture was incubated overnight at 37ºC. 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. This mixture was incubated overnight at 25ºC. For proteolysis with Asp-N endoprotease, 100 μl of reduced/alkylated BTN2A1 was mixed with 0.5 μl of Asp-N endoprotease (Roche Diagnostic) in a ratio of 1/200. This mixture was incubated overnight at 37ºC. For elastase proteolysis, 100 μl of reduced/alkylated BTN2A1 was mixed with 1 μl of elastase (Roche Diagnostic) in a ratio of 1/100. This mixture was incubated overnight at 37ºC. For thermolysin proteolysis, 100 μl of reduced/alkylated BTN2A1 was mixed with 2 μl of thermolysin (Roche Diagnostic) in a 1/50 ratio. This mixture was incubated overnight at 70ºC. After proteolytic incubation, formic acid was added to a final concentration of 1%. Upon completion of proteolysis, 10 μl of the resulting peptide solution was loaded into a nanoflow liquid chromatography system (Ultimate 3000-RSLC) with the following parameters: A: 95/05/0.1 H ₂ O/ACN/HCOOH v/v/v; B: 20/80/0.1 H ₂ O/ACN/HCOOH v/v/v; gradient 5-40% B in 35 minutes; introduced volume 10 µl; pre-column - internal diameter (ID) 300 µm ID, length 5 mm C18 PepMapTM, flow rate 20 µl/min; Column internal diameter (ID) - 75-μm ID, length 15 cm C18 PepMapRSLC, flow rate 200 nl/min.

Human BTN2A1 cross-reactivity assay

and cynomolgus orthologous protein by ELISA

The orthologous amino acid sequence of cynomolgus macaque (Macaca fascicularis) BTN2A1 (XM_015448906.1) was identified by BLAST searches using the amino acid sequence of human BTN2A1; The extracellular domain of the macaque protein was cloned into the pFUSE-hIgG1FC2 vector (InvivoGen) with EcoRI/EcoRV restriction sites. To construct the recombinant cynoBTN2A1-Fc fusion protein, the resulting plasmid pFUSE-hIgG1FC2-cynoBTN2A1 was transfected into Expi293F ^TM cells using ExpiFectamine™ 293 reagent (ThermoFisher) according to the manufacturer's instructions. On day 6, the culture supernatant of the transfected cells was collected and the protein was purified by affinity chromatography. Purified cynoBTN2A1-Fc protein was analyzed by SDS-PAGE and Western blotting to determine molecular weight and purity. The protein concentration of cynoBTN2A1-Fc was determined by the Bradford method using bovine serum albumin (BSA) as a standard. For ELISA analysis, cynoBTN2A1-Fc protein and recombinant human BTN2A1-Fc (huBTN2A1-Fc, GTP Technologies) were coated on plates (5 μg/ml in PBS x1) overnight at 4°C. The plates were washed 3 times with PBS, treated with BSA (2% v/v in PBS) for 1 hour at room temperature, and then this solution was discarded. 10-fold serial dilutions of reference monoclonal antibodies 101G5 and 107G3 or control human IgG1 were prepared in PBS with 2% BSA, starting from 1 μM to 1 pM;. 100 μl of each dilution was added to the cells and incubated for 90 min with mixing on a plate shaker. Each well was washed 3 times with PBS, then horseradish peroxidase-conjugated goat anti-mouse IgG (HRP; Jackson ImmunoResearch) diluted 1:10,000 in PBS with 2% BSA was added and incubated for 1 hour at room temperature. After incubation, the wells were washed 2 times with PBS and 2,2'-azino-bis-(3-ethylbenzthiozoline-6-sulfonate (1-step ABTS solution; ThermoFisher) was added to detect binding, after which the absorbance was determined at a wavelength of 405 nm using a spectrometer Spark (Tecan) Each experiment was repeated twice with all samples.

results

Identification of reference antibody 107G3 to BTN2A1

Reference antibody 107G3 to BTN2A1 was identified as follows. Mice were immunized with BTN2A1-Fc antigen; splenocytes were taken from animals with the highest titer of BTN2A1-specific serum and fused with myeloma cells to obtain hybridomas. From the supernatants from hybridoma cultures, those antibodies that had the highest affinity for BTN2A1 were selected, and they were subjected to the first screening for their ability to influence the secretion of IFN-γ by Vγ9/Vδ2-T cells. The clones selected in the first screen were subcloned and tested for their ability to induce IFN-γ secretion and Vγ9/Vδ2 cell degranulation, especially degranulation (Fig. 1c and 2); these data allowed the identification of the reference monoclonal antibody 107G3. The amino acid sequences of the VH and VL regions of these subclones were determined (see Table 10).

Anti-BTN2A1 Antibody 107G3 Induces Vγ9Vδ2-T Cell Degranulation towards Various Target Cancer Cells

Purified Vγ9/Vδ2 T cells from PBMCs of healthy donors were expanded and co-cultured with various cancer cell lines, including Daudi (Burkitt's lymphoma), Jurkat (acute T-cell leukemia), L-IPC (pancreatic adenocarcinoma), and MDA-MB- 134 (breast carcinoma) targets, in the presence or absence of hybridoma culture supernatant containing 107G3 anti-BTN2A1 antibodies. As shown in Figure 2 and Table 2, addition of hybridoma culture supernatant containing 107G3 anti-BTN2A1 antibodies resulted in the induction of Vγ9/Vδ2-T cell cytolytic function, which was measured as the relative number (percentage) of CD107 ⁺ cells degranulating in comparison with cultures of target cells alone or co-cultures of target cells with a control culture medium for hybridomas. Treatment of Vγ9/Vδ2-T lymphocytes with PMA/ionomycin resulted in maximal induction of their cytolytic function regardless of target cells, as expected.

The relative number (in percentage) of CD107 ⁺ cells induced by the hybridoma supernatant with 107G3 antibodies to BTN2A1 ranged from 71.1 ± 7.4% in the case of Daudi cells, to 17.1 ± 2.9% in the case of MDA-MB- cells 134 compared with 24.9 ± 4.7% and 4.9 ± 0.4% in the case of co-cultures of these cells with control hybridoma culture medium, respectively. In all co-cultures of Vγ9/Vδ2-T lymphocytes with cells of cancer lines that served as targets in this experiment, 107G3 antibodies to BTN2A1 caused degranulation of Vγ9/Vδ2-T cells 2-8 times higher than that in the same co-cultures in the presence of control hybridoma culture medium.

Purified 107G3 antibodies to BTN2A1 upon induction of degranulation were characterized by EC ₅₀ = 0.77 μg/ml (95% IC 0.32 - 13.22 μg/ml), which is confirmed by data on the relative number (percentage) of CD107 ⁺ cells in co-culture with Daudi cells as the target in the presence of increasing concentrations (from 0 to 18 μg/ml) of monoclonal antibody 107G3 to BTN2A1 (Fig. 2b).

Table 2

  Antibodies 107G3
to BTN2A1 Control
hybridoma supernatant EC ₅₀ with HEK-293T BTN2 KO + BTN2A1 cells 0.32
(95% IC 0.21-0.46) µg/ml Not applicable EC ₅₀ in functional analysis with Daudi cells 0.77
(95%IC 0.32 - 13.22) µg/ml Not applicable Functional analysis
with Daudi cells
(% CD107 ⁺ cells) 71.1 ± 7.4% 24.9 ± 4.7% Functional analysis
with MDA-MB-134 cells
(% CD107 ⁺ cells) 17.1 ± 2.9% 4.9 ± 0.4% Functional analysis
with L-IPC cells
(% CD107 ⁺ cells) 31.1 ± 3.6% 14.4 ± 0.5% Functional analysis
with Jurkat cages
(% CD107 ⁺ cells) 25.7 ± 5.1% 3.7 ± 0.4%

Antibody 107G3 to BTN2A1 recognizes BTN2A1 but not BTN3

To establish the specificity of mAb 107G3 to BTN2A1 in relation to only the BTN2A1 isoform, HEK-293T BTN2 KO cells, in which both BTN2 isoforms were inactivated by genomic editing using CRISPR-Cas9 technology, were transiently transfected with a plasmid encoding BTN2A1 as part of a CFP fusion protein. As can be seen from Fig. 3a, staining for purified anti-BTN2A1 mAb 107G3 was detected only in those HEK-293T BTN2 KO cells that were transfected with the plasmid encoding BTN2A1, but not in the HEK-293T BTN2 KO cells themselves.

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

Affinity of mAb 107G3 to BTN2A1 in cellulo

To quantify the affinity of mAb 107G3 to BTN2A1 for its target, HEK-293T BTN2 KO cells transfected with a plasmid encoding BTN2A1 were treated with purified mAb 107G3 to BTN2A1 or control monoclonal IgG1 antibody at increasing concentrations (from 5 ng/ml to 75 μg/ ml) (Fig. 3b). The obtained values of average fluorescence intensity were analyzed by nonlinear regression and it was found that for mAb 107G3 to BTN2A1 EC ₅₀ = 0.32 μg/ml (95% IC 0.21-0.46 μg/ml).

Preparation of chimeric monoclonal antibodies to BTN2A, determination of affinity and specificity for the BTN2A isoform

23 monoclonal antibodies were produced by transient expression in HEK-293T cells; At the same time, the production of antibodies occurred with different intensities. Most anti-BTN2A antibodies had high expression levels (>100 mg/ml, up to 430 mg/ml). One of the antibodies to BTN2A, namely mAb3 (see Table 3), was formed in HEK-293T cells very little - from 6 to 8 mg/ml. Analysis of its amino acid sequence showed that in part of the Fab there is an N-glycosylation site in the CDR1 region of the variable region of the heavy chain. Two other antibodies to BTN2A - mAb9 and mAb11 - also showed an N-glycosylation site in the Fab region (for mAb9 - in the CDR1_VH region; for mAb11 - in the CDR1_VL region). Six antibodies to BTN2A showed low purity (<95% in monomers), but only mAb1 and mAb3 had <90% purity (86% and 75%, respectively). After the final purification step, all monoclonal antibodies to BTN2A had very low endotoxin content (about 0.1 EU/mg; EU - endotoxin unit). Only mAb3 contained more endotoxin (0.73 EU/mg) than the resulting preparations of other antibodies, but still within the acceptable range, i.e., <1EU/mg. The affinity constants (K _D , k _on and k _off ) of these 23 monoclonal chimeric antibodies to BTN2A were determined with the BTN2A1 and BTN2A2 isoforms in the Octet system using biotinylated recombinant Fc fusion soluble proteins. Table 4 shows the K _D values for each anti-BTN2A mAb. In the case of mAb6 and mAb9, it was impossible to calculate K _D due to the lack of dissociation during the measurement period (k _off < ^10-7 s- ¹ ), which can be explained by the high avidity of these antibodies, which slowed down the dissociation of the complex with the target. Eight monoclonal antibodies to BTN2A bound only to the BTN2A1 isoform (mAb2, mAb3, mAb4, mAb5, mAb6, mAb8, mAb9 and mAb10), another eight monoclonal antibodies to BTN2A bound only to the BTN2A2 isoform (mAb16, mAb17, mAb18, mAb19, mAb20, mAb21, mAb22 and mAb23) and seven antibodies bound to both of these isoforms (mAb1, mAb7, mAb11, mAb12, mAb13, mAb14 and mAb15).

Expression of BTN2A1 and BTN2A2 on the plasma membrane of monocytes and NK cells

For therapeutic purposes, the authors tried to determine whether monoclonal antibodies to BTN2A in peripheral blood could be targeted by cells other than Vγ9/Vδ2 T cells, namely monocytes and NK cells. To do this, in an Octet assay, we used antibodies mAb5 and mAb17 to bind only BTN2A1 or BTN2A2, respectively, to phenotype monocytes and N cells from peripheral blood. As shown in FIG. 4, the plasma membrane of monocytes and NK cells was stained only in the case of mAb5 to BTN2A1, and in monocytes the staining was more intense. Consequently, BTN2A1, but not BTN2A2, is detected on the plasma membrane of monocytes and NK cells, which provides grounds for screening monoclonal antibodies that recognize BTN2A1 for their ability to modulate the functions of these immune cells.

Screening of monoclonal antibodies to BTN2A for their ability to modulate the differentiation of monocytes into macrophages

Monocytes, in response to signals from their microenvironment, differentiate into macrophages with the M1 or M2 phenotype. M1 macrophages exhibit pro-inflammatory and antitumor properties, whereas 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, monoclonal antibodies to BTN2A, recognizing only BTN2A1 or both isoforms - BTN2A1 and BTN2A2, were determined by their ability to interfere with the differentiation of monocytes into macrophages with the M2 phenotype in the presence of M-CSF in vitro. As a control for the divergence of macrophages according to the M1 and M2 phenotypes, we used M1 macrophages obtained in the presence of GM-CSF (CD14 ^+/ - CD163-) and M2 macrophages (CD14 ⁺ CD163 ⁺ ) obtained in the presence of M-CSF, both others without monoclonal antibodies. After 5 days of in vitro differentiation, the expression of CD14 and CD163 on the plasma membrane of macrophages M1, M2 and macrophages stimulated with M-CSF and differentiated in the presence of monoclonal antibodies to BTN2A or control IgG1 was determined by flow cytometry (see Table 4). As expected, cells with the M1 phenotype had low levels of CD14 expression and undetectable CD163 expression (see Table 5 and Figure 4), whereas M2 macrophages showed high levels of expression of both of these markers. Interestingly, anti-BTN2A mAb1, hereafter referred to as 101G5, caused the most significant reduction in CD14 CD163 expression in the presence of M-CSF, shifting M-CSF-induced macrophage differentiation toward an M1-like phenotype (see Table 4 and Fig. 4B ). The second best inhibitor of M-CSF-induced M2 macrophage differentiation was mAb2, also designated 107G3 (Table 4 and Figure 4B). This is opposite to the macrophage phenotype generated in the presence of M-CSF and control IgG1 antibody, and similar to the phenotype of untreated M2 macrophages.

Table 4. Effect of monoclonal antibodies to BTN2A on the expression of CD14 and CD163 after differentiation of monocytes into macrophages with the M2 fnotype

M2 phenotype markers (MFI; n=3) Cytokine determining
differentiation CD14 CD163 Average S.E.M. Average S.E.M. M1 GM-CSF 1047 463.6 64 17.27 M2 M-CSF 38878 3866 6405 1051 hIgG1 28070 2937 4524 839.4 mAb1
(101G5) 347 1892 38 60.87 mAb2
(107G3) 4408 8448 289 1407 mAb3 42997 3756 6757 1428 mAb4 43853 4650 6493 1129 mAb5 45119 4004 6450 949.7 mAb6 38277 4793 5299 932.3 mAb7 42071 3227 6337 1176 mAb8 39483 3564 6722 1199 mAb9 43203 4869 6432 1135 mAb10 45362 3078 6737 1097 mAb11 41519 3675 6605 1090 mAb12 44750 2845 6909 1113 mAb13 5684 9480 642 1173 mAb14 33889 10427 4614 1498 mAb15 17411 10372 2553 1429

SEM --- standard error of the mean, MFI - mean fluorescence intensity

hIgG1 - human IgG1

Fig. Figure 5 demonstrates the dose-dependent effect of inhibiting the expression of the M2 phenotype, determined by the expression of CD14 and CD163, by the reference monoclonal antibodies 101G5 and 107G3 to BTN2A compared with isotype control (Fig. 5A and 5B), as well as increasing the expression of PDL1 and CD86 characteristic of the phenotype M1 (Figs. 5C and 5D). The cytokine secretion profile of M2 macrophages is also different from that of M1 cells. Therefore, the secretion of IL-10 (an anti-inflammatory cytokine associated with the M2 phenotype) and TNFα (a pro-inflammatory cytokine associated with the M1 phenotype) after stimulation with lipopolysaccharide in the culture supernatants of M-CSF-induced macrophages in the presence and absence of reference monoclonal antibodies was further determined by ELISA. to BTN2A. As can be seen in FIG. 5E and 5F, reference monoclonal antibodies to BTN2A dose-dependently suppressed IL-10 secretion and enhanced TNFα secretion in contrast to isotype control. These observations confirm that antibodies 101G5 and 107G3 suppress M-CSF-induced differentiation of monocytes into macrophages with the M2 phenotype, shifting it towards an M1-like phenotype, as judged by phenotype and cytokine release. Additionally, these effects of antibodies 101G5 and 107G3 are dose dependent. The IC ₅₀ and EC ₅₀ values for each of these monoclonal antibodies are given in table. 5. Note that for all indicators, except for the PDL1 marker, the IC ₅₀ and EC ₅₀ values for 101G5 are less than for 107G3.

Table 5. _IC50 and _EC50 values of reference anti-BTN2A monoclonal antibodies for markers of M2 and M1 macrophage phenotypes and for cytokine secretion

Markers/
Cytokines Effect IC ₅₀ /EC ₅₀ (µg/ml) 101G5 107G3 CD14 Suppression 0.51 7.7 CD163 Suppression 0.43 6.2 CD86 Induction 2.55 25 PDL1 Induction 37.09 7 IL-10 Suppression 0.105 14.8 TNFα Induction 1.12 19.8

Other stimulating factors in the tumor microenvironment have been described that influence the formation of macrophages with the M2 phenotype (Mosser, Edwards, Nat. Rev. Immunol. 2008; Mantovani, Allavena, J. Exp. Med. 2015). Besides M-CSF, one of the most common stimulating agents that induces the formation of macrophages with the M2 phenotype is IL-4. The authors determined the effect of antibodies 101G5 and 107G3 on the differentiation of so-called pro-tumor macrophages M2+IL-4, formed from monocytes after stimulation with M-CSF and IL-4. After 45 days of culture under these conditions, antibodies 101G5 and 107G3 suppressed the expression of markers associated with the M2+IL-4 phenotype (CD14, CD163 and DC-SIGN; see Figs. 6A, 6B and 6D) and the secretion of IL-10 (Fig. 6F), while increasing the expression of markers associated with the M1 phenotype (CD86, PDL1) and TNFα secretion (Figs. 6C, 6E and 6G). Thus, in a protumor environment (M-CSF and IL-4), antibodies 101G4 and 107G3 suppress differentiation towards the M2+IL-4 phenotype and enhance differentiation towards the proinflammatory M1 phenotype.

In addition, the effect of antibodies 101G5 and 107G3 on cancer cell-induced differentiation of monocytes to form macrophages with the M2 phenotype was determined by culturing monocytes in the presence of supernatant from a culture of PANC-1 (pancreatic adenocarcinoma) cells. When antibodies 101G5 or 107G3 were added to such a monocyte culture, differentiation of monocytes toward the M2 phenotype was suppressed, as demonstrated by decreased expression of markers associated with the M2 phenotype (CD14, CD163) and reduction in IL-10 secretion (Fig. 7A-C) and increased expression of the proinflammatory cytokine TNFα, associated with the M1 phenotype (Fig. 7D).

Effect of reference antibodies 101G5 and 107G3 to BTN2A

to “reprogram” M2 macrophages towards the M1 phenotype

The ability of monoclonal antibodies 101G5 and 107G3 to revert differentiated macrophages with the M2 phenotype into cells with the M1 phenotype was determined. For this purpose, M2 macrophages, previously differentiated in the presence of M-CSF for 5 days, were seeded into plate wells coated with monoclonal antibodies 101G5 and 107G3 and cultured for another 2 or 4 days. Treatment of M2 macrophages with γ-interferon served as a positive control for M2->M1 reversion. As can be seen in FIG. 8, M2 macrophages cultured with reference antibodies 101G5 and 107G3 acquired an M1-like phenotype similar to that obtained by IFN-γ treatment. Specifically, treatment of M2 macrophages with the reference antibodies e 101G5 and 107G3 mAbs resulted in a decrease in the expression levels of CD14 (Fig. 8A) and CD163 (Fig. 8B), but an increase. CD86 expression (Fig. 8C). After treatment with the 101G5 antibody, a moderate increase in PDL1 expression was observed; after treatment with the 107G3 antibody, there was no increase in PDL1 expression at all (Fig. 8D). In addition, treatment of M2 macrophages with antibodies 101G5 and 107G3 suppressed IL-10 secretion (Fig. 8E) and enhanced TNFα secretion (Fig. 8F), indicating an M1 phenotype.

In fig. 8G-8I shows the dose-dependent effect of reference antibodies 101G5 and 107G3, consisting in “reprogramming” M2 macrophages to the M1 phenotype in comparison with isotype control, namely: suppression of CD163 expression (Fig. 8G), weakening of IL-10 secretion and increased secretion TNFα (Figures 8H and 8I). In table 6 shows the IC ₅₀ and EC ₅₀ values of each of these antibodies for markers of the M1 and M2 phenotypes; It can be seen that the monoclonal antibody 101G5 to BTN2A has the best activity in the sense of “reprogramming” M2 macrophages to the M1 phenotype.

Table 6. IC ₅₀ and EC ₅₀ values of reference monoclonal antibodies to BTN2A for markers of M2 macrophage transformation.

Markers Effect of reference antibody to BTN2A IC ₅₀ /EC ₅₀ (µg/ml) 101G5 107G3 CD14 Suppression 3.77 nd CD163 Suppression 6.79 15.46 CD86 Induction 1.14 nd IL-10 Suppression 0.87 3.03 TNFα Induction 6.44 80.33

nd - not defined

Reference monoclonal antibodies 101G5 and 107G3 to BTN2A reverse the suppression of T cell proliferation caused by M2 macrophages

The authors studied the ability of reference antibodies 101G5 and 107G3 to influence the functions of macrophages with the M2 phenotype, namely the M2 macrophage-mediated suppression of T-cell proliferation. To do this, allogeneic pre-activated (CD3 ⁺ ) T cells were co-cultured with M1, M2 or M2 macrophages obtained in the presence of the indicated antibodies. As expected, in coculture with conventional M2 macrophages there was a decrease in the number of (CD3 ⁺ ) T cells, as well as attenuation of T cell proliferation [determined by CellTrace violet (CTV) dye dilution] and IFNγ production compared to coculture containing M1 macrophages (Fig. 9A, C, G, E and I). In contrast, M2 macrophages obtained in the presence of antibodies 101G5 and 107G3 did not suppress T cell proliferation, as demonstrated by the higher relative number (percentage) and absolute number (CD3 ⁺ ) of T cells compared to coculture containing macrophages obtained in presence of control IgG1 antibodies (Figs. 9B, 9H and 9J). In addition, the relative number (percentage) and absolute number of T cells producing IFN-γ were also greater in co-cultures containing macrophages obtained in the presence of antibodies 101G5 and 107G3, compared with co-cultures containing macrophages obtained in the presence of control IgG1 antibodies (Fig. 9D and 9F).

Therefore, in contrast to M2 macrophages induced by M-CSF alone, macrophages formed in the presence of not only M-CSF but also antibodies 101G5 or 107G3 do not suppress the proliferation and helper (Th1) function (secretion of IFN-γ) allogeneic (CD3 ⁺ ) T cells, i.e. act like M1 macrophages.

Reference monoclonal antibodies 101G5 and 107G3 to BTN2A initiate NK cell activation and cytotoxicity

Since the BTN2A1 protein was found on the plasma membrane of NK cells, we examined the potential ability of antibodies 101G5 and 107G3 to influence NK cell activation. Purified NK cells were cultured for 5 days in the presence of antibody 101G5 or 107G3 and in the presence or absence of other activating agents (IL-2 and IL-15 or IL-2 alone, respectively). As can be seen in FIG. 10, both antibodies 101G5 and 107G3 increased the expression of CD69 on the plasma membrane of NK cells under experimental conditions (Fig. 10A). In addition, antibodies 101G5 and 107G3 also enhanced CD25 expression induced by exposure of NK cells to IL-2 and IL-15 (Fig. 10B). Because antibodies 101G5 and 107G3 were able to activate purified NK cells, we tested whether these antibodies also enhanced NK cell cytotoxicity. To do this, we determined the degranulation of NK cells [as the relative number (percentage) of CD107 cells] with cancer cells of the lines HL-60 (myelogenous leukemia), HT-29 (colon carcinoma), MDA-MB-231 (adenocracinoma of the breast) and A549 (lung adenocarcinoma) in the presence of antibody 101G5 or 107G3 and with or without stimulation with IL-2 and IL-15. As expected, in the presence of control IgG1 antibody, NK cell degranulation occurred only with HL-60 cells (Fig. 10C) and was enhanced by stimulation with IL-2 and IL-15 (Fig. 10D). With solid tumor cell lines HT-29, MDA-MB-231 and A549, moderate NK cell degranulation was also observed in the case of control IgG1 and IL-2+IL-15. In table Figure 7 shows data on the degranulation of NK cells with cancer cells of these and other (Raji, HCT116, DU-145) lines used in this experiment. Interestingly, reference monoclonal antibodies 101G5 and 107G3 enhanced NK cell degranulation with MDA-MB-231 and A549 solid tumor cells, and to a lesser extent with HT-29 cells without IL-2+IL-15 stimulation. The addition of IL-2 and IL-15 enhanced the effect of antibodies 101G5 and 107G3 in the case of cell lines MDA-MB-231, A549 and DU145 (see Table 7). Addition of reference monoclonal antibodies 101G5 and 107G3 did not lead to such enhancement in the case of HL-60 and Raji circulatory cancer cells (Table 7 and Figs. 10C and 10D). In addition, the reference monoclonal antibodies 101G5 and 107G3 were able to initiate NK cell degranulation in the case of A549 cells if these antibodies were preincubated with NK cells before coculture and were then not added to the coculture (Fig. 10E). It follows that the effect of antibodies 107G3 and 101G5 is to directly bind to NK cells, which initiates their cytotoxicity directed against cancer cells. Also, in the case of DU-145 cells (prostate adenocarcinoma), it was determined whether the increase in NK cell degranulation depends on the dose of antibodies 107G3 and 101G5. Indeed, antibodies 101G5 and 107G3 increased NK cell degranulation with DU-145 cells in a dose-dependent manner ( EC _{50 (no stimulation)} = 0.14 and 0.54 nm; EC _{50 (IL} - _2+IL - ₁₅₎ = 0.08 and 0.2 nm for 101G5 and 107G3, respectively).

Table 7. Degranulation of NK cells (relative percentage of CD107 ⁺ cells) with cancer cells of different lineages with and without IL-2 and IL-15 stimulation

Stimulation Target cells IgG1 101G5 107G3 Average S.E.M. Average S.E.M. Average S.E.M. No No 1.74 1.26 2.06 0.95 1.81 1.03 HL-60 24.57 0.71 22.43 1.31 27.23 2.29 Raji 8.70 1.43 9.19 0.84 11.68 0.56 HT-29 4.69 0.35 5.76 1.00 9.19 0.15 HCT116 2.79 0.46 3.98 1.81 5.62 0.47 MDA-MB-231 2.28 0.73 7.24 1.16 9.66 1.09 A549 1.68 0.07 6.78 2.44 12.57 1.15 DU-145 3.29 0.19 14.6 4.91 25.1 3.13 IL-2+IL-15 No 3.32 0.84 6.14 2.01 5.22 2.01 HL-60 65.83 1.09 57.37 3.18 58.57 3.18 Raji 21.33 2.31 28.83 0.00 24.50 0.00 HT-29 22.43 2.11 20.73 2.18 19.83 2.18 HCT116 25.53 2.62 27.70 1.93 26.20 1.93 MDA-MB-231 11.85 3.26 29.00 4.00 30.70 4.00 A549 14.53 2.64 24.57 1.38 32.03 1.38 DU-145 23.73 4.71 41.07 0.67 56.80 0.67

Finally, the authors tested the ability of antibodies 101G5 and 107G3 to enhance NK cell-mediated cancer cell death; To do this, we determined the relative number (in percentage) of cells containing caspases 3 and 7 after co-cultivation of purified NK cells with HL-60 cells (myelogenous leukemia) or with A459 cells (lung adenocarcinoma). As can be seen in FIG. 11, antibodies 101G5 and 107G3 enhanced NK cell-mediated death of A549 adenocarcinoma cells (approximately 2-fold), but not HL-60 leukemia cells. Taken together, these observations suggest that antibodies 101G5 and 107G3 enhance NK cell cytotoxicity, preferentially directed against solid tumors.

Reference monoclonal antibodies 101G5 and 107G3 to BTN2A

recognize different epitopes of BTN2A1

Both of these antibodies, 101G5 and 107G3, bind to BTN2A1 and have the ability to suppress the acquisition of the M2 phenotype by macrophages and enhance the activation and cytotoxicity of NK cells. The authors decided to test whether these two antibodies recognized the same epitope of the BTN2A1 protein. Epitope-specific antibody sorting was performed using the Octet system: antibodies 101G5 and 107G3 competed for binding to BTN2A1 “one after another.” As can be seen in FIG. 12, antibody 101G5 does not block binding of antibody 107G3 to BTN2A1 and vice versa; this indicates that these two antibodies do not recognize the same BTN2A1 epitope.

Mapping of epitopes recognized by monoclonal antibodies 101G5 and 107G3

To characterize BTN2A1 for epitope mapping, the protein was proteolyzed with trypsin, chymotrypsins, Asp-N endopeptidase, elastase, and thermolysin, and the resulting digests were analyzed on an nLC-LTQ-Orbitrap MS/MS. After tryptic hydrolysis, 32 peptides were identified in the amino acid sequence of BTN2A1, occupying 79.84% of the BTN2A1 polypeptide chain; after digestion with chymotrypsin, 27 peptides were identified, occupying 94.76% of the BTN2A1 polypeptide chain; after digestion with endopeptidase Asp-N, 2 peptides were identified, occupying 12.5% of the BTN2A1 polypeptide chain; after digestion with elastase, 33 peptides were identified, occupying 89.11% of the BTN2A1 polypeptide chain; after thermolysin digestion, 29 peptides were identified, occupying 78.23% of the BTN2A1 polypeptide chain. Based on the results obtained in this analysis, a “map” of the BTN2A1 polypeptide chain was compiled with overlapping regions - peptides resulting from the cleavage of BTN2A1 by these enzymes (Fig. 13). The combination of all these peptides covered 96.37% of the amino acid sequence of BTN2A1. To determine epitopes in the BTN2A1/107G3 and BTN2A1/101G5 complexes with high resolution, the complexes were incubated with deuterated cross-linkers and then digested with the indicated enzymes. After proteolysis of the BTN2A1/107G3 protein complex with trypsin, chymotrypsins, Asp-N endopeptidase, elastase and thermolysin, nLC-Orbitrap MS/MS analysis revealed 17 cross-linked peptides between the BTN2A1 antibody and the 107G3 antibody.

Table 8. Amino acid sequences and positions of cross-linked peptides after multienzyme proteolysis of the BTN2A1/107G3 complex

Amino acid sequence
(protein 1 - protein 2) Enzyme Protein 1 Protein 2 Position Protein 1 Position Protein 2 LTNYV-EDMEVRWFRSQFSPA-a4-b6 Elastase 107G3_VH BTN2A1 29-33 60-74 LTNYV-EDMEVRWFRSQFSPA-a2-b9 Elastase 107G3_VH BTN2A1 29-33 60-74 LTNYV-EDMEVRWFRSQFSPA-a4-b9 Elastase 107G3_VH BTN2A1 29-33 60-74 WTGGDTNYNS-RWFRSQFSPAV-a8-b4 Elastase 107G3_VH BTN2A1 52-61 65-75 YCQHSRDLPYAF-FRSQFSPAVF-a10-b3 Chemo-trypsin 107G3_VL BTN2A1 91-102 67-76 GLEWLGVIWTGGDTNYNSALKSR-SQFSPAVFVYKGGR-a18-b4 Trypsin 107G3_VH BTN2A1 44-66 69-82 LTNYV-EDMEVRWFRSQFSPA-a2-b13 Elastase 107G3_VH BTN2A1 29-33 60-74 TNYVI-EDMEVRWFRSQFSPA-a3-b13 Elastase 107G3_VH BTN2A1 30-34 60-74 YSYMHWYQQKPGQPPKL-FVYKGG-a2-b3 Elastase 107G3_VL BTN2A1 34-50 76-81 YCARGGQLGL-VYKGGRERTEEQMEEYRGRTTF-a4-b8 Chemo-trypsin 107G3_VH BTN2A1 94-103 77-98 KSRLS-RERTEEQMEEYRGRTTFV-a2-b4 Elastase 107G3_VH BTN2A1 64-68 82-99 ALKSR-VYKGGRERTEEQMEEYRGRTTF-a3-b9 Thermo-lysine 107G3_VH BTN2A1 62-66 77-98 SLTNYVISW-KGGRERTEEQMEEYRGRTTFVSKDISRGSVAL-a3-b17 Chemo-trypsin 107G3_VH BTN2A1 28-36 79-110 YCARGGQLGL-VYKGGRERTEEQMEEYRGRTTF-a4-b21 Chemo-trypsin 107G3_VH BTN2A1 94-103 77-98 GQRATISCRASKTVSSSGYSY-RGRTTFVSKDISRGSVAL-a13-b5 Chemo-trypsin 107G3_VL BTN2A1 16-36 93-110 TVSSSGYSYMHWYQQKPGQPPK-TTFVSK-a11-b5 Trypsin 107G3_VL BTN2A1 28-49 96-101 TVSSSGYSYMHWYQQKPGQPPK-TTFVSK-a8-b5 Trypsin 107G3_VL BTN2A1 28-49 96-101

Thus, the data obtained by the authors indicate that the interaction of BTN2A1 and antibody 107G3 involves amino acid residues of BTN2A1 occupying the following positions in its polypeptide chain: 65, 68, 69, 72, 78; 84, 85, 95, 97, 100. These results are illustrated in FIG. 14A and 15.

After multienzyme proteolysis of the BTN2A1/101G5 complex, nLC-Orbitrap MS/MS analysis revealed 14 cross-linked peptides between BTN2A1 and the 101G5 antibody.

Table 9. Amino acid sequences and positions of cross-linked peptides after multienzyme proteolysis of the BTN2A1/101G5 complex.

Amino acid sequence
(protein 1 - protein 2) Enzyme Protein 1 Protein 2 Position
Protein 1 Position
Protein 2 QSPEKSLEWIGEINPSTGGTTYNQKFK-DKSVR-a16-b2 Trypsin 101G5_VH BTN2A1 39-65 211-215 QSPEKSLEWIGEINPSTGGTTYNQKFK-DKSVR-a17-b2 Trypsin 101G5_VH BTN2A1 39-65 211-215 QSPEKSLEWIGEINPSTGGTTYNQKFK-DKSVR-a25-b2 Trypsin 101G5_VH BTN2A1 39-65 211-215 FTVYYM-IRDKSVRНМ SCS-a4-b5 Thermolysin 101G5_VH BTN2A1 29-34 209-220 FKAKATLTVDK-NM SCSINNTLLGQKK-a2-b3 Trypsin 101G5_VH BTN2A1 64-74 216-230 FKAKATLTVDK-HM SCSINNTLLGQKK-a4-b3 Trypsin 101G5_VH BTN2A1 64-74 216-230 TTYNQKFKAKA-VRНМ SCS-a6-b5 Elastase 101G5_VH BTN2A1 58-68 214-220 TTYNQKFKAKA-VRНМ SCS-a8-b5 Elastase 101G5_VH BTN2A1 58-68 214-220 INPSTGGTTYNQK-MSCSINNT-a10-b2 Thermolysin Chemo-trypsin 101G5_VH BTN2A1 51-63 217-224 INPSTGGTTYNQK-MSCSINNT-a8-b2 Thermolysin 101G5_VH BTN2A1 51-63 217-224 FKAKATLTVDK-HM SCSINNTLLGQKK-a4-b5 Trypsin 101G5_VH BTN2A1 64-74 216-230 LLIYRTSNLASGVPGR-SVRНМ SCSINNTLLGQK-a7-b12 Trypsin 101G5_VL BTN2A1 47-62 213-229 ISSNYLHWYRHKPGFSPK-MSCSINNTL-a2-b8 Thermolysin 101G5_VL BTN2A1 29-46 217-225 FKAKATLTVDK-NM SCSINNTLLGQKK-a2-b14 Trypsin 101G5_VH BTN2A1 64-74 216-230

Thus, the data obtained by the authors indicate that the interaction of BTN2A1 and antibody 101G5 involves amino acid residues of BTN2A1 occupying the following positions in its polypeptide chain: 212, 213, 218, 220, 224, 229. These results are illustrated in Fig. 14B and 16.

Reference monoclonal antibodies 101G5 and 107G3 to BTN2A show cross-reactivity with the cynomolgus macaque ortholog BTN2A1

Most nonhuman primates have orthologues of BTN2A1; they are found, for example, in the cynomolgus macaque (Macaca fascicularis). To determine the cross-reactivity of monoclonal antibodies 101G5 and 107G3 with the cynoBTN2A1 orthologous cynomolgus protein [cynoBTN2A1; registration number in the US National Center for Biotechnology Information (NCBI) database XM_015448906.1; identity with human (hu) BTN2A1 93.31%], the authors obtained a recombinant Fc-fusion protein containing the ectodomain of the cynoBTN2A1 protein (cynoBTN2A1-Fc) and determined the binding of reference monoclonal antibodies to this protein by ELISA. An ELISA assay was also performed with the recombinant huBTN2A1-Fc fusion protein to compare the binding affinity of the reference antibodies to human BTN2A1 and its simian orthologue. As can be seen in FIG. 13, both antibody 101G5 and 107G3 had the ability to bind to the ectodomain of cynoBTN2A1, which was characterized by EC ₅₀ = 0.60 and 0.57 nm, respectively; these values are comparable to the EC ₅₀ for the binding of these antibodies to huBTN2A1 (0.82 and 0.56 nm, respectively).

Table 10. Brief description of amino acid and nucleotide sequences necessary for the practical implementation of this invention

Links

Various documents have been cited throughout this application to describe the prior art to which this invention relates. The contents of the related works are incorporated herein by reference.

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List of sequences

<110> IMCHEK THERAPUTICS SAS

INSERM

SNRS

UNIVERSITY DE HAIM-MARCAIL

ENSTITUE JEAN PAOLI E IRENE CALMETTE

<120> ANTIBODIES HAVING SPECIFICITY FOR BTN2 AND USES THEREOF

<130> BCT200062 QT

<150>EP19305345.1

<151> 2019-03-20

<150>EP19219691.3

<151> 2019-12-24

<160> 35

<170> Patent In version 3.5

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Met Ala Val Leu Ala Leu Leu Leu Cys Leu Met Thr Phe Pro Ser Cys

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Ala Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala

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Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

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Thr Asn Tyr Val Ile Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

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Glu Trp Leu Gly Val Ile Trp Thr Gly Gly Asp Thr Asn Tyr Asn Ser

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Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln

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Val Phe Leu Lys Met Asn Ser Leu Gln Thr Gly Asp Thr Ala Arg Tyr

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Tyr Cys Ala Arg Gly Gly Gln Leu Gly Leu Arg Gly Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ala

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Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Thr

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Val Ser Ser Ser Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro

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Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser

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Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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aactatgtta taagc

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ggaaagggtc tggagtggct tggagtaatt tggactggtg gagacacaaa ttataattca

240

gctctcaaat ccagactgag catcagcaaa gacaactcca agagtcaagt tttcttaaaa

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Met Glu Ser Ala Ala Ala Leu His Phe Ser Arg Pro Ala Ser Leu Leu

1 5 10 15

Leu Leu Leu Leu Ser Leu Cys Ala Leu Val Ser Ala Gln Phe Ile Val

20 25 30

Val Gly Pro Thr Asp Pro Ile Leu Ala Thr Val Gly Glu Asn Thr Thr

35 40 45

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50 55 60

Arg Trp Phe Arg Ser Gln Phe Ser Pro Ala Val Phe Val Tyr Lys Gly

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Gly Arg Glu Arg Thr Glu Glu Gln Met Glu Glu Tyr Arg Gly Arg Thr

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His Asn Ile Thr Ala Gln Glu Asn Gly Thr Tyr Arg Cys Tyr Phe Gln

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Glu Gly Arg Ser Tyr Asp Glu Ala Ile Leu His Leu Val Val Ala Gly

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Leu Gly Ser Lys Pro Leu Ile Ser Met Arg Gly His Glu Asp Gly Gly

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Ile Arg Leu Glu Cys Ile Ser Arg Gly Trp Tyr Pro Lys Pro Leu Thr

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Val Trp Arg Asp Pro Tyr Gly Gly Val Ala Pro Ala Leu Lys Glu Val

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Ser Met Pro Asp Ala Asp Gly Leu Phe Met Val Thr Thr Ala Val Ile

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Ile Arg Asp Lys Ser Val Arg Asn Met Ser Cys Ser Ile Asn Asn Thr

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Leu Leu Gly Gln Lys Lys Glu Ser Val Ile Phe Ile Pro Glu Ser Phe

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Met Pro Ser Val Ser Pro Cys Ala Val Ala Leu Pro Ile Ile Val Val

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Ile Leu Met Ile Pro Ile Ala Val Cys Ile Tyr Trp Ile Asn Lys Leu

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305 310 315 320

Arg Arg Thr Phe Leu His Ala Val Asp Val Val Leu Asp Pro Asp Thr

325 330 335

Ala His Pro Asp Leu Phe Leu Ser Glu Asp Arg Arg Ser Val Arg Arg

340 345 350

Cys Pro Phe Arg His Leu Gly Glu Ser Val Pro Asp Asn Pro Glu Arg

355 360 365

Phe Asp Ser Gln Pro Cys Val Leu Gly Arg Glu Ser Phe Ala Ser Gly

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Gly Val Cys Arg Asp Ser Val Glu Arg Lys Gly Glu Val Leu Leu Ile

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Pro Gln Asn Gly Phe Trp Thr Leu Glu Met His Lys Gly Gln Tyr Arg

420 425 430

Ala Val Ser Ser Pro Asp Arg Ile Leu Pro Leu Lys Glu Ser Leu Cys

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Arg Val Gly Val Phe Leu Asp Tyr Glu Ala Gly Asp Val Ser Phe Tyr

450 455 460

Asn Met Arg Asp Arg Ser His Ile Tyr Thr Cys Pro Arg Ser Ala Phe

465 470 475 480

Ser Val Pro Val Arg Pro Phe Phe Arg Leu Gly Cys Glu Asp Ser Pro

485 490 495

Ile Phe Ile Cys Pro Ala Leu Thr Gly Ala Asn Gly Val Thr Val Pro

500 505 510

Glu Glu Gly Leu Thr Leu His Arg Val Gly Thr His Gln Ser Leu

515 520 525

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Met Glu Pro Ala Ala Ala Leu His Phe Ser Leu Pro Ala Ser Leu Leu

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Leu Leu Leu Leu Leu Leu Leu Leu Ser Leu Cys Ala Leu Val Ser Ala

20 25 30

Gln Phe Thr Val Val Gly Pro Ala Asn Pro Ile Leu Ala Met Val Gly

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Glu Asn Thr Thr Leu Arg Cys His Leu Ser Pro Glu Lys Asn Ala Glu

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Asp Met Glu Val Arg Trp Phe Arg Ser Gln Phe Ser Pro Ala Val Phe

65 70 75 80

Val Tyr Lys Gly Gly Arg Glu Arg Thr Glu Glu Gln Met Glu Glu Tyr

85 90 95

Arg Gly Arg Ile Thr Phe Val Ser Lys Asp Ile Asn Arg Gly Ser Val

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Ala Leu Val Ile His Asn Val Thr Ala Gln Glu Asn Gly Ile Tyr Arg

115 120 125

Cys Tyr Phe Gln Glu Gly Arg Ser Tyr Asp Glu Ala Ile Leu Arg Leu

130 135 140

Val Val Ala Gly Leu Gly Ser Lys Pro Leu Ile Glu Ile Lys Ala Gln

145 150 155 160

Glu Asp Gly Ser Ile Trp Leu Glu Cys Ile Ser Gly Gly Trp Tyr Pro

165 170 175

Glu Pro Leu Thr Val Trp Arg Asp Pro Tyr Gly Glu Val Val Pro Ala

180 185 190

Leu Lys Glu Val Ser Ile Ala Asp Ala Asp Gly Leu Phe Met Val Thr

195 200 205

Thr Ala Val Ile Ile Arg Asp Lys Tyr Val Arg Asn Val Ser Cys Ser

210 215 220

Val Asn Asn Thr Leu Leu Gly Gln Glu Lys Glu Thr Val Ile Phe Ile

225 230 235 240

Pro Glu Ser Phe Met Pro Ser Ala Ser Pro Trp Met Val Ala Leu Ala

245 250 255

Val Ile Leu Thr Ala Ser Pro Trp Met Val Ser Met Thr Val Ile Leu

260 265 270

Ala Val Phe Ile Ile Phe Met Ala Val Ser Ile Cys Cys Ile Lys Lys

275 280 285

Leu Gln Arg Glu Lys Lys Ile Leu Ser Gly Glu Lys Lys Val Glu Gln

290 295 300

Glu Glu Lys Glu Ile Ala Gln Gln Leu Gln Glu Glu Leu Arg Trp Arg

305 310 315 320

Arg Thr Phe Leu His Ala Ala Asp Val Val Leu Asp Pro Asp Thr Ala

325 330 335

His Pro Glu Leu Phe Leu Ser Glu Asp Arg Arg Ser Val Arg Arg Gly

340 345 350

Pro Tyr Arg Gln Arg Val Pro Asp Asn Pro Glu Arg Phe Asp Ser Gln

355 360 365

Pro Cys Val Leu Gly Trp Glu Ser Phe Ala Ser Gly Lys His Tyr Trp

370 375 380

Glu Val Glu Val Glu Asn Val Met Val Trp Thr Val Gly Val Cys Arg

385 390 395 400

His Ser Val Glu Arg Lys Gly Glu Val Leu Leu Ile Pro Gln Asn Gly

405 410 415

Phe Trp Thr Leu Glu Met Phe Gly Asn Gln Tyr Arg Ala Leu Ser Ser

420 425 430

Pro Glu Arg Ile Leu Pro Leu Lys Glu Ser Leu Cys Arg Val Gly Val

435 440 445

Phe Leu Asp Tyr Glu Ala Gly Asp Val Ser Phe Tyr Asn Met Arg Asp

450 455 460

Arg Ser His Ile Tyr Thr Cys Pro Arg Ser Ala Phe Thr Val Pro Val

465 470 475 480

Arg Pro Phe Phe Arg Leu Gly Ser Asp Asp Ser Pro Ile Phe Ile Cys

485 490 495

Pro Ala Leu Thr Gly Ala Ser Gly Val Met Val Pro Glu Glu Gly Leu

500 505 510

Lys Leu His Arg Val Gly Thr His Gln Ser Leu

515 520

<210> 19

<211> 137

<212>PRT

<213> Mus musculus

<400> 19

Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Val Tyr Tyr Met Leu Trp Val Lys Gln Ser Pro Glu Lys Ser Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn

65 70 75 80

Gln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Pro Ser Phe Tyr Ala Leu Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 20

<211> 130

<212>PRT

<213> Mus musculus

<400> 20

Met Asp Phe Gln Met Gln Ile Ile Ser Leu Leu Leu Ile Ser Val Thr

1 5 10 15

Val Ile Val Ser His Gly Glu Ile Val Leu Thr Gln Ser Pro Thr Thr

20 25 30

Met Ala Ala Ser Pro Gly Glu Lys Ile Thr Ile Thr Cys Ser Ala Thr

35 40 45

Ser Ser Ile Ser Ser Asn Tyr Leu His Trp Tyr Arg His Lys Pro Gly

50 55 60

Phe Ser Pro Lys Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly

65 70 75 80

Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

85 90 95

Thr Ile Gly Thr Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln

100 105 110

Gln Gly Ser Ser Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys

130

<210> 21

<211> 5

<212>PRT

<213> Mus musculus

<400> 21

Val Tyr Tyr Met Leu

15

<210> 22

<211> 17

<212>PRT

<213> Mus musculus

<400> 22

Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Ala

<210> 23

<211> 9

<212>PRT

<213> Mus musculus

<400> 23

Gly Pro Ser Phe Tyr Ala Leu Asp Tyr

15

<210> 24

<211> 12

<212>PRT

<213> Mus musculus

<400> 24

Ser Ala Thr Ser Ser Ile Ser Ser Asn Tyr Leu His

1 5 10

<210> 25

<211> 7

<212>PRT

<213> Mus musculus

<400> 25

Arg Thr Ser Asn Leu Ala Ser

15

<210> 26

<211> 9

<212>PRT

<213> Mus musculus

<400> 26

Gln Gln Gly Ser Ser Ile Pro Arg Thr

15

<210> 27

<211> 15

<212> DNA

<213> Mus musculus

<400> 27

gtctactaca tgctc

15

<210> 28

<211> 51

<212> DNA

<213> Mus musculus

<400> 28

gagattaatc ctagcactgg tggtactacc tacaaccaga agttcaaggc c

51

<210> 29

<211> 27

<212> DNA

<213> Mus musculus

<400> 29

ggcccgagct tttatgctct ggactac

27

<210> 30

<211> 36

<212> DNA

<213> Mus musculus

<400> 30

agtgccacct ctagtataag ttccaattac ttgcat

36

<210> 31

<211> 21

<212> DNA

<213> Mus musculus

<400> 31

aggacatcca atctggcttc t

21

<210> 32

<211> 27

<212> DNA

<213> Mus musculus

<400> 32

cagcagggta gtagtatacc acgcacg

27

<210> 33

<211> 411

<212> DNA

<213> Mus musculus

<400> 33

atgggatgga actggatctt tattttaatc ctgtcagtaa ctacaggtgt ccactctgag

60

gtccagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc

120

tgcaaggctt ctggttactc attcactgtc tactacatgc tctgggtgaa acaaagtcct

180

gaaaagagcc ttgagtggat tggagagatt aatcctagca ctggtggtac tacctacaac

240

cagaagttca aggccaaggc cacattgact gtagacaaat cctccagcac agcctacatg

300

cagctcaaga gcctgacatc tgaggactct gcagtctatt actgtgcaag gggcccgagc

360

ttttatgctc tggactactg gggtcaagga acctcagtca ccgtctcctc a

411

<210> 34

<211> 390

<212> DNA

<213> Mus musculus

<400> 34

atggattttc agatgcagat tatcagcttg ctgctaatca gtgtcacagt catagtgtct

60

catggagaaa ttgtgctcac ccagtctcca accaccatgg ctgcatctcc cggggagaag

120

atcactatca cctgcagtgc cacctctagt ataagttcca attacttgca ttggtatcga

180

cataagccag gattctcccc taaactcttg atttatagga catccaatct ggcttctgga

240

gtcccaggtc gcttcagtgg cagtgggtct gggacctctt actctctcac aattggcacc

300

atggaggctg aagatgttgc cacttactac tgccagcagg gtagtagtat accacgcacg

360

ttcggagggg ggaccaagct ggaaataaaa

390

<210> 35

<211> 1083

<212>PRT

<213> Macaca fascicularis

<400> 35

Met Gln Arg Gln Phe Ser Lys Ala Ser Arg Pro Cys Leu Pro Trp Val

1 5 10 15

Leu Met Glu Pro Ala Ala Ala Leu His Phe Ser Leu Pro Ala Ser Leu

20 25 30

Ile Leu Leu Leu Leu Leu Leu Arg Leu Cys Ala Leu Val Ser Ala Gln

35 40 45

Phe Thr Val Val Gly Pro Thr Asp Pro Ile Leu Ala Met Val Gly Glu

50 55 60

Asn Thr Thr Leu Arg Cys His Leu Ser Pro Glu Lys Asn Ala Glu Asp

65 70 75 80

Met Glu Val Arg Trp Phe Arg Ser Gln Phe Ser Pro Ala Val Phe Val

85 90 95

Tyr Lys Gly Gly Arg Glu Arg Thr Glu Glu Gln Met Glu Glu Tyr Arg

100 105 110

Gly Arg Thr Thr Phe Val Ser Lys Asp Ile Ser Arg Gly Ser Val Ala

115 120 125

Leu Ile Ile His Asn Val Thr Ala Gln Glu Asn Gly Thr Tyr Arg Cys

130 135 140

Tyr Phe Gln Glu Gly Arg Ser Tyr Asp Glu Ala Ile Leu His Leu Met

145 150 155 160

Val Ala Gly Leu Gly Ser Lys Pro Leu Val Glu Met Arg Gly His Glu

165 170 175

Asp Gly Gly Ile Arg Leu Glu Cys Ile Ser Arg Gly Trp Tyr Pro Lys

180 185 190

Pro Leu Thr Val Trp Arg Asp Pro Tyr Gly Arg Val Val Pro Ala Leu

195 200 205

Lys Glu Val Phe Pro Pro Asp Thr Asp Gly Leu Phe Met Val Thr Thr

210 215 220

Ala Val Ile Ile Arg Asp Lys Ser Met Arg Asn Met Ser Cys Ser Ile

225 230 235 240

Ser Asp Thr Leu Leu Gly Gln Lys Lys Glu Ser Val Ile Phe Ile Pro

245 250 255

Glu Ser Phe Met Pro Ser Val Ser Pro Cys Val Val Ala Leu Pro Ile

260 265 270

Ile Val Val Phe Leu Met Ile Ile Ile Ala Val Cys Ile Tyr Trp Ile

275 280 285

Asn Arg Leu Gln Lys Glu Thr Lys Ile Leu Ser Gly Glu Lys Glu Ser

290 295 300

Glu Arg Lys Thr Arg Glu Ile Ala Val Lys Glu Leu Lys Lys Glu Arg

305 310 315 320

Val Gln Lys Glu Lys Glu Leu Gln Val Lys Glu Gln Leu Gln Glu Glu

325 330 335

Leu Arg Trp Arg Arg Thr Val Leu His Ala Val Asp Val Val Leu Asp

340 345 350

Pro Asp Thr Ala His Pro Asp Leu Leu Leu Ser Glu Asp Arg Arg Ser

355 360 365

Val Arg Arg Cys Pro Leu Gly His Leu Gly Glu Ser Val Pro Asp Asn

370 375 380

Pro Glu Arg Phe Asn Ser Glu Pro Cys Val Leu Gly Arg Glu Ser Phe

385 390 395 400

Ala Ser Gly Lys His Tyr Trp Glu Val Glu Val Glu Asn Val Ile Glu

405 410 415

Trp Thr Val Gly Val Cys Arg Asp Ser Val Glu Arg Lys Glu Glu Val

420 425 430

Leu Leu Arg Pro Arg Asn Gly Phe Trp Thr Leu Glu Met Cys Lys Gly

435 440 445

Gln Tyr Arg Ala Leu Ser Ser Pro Lys Arg Ile Leu Pro Leu Lys Glu

450 455 460

Ser Leu Cys Arg Val Gly Val Phe Leu Asp Tyr Glu Ala Gly Asp Val

465 470 475 480

Ser Phe Tyr Asn Met Arg Asp Arg Ser His Ile Tyr Thr Cys Pro Arg

485 490 495

Leu Ala Phe Ser Val Pro Val Arg Pro Phe Phe Arg Ile Gly Ser Asp

500 505 510

Asp Ser Pro Ile Phe Ile Cys Pro Ala Leu Thr Gly Ala Ser Gly Ile

515 520 525

Thr Val Pro Glu Glu Gly Leu Ile Leu His Arg Val Gly Thr Asn Gln

530 535 540

Ser Leu Met Pro Val Gly Thr Arg Cys Tyr Gly His Gly Met Arg Pro

545 550 555 560

Thr Gly Phe Ile Arg Met Arg Glu Glu Arg Gly Ile His Arg Thr Thr

565 570 575

Arg Glu Glu Arg Glu Pro Asp Met Gln Asn Phe Asp Leu Gly Ala His

580 585 590

Trp Ser Asn Asn Leu Pro Ser Ala Arg Ser Arg Glu Phe Leu Asn Ser

595 600 605

Asp Leu Val Pro Asp His Ser Leu Glu Ser Pro Val Thr Pro Gly Leu

610 615 620

Ala Asn Lys Thr Gly Glu Pro Gln Ala Glu Val Thr Cys Leu Cys Phe

625 630 635 640

Ser Leu Pro Ser Ser Glu Leu Arg Ala Phe Pro Ser Thr Ala Thr Asn

645 650 655

His Asn His Lys Ala Thr Ala Leu Gly Ser Asp Leu His Ile Glu Val

660 665 670

Lys Gly Tyr Glu Asp Gly Gly Ile His Leu Glu Cys Arg Ser Thr Gly

675 680 685

Trp Tyr Pro Gln Pro Gln Ile Gln Trp Ser Asn Thr Lys Gly Gln His

690 695 700

Ile Pro Ala Val Lys Ala Pro Val Val Ala Asp Gly Val Gly Leu Tyr

705 710 715 720

Ala Val Ala Ala Ser Val Ile Met Arg Gly Ser Ser Gly Glu Gly Val

725 730 735

Ser Cys Ile Ile Arg Asn Ser Leu Leu Gly Leu Glu Lys Thr Ala Ser

740 745 750

Ile Ser Ile Thr Asp Pro Phe Phe Arg Asn Ala Gln Pro Trp Ile Ala

755 760 765

Ala Leu Ala Gly Thr Leu Pro Ile Ser Leu Leu Leu Leu Ala Gly Ala

770 775 780

Ser Tyr Phe Leu Trp Arg Gln Gln Lys Glu Lys Ile Ala Leu Ser Arg

785 790 795 800

Glu Thr Glu Arg Glu Arg Glu Met Lys Glu Met Gly Tyr Ala Ala Thr

805 810 815

Lys Gln Glu Ile Ser Leu Arg Gly Gly Glu Lys Ser Leu Ala Tyr His

820 825 830

Gly Thr His Ile Ser Tyr Leu Ala Ala Pro Glu Arg Trp Glu Met Ala

835 840 845

Val Phe Pro Asn Ser Gly Leu Pro Arg Cys Leu Leu Thr Leu Ile Leu

850 855 860

Leu Gln Leu Pro Lys Leu Asp Ser Ala Pro Phe Asp Val Ile Gly Pro

865 870 875 880

Pro Glu Pro Ile Leu Ala Val Val Gly Glu Asp Ala Glu Leu Pro Cys

885 890 895

Arg Leu Ser Pro Asn Ala Ser Ala Glu His Leu Glu Leu Arg Trp Phe

900 905 910

Arg Lys Lys Val Ser Pro Ala Val Leu Val His Arg Asp Gly Arg Glu

915 920 925

Gln Glu Ala Glu Gln Met Pro Glu Tyr Arg Gly Arg Ala Thr Leu Val

930 935 940

Gln Asp Gly Ile Ala Glu Gly Arg Val Ala Leu Arg Ile Arg Gly Val

945 950 955 960

Arg Val Ser Asp Asp Gly Glu Tyr Thr Cys Phe Phe Arg Glu Asp Gly

965 970 975

Ser Tyr Glu Glu Ala Leu Val His Leu Lys Val Ala Ala Leu Gly Ser

980 985 990

Asp Pro His Ile Ser Met Gln Val Gln Glu Asn Gly Glu Ile Trp Leu

995 1000 1005

Glu Cys Thr Ser Val Gly Trp Tyr Pro Glu Pro Gln Val Gln Trp

1010 1015 1020

Arg Thr Ser Lys Gly Glu Lys Phe Pro Ser Thr Ser Glu Ser Arg

1025 1030 1035

Asn Pro Asp Glu Glu Gly Leu Phe Thr Val Ala Ala Ser Val Ile

1040 1045 1050

Ile Arg Asp Thr Ser Val Lys Asn Val Ser Cys Tyr Ile Gln Asn

1055 1060 1065

Leu Leu Leu Gly Gln Glu Lys Glu Val Glu Ile Phe Ile Pro Gly

1070 1075 1080

<---

Claims (35)

1. Antibody to butyrophilin-2A1 (BTN2A1), characterized by the fact that it contains: heavy chain variable region CDR1 region containing the sequence SEQ ID NO:3, heavy chain variable region CDR2 region containing the sequence SEQ ID NO:4, heavy chain variable region CDR3 region containing the sequence SEQ ID NO:5, light chain variable region CDR1 region chain containing the sequence of SEQ ID NO:6, a light chain variable region CDR2 region containing the sequence SEQ ID NO:7 and a light chain variable region CDR3 region containing the sequence SEQ ID NO:8, or heavy chain variable region CDR1 region containing the sequence SEQ ID NO:21, heavy chain variable region CDR2 region containing the sequence SEQ ID NO:22, heavy chain variable region CDR3 region containing the sequence SEQ ID NO:23, light chain variable region CDR1 region chain comprising the sequence of SEQ ID NO:24, a light chain variable region CDR2 region containing the sequence SEQ ID NO:25, and a light chain variable region CDR3 region containing the sequence SEQ ID NO:26. 2. An antibody according to claim 1, which has at least one of the following functions: (i) inhibits the polarization of monocytes into M2 macrophages; (ii) causes a reversion of M2 macrophages towards antitumor M1 macrophages; (iii) directly triggers NK cell activation; (iv) enhances NK cell-mediated cytotoxicity. 3. An antibody according to claim 1 or 2, which directly triggers NK cell activation and/or enhances NK cell-mediated cytotoxicity. 4. Antibody according to any one of paragraphs. 1-3, which competes for binding to BTN2A1 - with a reference monoclonal antibody comprising (i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO:1, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO:2, or - with a reference monoclonal antibody comprising (i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO:19, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO:20. 5. Antibody according to any one of paragraphs. 1-4, which binds to an epitope containing amino acid residues at positions 65, 68, 69, 72, 78; 84, 85, 95, 97 and 100 of SEQ ID NO:17. 6. Antibody according to any one of paragraphs. 1-5, which binds to an epitope containing amino acid residues at positions 212, 213, 218, 220, 224 and 229 of the sequence SEQ ID NO:17. 7. Antibody according to any one of paragraphs. 1-6, which does not show cross-reactivity with human BTN3 isoforms and/or shows cross-reactivity with the cynomolgus protein, which is an ortholog of BTN2A1. 8. An antibody according to any of the previous paragraphs, which contains: - a heavy chain variable region containing an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:1, and a light chain variable region containing an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:2, or - a heavy chain variable region containing an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:19, and a light chain variable region containing an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:20. 9. Antibody according to any one of paragraphs. 1-7, which additionally has at least one of the following functions: i. activates the secretion of cytolytic agents by Vγ9Vδ2-T cells, ii. activates the cytolytic function of Vγ9Vδ2-T cells and/or iii. activates the proliferation of Vγ9Vδ2-T cells. 10. The antibody of claim 9, which competes for binding to BTN2A1 with a reference mouse monoclonal antibody comprising (i) a heavy chain variable region containing the amino acid sequence of SEQ ID NO:1, and (ii) a light chain variable region containing the amino acid sequence of SEQ ID NO:2. 11. Antibody according to any one of paragraphs. 9 or 10, which includes: a heavy chain variable region containing an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:1, and a light chain variable region containing an amino acid sequence at least 90% identical to the sequence of SEQ ID NO:2. 12. Antibody according to any one of paragraphs. 9-11, specific for the BTN2A1 isoform of the human protein. 13. An antibody according to any of the previous paragraphs, which is human, chimeric or humanized. 14. A nucleic acid molecule encoding the heavy chain and light chain of an antibody according to any one of claims. 1-13. 15. A host cell containing the nucleic acid molecule according to claim 14, for use in producing an antibody according to any one of claims. 1-13. 16. Antibody according to any one of paragraphs. 1-13 for use in therapy. 17. Antibody according to any one of paragraphs. 1-13 for use in the treatment of cancer or infectious diseases. 18. Pharmaceutical composition containing an antibody according to any one of paragraphs. 1-13 and at least a pharmaceutically acceptable carrier, for use in enhancing macrophage-mediated immunity in a patient having cancer or an infectious disease. 19. A method of enhancing macrophage-mediated immunity in a patient with cancer or an infectious disease, including the step of activating the BTN2A1 protein expressed on the plasma membrane of monocytes and macrophages. 20. The method according to claim 19, including administering to the patient an effective amount of an antibody according to any one of claims. 1-13. 21. The method according to claim 19 or 20 for the treatment of cancer or an infectious disease.

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