Butyrophilin (BTN) 3A activating antibodies for use in methods of treating infectious disorders

Abstract

The present disclosure relates to methods of treating infectious disorders. In particular, the present disclosure provides BTN3A activating antibodies and uses thereof in treating infectious disorders, e.g., caused by SARS-Cov2 or Coxiella burnetii infection, in a human subject in need of such treatment.

Description

[Technical field]

[0001] The present disclosure relates to methods of treating infectious disorders. In particular, the present disclosure provides BTN3A activating antibodies and uses thereof in treating infectious disorders, such as disorders caused by SARS-Cov2 or Coxiella burnetii infection, in a human subject in need of such treatment. Background

[0002] The global pandemic of coronavirus disease 2019, caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted to date in over 220 million cases worldwide and over 4.8 million deaths. While some infected individuals remain asymptomatic and some develop a mild respiratory illness that dissipates without or with little medical attention, others (10–20% of symptomatic individuals) experience severe disease that often results in a sudden deterioration approximately 11 days after the onset of the first symptoms (Chen et al., 2020), associated with multi-organ complications that can lead to respiratory failure and death of the patient (Gupta et al., 2020; Schultze and Aschenbrenner, 2021). Vaccination remains an asset against the virus and viral variants to prevent the disease, but currently only symptomatic treatment is offered. In this context, efforts must continue in the fight against the disease, which requires the development of drug treatments. In another context, immunotherapy may be considered in the case of Q fever. This worldwide zoonotic disease is caused by the bacterium Coxiella burnetii, which in humans causes a primary infection (acute Q fever) that is symptomatic in 40% of cases. Progression to a localized persistent infection is also observed in 1-5% of cases, mainly with cardiovascular and osteoarticular symptoms. Currently, the main treatment for acute Q fever is doxycycline. In some cases, such as endocarditis, prolonged treatment with doxycycline and hydroxychloroquine for several months is prescribed.

[0003] Current clinical management of COVID-19 consists of prevention, control measures such as social distancing, and supportive care including supplemental oxygen and mechanical ventilatory support when indicated. To date, the FDA has approved one drug, remdesivir (Veklury), for the treatment of COVID-19 in hospitalized patients aged 12 years or older who weigh at least 40 kg.

[0004] Human Vγ9Vδ2 T cells, which account for 2-5% of peripheral blood T cells, have been reported for patients with mycobacterial diseases, listeriosis, salmonellosis, brucellosis, tularemia, legionellosis and Q fever (Morita et al Immunol Rev 2007;215:59-76; Balbi et al. Am Rev Respir Dis 1993;148:1685-90; Jouen-Beades F,et al. Infect Immun 1997;65:4267-72; Hara T,Mizuno Y,Takaki K,et al. J Clin Invest 1992;90:204-10; Bertotto A,Gerli R,Spinozzi F,et al. Eur J Immunol 1993;23:1177-80; Poquet Y,Kroca M,Halary F,et al. Infect Immun 1998;66:2107-14; Kroca M, Johansson A, Sjostedt A, Tarnvik A. Clin Diagn Lab Immunol 2001;8:949-54; Schneider T, Jahn HU, Liesenfeld O,et al. Clin Infect Dis 1997;24:261-4), have been found to be expanded by a wide range of microbial agents during infection and to represent >50% of the circulating T cell pool (Chen ZW. Cell Mol Life Sci 2011;68:2409-1). Furthermore, localized expansion of Vγ9Vδ2 T cells has also been reported in bronchoalveolar lavage fluid from patients with active pulmonary tuberculosis and in cerebrospinal fluid from patients with bacterial meningitis (Chen ZW, Letvin NL. Microbes Infect 2003;5:491-8; Dieli F, Sireci G, Di Sano C, et al. Mol Med 1999;5:301-12; Caccamo N, La Mendola C, Orlando V, et al. Blood 2011;118:129-38).

[0005] Two direct antibacterial actions of Vγ9Vδ2 T cells against various viruses, protozoa and bacteria have been reported, including cytotoxic activity against pathogen-infected cells and cell-mediated non-cytolytic activity based on cytokine production (Bonneville M, Scotet E. Curr Opin Immunol 2006;18:539-46; Chen ZW. Cell Mol Immunol 2013;10:58-64; Poccia F, Agrati C, Martini F, Capobianchi MR, Wallace M, Malkovsky M. Microbes Infect 2005;7:518-28; Dong P, Ju X, Yan Y, et al. Front Immunol 2018;9. DOI:10.3389 / fimmu.2018.02812). In vitro studies have shown that gamma T cells can effectively kill intracellular pathogens such as M. tuberculosis, L. monocytogenes, and Brucella suis. Ryan-Payseur B,Frencher J,Shen L,Chen CY,Huang D,Chen ZW.J Immunol 2012;189:1285-93;Spencer CT,Abate G,Sakala IG,et al.PLOS Pathog 2013;9:e1003119;Dieli F,Troye-Blomberg M,Ivanyi J,et al.J Infect Dis 2001;184:1082-5;Martino A,Casetti R,Sacchi A,Poccia FJ Immunol Baltim Md 1950 2007;179:3057-64;Oliaro J,Dudal S,Liautard J,Andrault JB,Liautard JP,Lafont VJ Leukoc Biol 2005;77:652-60.

[0006] Several lines of evidence highlight the key role of Vγ9Vδ2 T cells in Q fever, an infectious disease caused by the intracellular parasitic bacterium Coxiella burnetii: (1) the number and percentage of Vγ9Vδ2 T cells increase during the acute phase of the disease, and (2) they are found to have significant expression of the activation marker HLA-DR but not CD25. Schneider T,Jahn HU,Liesenfeld O,et al.Clin Infect Dis 1997;24:261-4.

[0007] WO 2012 / 080351 reports BTN3A activating antibodies, such as murine mAb7.2 or mAb20.1, with the ability to induce proliferation and cytokine secretion of Vy9V52 T cells.

[0008] WO 2020 / 025703 further reports certain humanized BTN3A activating antibodies, particularly for use in the treatment of cancer disorders.

[0009] WO 2020 / 136218 also discloses fragments derived from the Fab fragment of the anti-BTN3A antibody mAb103.2, and the use of fragments derived from the Fab fragment as BTN3A activating antibodies for inducing proliferation and cytokine secretion of Vγ9Vδ2 T cells.

[0010] However, to our knowledge, there is no convincing evidence for the use of Vγ9Vδ2 T cell activating compounds, in particular BTN3A activating antibodies, to treat infectious disorders caused by Coxiella burnetii or SARS-Cov2.

[0011] The inventors surprisingly found that treatment with BTN3A-activating antibodies, such as treatment with mAb20.1, enhances Vγ9Vδ2 T cell responses against cells infected with SARS-CoV-2 and Coxiella burnetii, specifically reducing viral and bacterial burden, respectively.

[0012] A first aspect of the disclosure relates to a BTN3A activating antibody for use in the treatment of an infectious disorder in a human subject in need thereof, in particular for treating a disorder caused by SARS-Cov2 infection or Coxiella Burnetii infection.

[0013] In a specific embodiment, the BTN3A activating antibody is mAb20.1 or a humanized form of mAb20.1.

[0014] Another aspect of the present disclosure relates to novel humanized forms of mAb20.1 or pharmaceutical compositions of said novel humanized forms, in particular the use of said novel humanized forms and said pharmaceutical compositions in the treatment of disorders caused by SARS-Cov2 infection or Coxiella Burnetii infection.

[0015] Other specific aspects and particular embodiments are disclosed below. [Brief description of the drawings]

[0016] [Figure 1A] Expression of BTN2A and BTN3A in response to SARS-CoV-2 infection in monocytes, MDMs and lung epithelial cell lines [Figure 1B]Expression of BTN2A and BTN3A in response to SARS-CoV-2-infection in monocytes, MDMs and lung epithelial cell lines. (A, B) Monocytes, MDMs, BEAS-2B and MRC-5 cells were stimulated with SARS-CoV-2 IHU-MI6 strain (1 MOI) for 24 h. The mean fluorescence intensity (MFI) of BTN2A (A) and BTN3A (B) expression was examined in healthy donor monocytes and MDMs, and in the lung epithelial cell lines BEAS-2B and MRC-5 (n=3). Gene expression of the two BTN2A isoforms (A1, A2) (A) and the three BTN3A isoforms (A1, A2, A) (B) was examined by qRT-PCR after normalization using housekeeping genes as endogenous controls. The relative expression of the investigated genes at 24 h of stimulation was assessed in monocytes and MDMs (n=6), as well as in BEAS-2B and MRC-5 cells (n=3). [Diagram 2] Impact of SARS-CoV-2 on Vγ9Vδ2 T cell viability. Vγ9Vδ2 T cells (isolated from three healthy volunteers) were stimulated with SARS-CoV-2 IHU-MI6 strain (0.25, 0.5 or 1 MOI). After 24 h, Vγ9Vδ2 T cell viability was assessed by flow cytometry as the percentage of live cells in the Vγ9Vδ2 T cell population. Values ​​represent the mean ± standard error of the mean. [Figure 3A] Assessment of anti-SARS-CoV-2 responses of Vγ9Vδ2 T lymphocytes. [Figure 3B]Evaluation of anti-SARS-CoV-2 responses of Vγ9Vδ2 T lymphocytes. (A, B) Monocytes, MDM, BEAS-2B and MRC-5 cells (n=6) were stimulated with SARS-CoV-2 IHU-MI6 strain (1 MOI) and co-cultured with Vγ9Vδ2 T cells in the presence of anti-BTN3A 20.1 Ab (0, 0.1, 1 or 10 μg / ml) at a 1:1 effector-to-target (E:T) ratio for 24 h. (A) After 24 h, SARS-CoV-2 viral load was quantified by RT-PCR. Data were analyzed by the following formula 2^-(Ct anti-BTN3A ICT01-Ct diluent) and expressed as viral copies %. Cytotoxicity was assessed by flow cytometry as the percentage of caspase 3 / 7+ cells in the target cell population. (B) Culture supernatants from co-cultures were analyzed for the presence of IFN-γ by ELISA. Values ​​represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 4A] C. burnetii infection modulates the expression of BTN3A and BTN2A. [Figure 4B] C. burnetii infection modulates the expression of BTN3A and BTN2A. [Figure 4C] C. burnetii infection modulates the expression of BTN3A and BTN2A. [Figure 4D] C. burnetii infection modulates the expression of BTN3A and BTN2A. [Figure 4E] C. burnetii infection modulates the expression of BTN3A and BTN2A. [Figure 4F]C. burnetii infection modulates the expression of BTN3A and BTN2A. Monocytes isolated from healthy donors (n=4) were infected with C. burnetii strains (50 MOI) for 24 h. (A) Relative gene expression of BTN3A isoforms (A1, A2, A3) and (B) protein expression of BTN3A were examined by qRT-PCR and flow cytometry, respectively. (D) Relative gene expression of BTN2A isoforms (A1, A2) and (E) protein expression of BTN2A were examined by qRT-PCR and flow cytometry, respectively. (C) Protein expression of BTN3A and (F) BTN2A were examined in PBMCs from Q fever patients (n=6) or healthy donors (n=3). Values ​​represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 5A] Involvement of BTN3A and BTN2A in C. burnetii infection. [Figure 5B] Involvement of BTN3A and BTN2A in C. burnetii infection. CRISPR-Cas9-mediated inactivation of BTN3A or BTN2A was performed in THP-1 cell lines. THP-1 cells transduced with guides targeting all BTN2A isoforms (BTN2AKO) or all BTN3A isoforms (BTN3AKO), or with an irrelevant CRISPR guide for control cells (mock), were infected with C. burnetii NM1 (50 MOI) (n=3). (A) 4 and 24 hours after infection, bacterial DNA copy numbers in THP-1 cells were assessed by qPCR. (B) THP-1 cells were incubated with C. burnetii for 4 hours (day 0), then washed to remove free bacteria and incubated for 4 days. Every day, bacterial DNA copy numbers were assessed by qPCR. [Figure 6A] Involvement of BTN3A and BTN2A in the inflammatory response to C. burnetii infection. [Figure 6B]Involvement of BTN3A and BTN2A in the inflammatory response to C. burnetii infection. THP-1 cells transduced with an irrelevant CRISPR guide (mock) or guides targeting all BTN2A isoforms (BTN2AKO) or all BTN3A isoforms (BTN3AKO) were infected with C. burnetii NM1 (100 MOI) (n=3). 24 h post-infection, expression of genes involved in inflammatory (TNF, IL1B, IL6, IFNG, CXCL10) or immunoregulatory (IL10, TGFB1, IL1RA, CD163) responses was examined by quantitative reverse transcription polymerase chain reaction after normalization using the housekeeping actin gene as an endogenous control. Data are presented as (A) relative expression (RQ) of the investigated genes. (B) 24 h after infection, the release of TNF-α, IL-1β, IFN-γ, IL-6, IL-10, and TGF-β in the culture supernatant was evaluated by ELISA assay. Values ​​represent the mean ± SEM. *p<0.05 and **p<0.01. [Figure 7A] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7B] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7C] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7D] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7E] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7F] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7G]Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. [Figure 7H] Infection with C. burnetii leads to BTN3A- and BTN2-dependent activation of Vγ9Vδ2 T lymphocytes. (A) Monocytes isolated from healthy donors (n=3) pre-infected with C. burnetii NM1 (50 or 100 MOI) for 24 h were co-cultured with expanded Vγ9Vδ2 T cells from healthy donors (E:T ratio 1:1). Degranulation of Vγ9Vδ2 T cells (%CD107ab+ cells) was assessed by flow cytometry 4 h after co-culture. (B, C, F) Monocytes isolated from healthy donors (n=4), previously infected for 24 h with C. burnetii strains (50 MOI), were co-cultured with expanded Vγ9Vδ2 T cells from healthy donors (E:T ratio 1:1) in the presence of (B) anti-BTN2A antibody (clone 7.48), (C) anti-BTN3A antibody (clone 103.2) or (F) anti-BTN3A antibody (clone 20.1) (10 μg / ml). Degranulation of Vγ9Vδ2 T cells (% CD107ab+ cells) was assessed by flow cytometry after 4 h of co-culture. (G) Cytotoxicity was assessed by flow cytometry as the percentage of caspase 3 / 7+ cells in the target cell population after 4 h of co-culture in the presence of the reference anti-BTN3A 20.1 antibody (10 μg / ml). (D, E, H) Expanded Vγ9Vδ2 T cells from healthy donors were co-cultured with PBMCs from Q fever patients (n=6) or healthy donors (n=3) in the presence of (D) anti-BTN2A (clone 7.48), (E) anti-BTN3A (clone 103.2) or (F) anti-BTN3A (clone 20.1) (10 μg / ml) (E:T ratio 1:1). Degranulation of Vγ9Vδ2 T cells (%CD107ab+ cells) was assessed by flow cytometry 4 h after co-culture. Values ​​represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 8A] ref 20.1 BTN3A-activating antibodies increase the antibacterial activity of Vγ9Vδ2 T cells against C. burnetii-infected monocytes. [Figure 8B] Reference 20.1 BTN3A activating antibodies increase the antibacterial activity of Vγ9Vδ2 T cells against monocytes infected with C. burnetii. (A, B) Monocytes isolated from healthy donors (n=4) pre-infected for 24 h with C. burnetii NM1 (50 MOI) were co-cultured (E:T ratio 1:1) with expanded Vγ9Vδ2 T cells from healthy donors in the presence of anti-BTN3A 20.1 antibodies (0–10 μg / ml). After 4 h of co-culture, C. burnetii load was measured by (A) flow cytometry and (B) qPCR. Values ​​represent the mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001. [Figure 9A] Reference 20.1 BTN3A activating antibodies increase secretion of cytokines and cytotoxic molecules in Vγ9Vδ2 T cell / infected monocyte co-cultures. [Figure 9B] Reference 20.1 BTN3A activating antibodies increase secretion of cytokines and cytotoxic molecules in Vγ9Vδ2 T cell / infected monocyte co-cultures. Monocytes isolated from healthy donors (n=4), previously infected for 24 h with C. burnetii NM1 (50 MOI), were co-cultured (E:T ratio 1:1) with expanded Vγ9Vδ2 T cells from healthy donors in the presence of anti-BTN3A antibodies (clone 20.1) (0–10 μg / ml). After 4 h of co-culture, culture supernatants were analyzed for the presence of cytokines (A, left panel) and cytotoxic molecules (B, right panel) by ELISA assay. Values ​​represent the mean ± standard error. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Detailed Description

[0017] definition In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0018] The term "agonist" is used herein to refer to a molecule (e.g., a small organic molecule or a polypeptide, such as an antibody) that binds to a receptor and activates the receptor to produce a biological response. A selective agonist is selective for a particular type of receptor. Binding to the receptor can be specific binding, e.g., as determined by surface plasmon resonance at biologically relevant concentrations.

[0019] The terms "polypeptide," "protein," or "peptide" as used herein refer to any chain of amino acid residues, regardless of the length or post-translational modification (such as glycosylation) of the "polypeptide," "protein," or "peptide."

[0020] As used herein, the term "BTN3A" has the general meaning of "BTN3A" in the art. In certain embodiments, "BTN3A" refers to a human BTN3A polypeptide, including any of BTN3A1 of SEQ ID NO: 32, BTN3A2 of SEQ ID NO: 33, or BTN3A3 of SEQ ID NO: 34.

[0021] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. The terms "antibody" or "immunoglobulin" have the same meaning and are used equally in this disclosure. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies. The term "antibody" as used herein also includes bispecific or multispecific molecules. An antibody can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds at least two different binding sites or target molecules. An antibody can in fact be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds more than two different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the present disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results. In addition, for embodiments in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity in addition to the first and second target epitopes.

[0022] In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain contains two domains, the variable domain (VL) and the constant domain (CL). The heavy chain contains four domains, the variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The light chain (CL) and heavy chain (CH) constant region domains confer important biological properties such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR).

[0023] Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable part. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues mainly from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or may affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs designated as L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequence between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0024] Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the sequence of the sequence of sequence ID (SEQ ID). The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to truncations of or insertions into structural components, whether framework or complementarity determining regions (CDRs) of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by alignment of the homologous residues in the sequence of the antibody with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0025] The non-CDR regions of a mammalian antibody can be replaced with similar regions of a homospecific or heterospecific antibody, usually while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies, in which non-human CDRs are covalently linked to human FR and / or Fc / pFc' regions to produce a functional antibody.

[0026] As used herein, "humanization" describes an antibody in which some, most or all of the amino acids outside the CDR region are replaced with corresponding amino acids from human immunoglobulin molecules to reduce immunogenicity in human subjects. Humanization methods include, but are not limited to, those described in U.S. Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are incorporated herein by reference. The above U.S. Patent Nos. 5,585,089 and 5,693,761, as well as WO 90 / 07861, also propose four possible criteria that can be used when designing humanized antibodies. The first suggestion was to use a framework for the acceptor from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin being humanized, or to use a consensus framework from many human antibodies. The second suggestion was that if an amino acid in the framework of a human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, the donor amino acid rather than the acceptor amino acid can be selected. The third suggestion was that the donor amino acid rather than the acceptor amino acid can be selected at positions directly adjacent to the three CDRs of the humanized immunoglobulin chain. The fourth suggestion was to use donor amino acids at framework positions that are predicted to have side chain atoms within 3 Å of the CDRs in the three-dimensional model of the antibody and are predicted to be able to interact with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art can use to create humanized antibodies. One skilled in the art will be familiar with other methods for humanizing antibodies. In some humanized forms of antibodies, some, most or all of the amino acids outside the CDR regions may be replaced with amino acids from a human immunoglobulin molecule, while some, most or all of the amino acids within one or more CDR regions remain unchanged.Some additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not destroy the antibody's ability to bind to a given antigen. Suitable human immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody usually retains a similar antigen specificity as the original antibody. However, using certain methods of humanization, the binding affinity and / or specificity of the antibody may be increased using the method of "directed evolution" as described in Wu et al., Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

[0027] Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for most of the human immunoglobulin heavy and light chain loci.See, for example, U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.These animals are genetically modified so that there is a functional deficiency in the production of endogenous (e.g., mouse) antibodies.These animals are further modified to contain all or part of the human germline immunoglobulin loci, so that immunization of these animals results in the production of fully human antibodies against the antigen of interest. After immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke a human anti-mouse antibody (KAMA) response when administered to humans.

[0028] There are also in vitro methods for selecting human antibodies from human antibody libraries. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) or in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0029] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment", as used herein, refers to a full-length antibody or one or more fragments thereof that retain the ability to specifically bind to an antigen (e.g., a BTN3A protein as defined above) and the BTN3A-activating properties of the antibody. In certain embodiments, a BTN3A-activating antibody for use in the treatment of an infectious disorder as disclosed herein is an antibody fragment, more specifically any protein that comprises the antigen-binding domain of a BTN3A-activating antibody as disclosed herein. Well-known antibody fragments include: a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of the VH domain (Ward et al., 1989 Nature 133:1311-1323). 341:544-546), or any fusion protein containing such an antigen-binding fragment; diabody, which refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to create two antigen-binding sites. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be linked by a synthetic linker that allows them to be made as a single-chain protein using recombinant methods, in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 (See Proc. Natl. Acad. Sci. 85:5879-5883.) Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment of an antibody" (also abbreviated herein as antibody fragment).More generally, antibody fragments as intended herein also encompass single-domain antibodies, which are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Pat. No. 6,248,516). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Well-suited antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells as described herein.

[0030] The term "monoclonal antibody" as used herein refers to a preparation of monospecific antibody molecules. A monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity with variable and constant regions derived from or based on human germline immunoglobulin sequences, or derived from completely synthetic sequences. The method of preparing a monoclonal antibody does not relate to the binding specificity.

[0031] A "recombinant antibody" is an antibody that is produced, expressed, generated, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; an antibody isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; or an antibody produced, expressed, generated, or isolated in any other manner in which a particular immunoglobulin gene sequence (e.g., a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies. In some embodiments, a recombinant human antibody for use according to the present disclosure has the same amino acid sequence as a corresponding naturally occurring human antibody, but is structurally different from said naturally occurring human antibody. For example, in some embodiments, the glycosylation pattern differs as a result of the recombinant production of the recombinant human antibody. In some embodiments, the recombinant human antibody is chemically modified by the addition or deletion of at least one covalent chemical bond relative to the structure of a human antibody that occurs naturally in humans.

[0032] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of antibodies that specifically bind to other antigens other than BTN3A). However, an isolated antibody that specifically binds to BTN3A may have cross-reactivity to other antigens, such as related BTN3A molecules from other species. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0033] The phrases "antibody that recognizes an antigen" and "antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen." The term "antibody against BTN3A" or "BTN3A antibody" is also used herein simply to mean "an antibody that recognizes BTN3A."

[0034] As used herein, the term "activating antibody" refers to an antibody capable of directly or indirectly inducing an immune function of an effector cell. In particular, as used herein, a BTN3A activating antibody has at least the ability to induce activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay (such a degranulation assay is described in the Examples below).

[0035] As used herein, the term "binding" in the context of antibody binding to a given antigen or epitope, particularly BTN3A, refers to a binding rate of approximately 10 to 20% as determined, for example, by surface plasmon resonance (SPR) technology on a BIAcore instrument, typically using a soluble form of the antigen as the ligand and the antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K below M D BIACORE® (GE Healthcare, Piscataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used for epitope binning panels of monoclonal antibodies. Typically, an antibody binds to a given antigen with an affinity corresponding to a K relative to binding to nonspecific antigens that are neither identical nor closely related (e.g., BSA, casein). D at least 10 times lower, e.g. at least 100 times lower, e.g. at least 1,000 times lower, e.g. at least 10,000 times lower, e.g. at least 100,000 times lower, K D The antibody binds with an affinity corresponding to the K D is very low (i.e., the antibody has high affinity), the K D is typically a K DAn antibody is said to essentially not bind to an antigen or epitope if such binding is either not detectable (e.g., using plasmon resonance (SPR) technology on a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte) or is 100-fold, 500-fold, 1000-fold or more than 1000-fold lower than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

[0036] The term "affinity," as used herein in the context of antibodies, refers to the strength of the binding of an antibody to an epitope.

[0037] The terms "Kon" or "Kass" (Ka), as used herein, are intended to refer to the association rate of a particular antibody-antigen interaction, while the terms "Kdis" (Kd) or "Koff", as used herein, are intended to refer to the dissociation rate of a particular antibody-antigen interaction.

[0038] "K D As used herein, the term " off and k on (i.e., koff / kon) and expressed as a molar concentration (M). D Since the value is related to the concentration of the antibody (the amount of antibody needed for a particular experiment), K D The lower the value (lower concentration), the higher the affinity of the antibody. D The K value can be determined using methods well established in the art. DPreferred methods for determining K values ​​can be found in Harlow, et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. DMethods for determining affinity are by using surface plasmon resonance or by using biosensor systems such as the Biacore® (for more information on affinity evaluation, see also Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using BIACORE®. Anal Biochem. 2001 Sep 15;296(2):197-207) or Octet® systems. The Octet® platform is based on the Biolayer Interferometry (BLI) technology. The principle of the BLI technology is based on the optical interference pattern of white light reflected from two surfaces (a layer of immobilized proteins and an internal reference layer). Binding between a ligand immobilized on the biosensor chip surface and an analyte in solution causes an increase in optical thickness at the biosensor chip, resulting in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of the biological layer, and when this shift is measured over a period of time and the magnitude of the shift is plotted as a function of time, a classical association / dissociation curve is obtained. This interaction is measured in real time, allowing the binding specificity, association and dissociation rates, and concentration to be monitored. (See Abdiche et al. 2008). Affinity measurements are typically performed at 25°C.

[0039] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen, such as BTN3A. In some embodiments, it is intended to refer to an antibody that binds to human BTN3A expressed on peripheral blood marrow cells (PBMCs) with an EC50 of preferably less than 50 μg / ml, more preferably less than 10 μg / ml, as determined by flow cytometry, as described in the Examples. In other embodiments, the antibody has a K of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less, as measured by SPR measurements, as described in the Examples. D The antigen binds to the recombinant polypeptide.

[0040] An antibody that "cross-reacts with antigens other than BTN3A" has a K of 10 nM or less, 1 nM or less, or 100 pM or less. D An antibody that "does not cross-react with a specific antigen" is intended to refer to an antibody that binds to an antigen other than BTN3A at a K D , or a K of 10 μM or higher D It is intended to refer to an antibody that binds to its antigen at a specific site. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding to these proteins in standard binding assays.

[0041] Specificity can further be demonstrated, for example, by a ratio of affinity / avidity of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater for binding to a specific antigen (in this case the specific antigen is a BTN3A polypeptide) versus non-specific binding to other unrelated molecules.

[0042] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0043] As used herein, the term "optimized" means that the nucleotide sequence has been modified to encode an amino acid sequence using preferred codons in the producing cell or organism, typically a eukaryotic cell, such as a Chinese hamster ovary cell (CHO) or a human cell. An optimized nucleotide sequence has been engineered to retain the amino acid sequence originally encoded by the starting nucleotide sequence entirely or as much as possible. The amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0044] The term "identity" as used herein with respect to polypeptide sequences refers to the identity of amino acid sequences between two molecules. If an amino acid position in both molecules is occupied by the same amino acid, the molecules are identical at that position. The identity between two polypeptides is a direct function of the number of identical positions. In general, the sequences are aligned (including gaps, if necessary) to obtain the highest match. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

[0045] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) as implemented in the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using published techniques and widely available computer programs, such as the Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) algorithms available in the BLASTP, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990), or GAP program of the GCG software package (Devereux et al., Nucleic Acids Res. 12:387, 1984, generally available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0046] Generally, although not necessarily, it is preferred that amino acid substitutions with respect to a reference polypeptide, such as a CDR region, be conservative amino acid substitutions.

[0047] As used herein, "conservative amino acid substitution" means that a given amino acid may be replaced by a residue having similar physicochemical characteristics, such as replacing one aliphatic residue with another aliphatic residue (e.g., Ile, Val, Leu, or Ala for each other), or replacing one polar residue with another polar residue (e.g., between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions are known, such as replacement of entire regions with similar hydrophobic characteristics. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, such as antigen binding activity and specificity, of the native or reference polypeptide is retained. Amino acids may be grouped according to the similarity of the properties of their side chains as follows (A. L. Lehninger, Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.Particular conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0048] The percent identity between two nucleotide amino acid sequences can also be determined using an algorithm such as the BLASTN program for nucleic acid sequences, which uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0049] Activating BTN3A antibodies for use in the present disclosure The present disclosure relates to the use of a BTN3A activating antibody in a method of treating an infectious disorder, more specifically a disorder caused by Coxiella burnetii infection, e.g., Q fever, or a disorder caused by SARS-Cov2, e.g., COVID-19, in a subject in need of such treatment.

[0050] In certain embodiments, BTN3A activating antibodies for use in accordance with the present disclosure exhibit the following properties: (i) an activating antibody has a K of 10 nM or less, as measured by SPR, e.g., as described in the Examples; D and preferably a K of 1 nM or less D binds to BTN3A at ; (ii) The activating antibody has an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50binds to human PBMCs at (iii) an activating antibody has an EC of less than 5 μg / ml, preferably less than 1 μg / ml, as measured in a degranulation assay, e.g., as described in the Examples; 50 In co-culture with cells expressing BTN3, it induces activation of γδ T cells, typically Vγ9Vδ2 T cells.

[0051] In certain embodiments that can be combined with the following specific embodiments, the BTN3A activating antibody for use in the present disclosure is an antibody fragment of a specific antibody as disclosed below. Antibody fragments include, but are not limited to, for example, Fab, Fab', Fab'-SH, F(ab')2, Fv, unibody, and scFv fragments, diabodies, single domains or nanobodies and other fragments. Preferably, the activating antibody is a monovalent antibody, such as a Fab of a scFv fragment. In particular, the activating antibody is a monovalent BTN3A activating antibody, such as a Fab fragment having all six CDRs of mAb103.2.

[0052] In some embodiments, the antibody for use in the present disclosure is a chimeric, humanized, or human antibody. In a preferred embodiment of the present disclosure, the BTN3A activating antibody is a humanized antibody. Typically, the non-human antibody is humanized to reduce immunogenicity to humans while having at least the same affinity (or better affinity) as the parent non-human antibody. More specifically, the BTN3A activating antibody is a humanized form of antibody 20.1 or 7.2 disclosed in WO2012080351. In certain embodiments, the humanized antibody comprises all six CDRs of a non-human antibody, such as mouse mAb 20.1 or 7.2, and FRs (or portions thereof) derived from a mouse antibody sequence with one or more mutations to reduce immunogenicity. In other specific embodiments, the activating antibody is a monovalent humanized BTN3A activating antibody, such as a humanized Fab fragment with all six CDRs of mAb 103.2.

[0053] A humanized antibody optionally also comprises at least a portion of a human constant region. Preferably, a recombinant antibody according to this disclosure is a humanized silenced antibody, typically a humanized silenced IgG1 or IgG4 antibody.

[0054] As used herein, the term "silent" antibody refers to an antibody that exhibits no or low FcγR and / or C1q binding as measured in a binding assay, such as those described in WO2020 / 025703. In one embodiment, the term "no or low FcγR and / or C1q binding" means that the silent antibody exhibits at least 50% less, e.g., 80% less, FcγR and / or C1q binding than that observed with a corresponding antibody having a wild-type human IgG1 or IgG4 isotype.

[0055] Examples of BTN3A activating antibodies are described in the following paragraphs. In some embodiments, the BTN3A activating antibody is selected from the group consisting of BTN3A activating antibodies such as those described in International Patent Application Nos. 2012 / 080769, 2012 / 080351, and 2020 / 025703 and 2020 / 136218.

[0056] In some specific embodiments, the BTN3A activating antibody is selected from the humanized antibodies described in WO2020025703, or is a humanized version of the BTN3A activating antibodies described in WO2012 / 080769, WO2012 / 080351, and WO2020 / 136218.

[0057] In some embodiments, the BTN3A antibody may be selected from mAb20.1 and mAb7.2, which may be obtained from one of the hybridomas accessible under CNCM deposit numbers I-4401 and I-4402, or their humanized versions, as described in International Publication Nos. 2012080769 and 2012080351.

[0058] In some embodiments, the BTN3A activating antibody comprises the six CDRs (CDR1 (also referred to as HCDR1), VH CDR2 (also referred to as HCDR2), VH CDR3 (also referred to as HCDR1), VL CDR1 (also referred to as LCDR1), VL CDR2 (also referred to as LCDR2), VL CDR3 (also referred to as HCDR3)) of antibodies 20.1 or 7.2 as described in WO2012080769 and WO2012080351, or any of mAbs 1-5 as described in WO2020025703, or a monovalent fragment of mAb 103.2 as described in WO2020 / 136218.

[0059] In certain embodiments, a BTN3A activating antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as shown in Table 1 below.

[0060] [Table 1]

[0061] In some embodiments, an antibody for use as disclosed herein comprises six CDR regions that are 100% identical to the six CDR regions of mAb20.1, mAb7.2 or humanized mAb103.2 as set forth in Table 1, particularly mAb20.1.

[0062] Other antibodies as disclosed herein include those that have been mutated by amino acid deletion, insertion or substitution, but have amino acids that have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity in the CDR regions compared to the six CDR regions of antibodies 20.1 or 7.2 described in WO2012080769 and WO2012080351, or humanized mAb 103.2 described in WO2020 / 136218, particularly compared to the six CDR regions defined in Table 1.

[0063] BTN3A activating antibodies for use in this disclosure also include those having at least 90%, particularly at least 95, 96, 97, 98, 99 or 100% identity with the VH and VL regions defined in Table 2. More specifically, BTN3A activating antibodies for use in this disclosure include selected humanized recombinant antibodies mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6, which are structurally characterized by their variable heavy and light chain amino acid sequences and human constant regions (isotypes) as set forth in Table 2 below.

[0064] [Table 2]

[0065] The corresponding amino acid and nucleotide coding sequences of the constant isotype regions of IgG1, IgG4 and their mutated versions IgG1 L247F / L248E / P350S and IgG4 S241P / L248E used to generate mAb1-mAb6 are well known in the art (Oganesyan et al., 2008; Reddy et al., 2000).

[0066] The C-terminal lysine found in IgG may be naturally cleaved and this modification does not affect the properties of the antibody, therefore this residue may additionally be deleted in the constructs of mAb1-mAb6.

[0067] The full-length light and heavy chains and coding sequences for making preferred humanized antibodies mAb1 and mAb3 for use in the present disclosure are shown in Table 3 below.

[0068] [Table 3]

[0069] Functional variants of the BTN3A activating fragment of mAb20.1, mAb7.2 or mAb103.2 for use according to the present disclosure Epitope mapping analysis indicates that reference mAb20.1 binds to residues 79, 83 and 88 of human BTN3A1 of SEQ ID NO: 32. Thus, the present disclosure encompasses the use of BTN3A antibodies that bind to an epitope comprising amino acid residues located at positions 79-88 of SEQ ID NO: 32 and have one or more of the functional properties as defined above and further reaffirmed below, in particular one or more of the functional properties of reference mAb20.1 or the humanized form of reference mAb20.1, mAb3.

[0070] Also as shown by epitope mapping analysis, reference mAb7.2 binds to residues at positions 73, 79, 83, 88, 90, 93 of human BTN3A1 of SEQ ID NO: 32. Thus, the present disclosure encompasses the use of BTN3A antibodies that bind to an epitope comprising amino acid residues located at positions 73-93 of SEQ ID NO: 32, most particularly an epitope comprising amino acid residues at positions 73, 79, 83, 88, 90 and 93 of SEQ ID NO: 32, and that have one or more of the functional properties as defined above and further reaffirmed below, in particular one or more of the functional properties of reference mAb7.2 or the humanized form mAb1 of reference mAb7.2.

[0071] In yet other embodiments, functional variant antibodies of the present disclosure have full-length heavy and light chain amino acid sequences, or variable region heavy and light chain amino acid sequences, or all six CDR region amino acid sequences that are homologous, or more specifically identical, to the corresponding amino acid sequences of any one of the above reference antibodies mAb20.1 or mAb7.2 or the humanized forms of mAb20.1 or mAb7.2 (mAb3 or mAb1, respectively), and such functional variant antibodies retain the desired functional properties of said reference antibodies.

[0072] In yet other embodiments, a functional variant antibody of the present disclosure is a BTN3A-activating fragment of mAb103.2, such as a Fab fragment having variable region heavy and light chain amino acid sequences, or all six CDR region amino acid sequences that are homologous, or more specifically identical, to the corresponding amino acid sequences of any of the above reference antibodies mAb103.2 or humanized forms of mAb103.2, and such functional variant antibodies retain the desired functional properties of said reference antibody.

[0073] Functional variants of the reference mAb20.1 antibody or a humanized form of the mAb20.1 antibody, mAb3, or the reference antibody mAb7.2, or a humanized form of mAb7.2, mAb1, or a reference activation fragment of mAb103.2 or a humanized form of said activation fragment, in particular a VH and VL, or a functional variant having all six CDRs, as used in the context of the monoclonal antibodies of the present disclosure, may have the affinity (K typically measured by surface plasmon resonance (SPR)) of the parent antibody (e.g., mAb3 or mAb1). D still permitting the antibody to retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the total amino acid sequence (as assessed by ) and, in some cases, such functional variants may associate with greater affinity, selectivity and / or specificity than the reference antibody (e.g., a Fab fragment of mAb3 or mAb1 or mAb103.2). Typically, the desired functional property of the reference antibody, where the reference antibody is a Fab fragment of mAb3 or mAb1 or mAb103.2, or of any of the exemplary reference antibodies disclosed herein, may be selected from the group consisting of: (i) specificity for BTN3A1, in particular binding characteristics to human BTN3A1 as measured by a surface plasmon resonance (SPR) assay, e.g., as described in the Examples; (ii) in vitro induction of activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, as measured in a degranulation assay, e.g., as described in the Examples; (iii) reduction of C. burnetii bacterial burden in monocytes in the presence of Vγ9Vδ2 T cells; (iv) increased in vitro cytotoxic activity of Vγ9Vδ2 T cells against C. burnetii -infected monocytes after 4 h of coculture; (v) increased in vitro degranulation of Vγ9Vδ2 T cells against peripheral blood mononuclear cells from C. burnetii-infected patients; and / or (vi) An in vitro increase in the cytotoxic activity of Vγ9V52 T cells against cells infected with SARS-Cov2, for example, when measured in vitro in co-cultures of infected cells with Vγ9V52 T cells.

[0074] Typically, the functional properties according to points (i) to (vi) above of the functional variant of the activation fragment of reference mAb3 or 1 or mAb103.2 are substantially equal to or better than the corresponding functional properties of the activation fragment of the corresponding reference antibody mAb3 or mAb1 or mAb103.2 as described above. By substantially equal, it is intended herein that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding functional properties of the activation fragment of reference mAb3 or mAb1 or mAb103.2.

[0075] In certain embodiments, a BTN3A activating antibody for use in accordance with the present disclosure is a humanized form of mAb20.1 or mAb3, or a humanized form of mAb7.2 or mAb1, or an activating fragment of mAb103.2 or a functional variant of a humanized form thereof, having no more than one, two, three or four amino acid changes (including deletions, insertions or substitutions) in one or more CDRs compared to the CDR sequences of antibody 20.1 or 7.2 or the activating fragment of mAb103.2, respectively, or more specifically compared to the CDR sequences of mAb20.1. For example, the present disclosure provides a method for the production of a variable heavy chain (V H ) and variable light chain (V L ) sequence, wherein the CDR sequences, i.e. the six CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, share at least 60, 70, 90, 95 or 100 percent sequence identity with the corresponding CDR sequences of the mAb20.1 or humanized form mAb3 of mAb20.1 reference antibody, as defined in SEQ ID NOs: 5-10, said functional variant antibody specifically binds to BTN3A, and the antibody exhibits at least one of the following functional properties i) to iii): (i) specificity for BTN3A1, in particular a K of 10 nM or less as measured by surface plasmon resonance (SPR), e.g., as described in the Examples; D , preferably 5 nM or less K D , or a K of 5 nM or less D Binding properties to human BTN3A1; (ii) an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 to bind to human PBMC; (iii) induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0076] Most preferably, the functional variant antibody exhibits properties i) to iii). The present disclosure also relates to a variable heavy chain (V H ) and variable light chain (V L ) sequence, wherein the CDR sequences, i.e. the six CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, share at least 60, 70, 90, 95 or 100 percent sequence identity with the corresponding CDR sequences of the mAb7.2 or mAb1 reference antibody, as defined in SEQ ID NOs: 11-16, said functional variant antibody specifically binds to BTN3A1, and the antibody exhibits at least one of the following functional properties: (i) specificity for BTN3A1, in particular a K of 10 nM or less as measured by surface plasmon resonance (SPR), e.g., as described in the Examples; D , preferably 5 nM or less K D , or a K of 5 nM or less D Binding properties to human BTN3A1; (ii) an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 to bind to human PBMC; (iii) induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0077] Preferably, the functional variant exhibits all of the functional activities i) to iii). The present disclosure also relates to a variable heavy chain (V H ) and variable light chain (V L ) sequence, wherein the CDR sequences, i.e. the six CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, share at least 60, 70, 90, 95 or 100 percent sequence identity with the corresponding CDR sequences of the reference Fab fragment of mAb103.2, as defined in SEQ ID NOs: 17-22, said functional variant antibody specifically binds to BTN3A1, and the antibody exhibits at least one of the following functional properties: (i) specificity for BTN3A1, in particular a K of 10 nM or less as measured by surface plasmon resonance (SPR), e.g., as described in the Examples; D , preferably 5 nM or less K D , or a K of 5 nM or less D Binding properties to human BTN3A1; (ii) an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 to bind to human PBMC; (iii) induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0078] Preferably, the functional variant exhibits all of the functional activities i) to iii). The present invention further relates to functional variant antibodies of the mAb3 reference antibody comprising heavy and light chain variable regions that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99% or 100% identical to the corresponding heavy and light chain variable regions of said mAb3 reference antibody as defined in SEQ ID NOs: 1 and 2, respectively, wherein the functional variant antibodies specifically bind to BTN3A and exhibit at least one of the following functional properties: (i) specificity for BTN3A1, in particular a K of 10 nM or less as measured by surface plasmon resonance (SPR), e.g., as described in the Examples; D , preferably 5 nM or less K D , or a K of 5 nM or less D Binding properties to human BTN3A1; (ii) an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 to bind to human PBMC; (iii) induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0079] Preferably, the functional variant antibody exhibits properties i) to iii). The present invention further relates to functional variant antibodies of the mAb1 reference antibody comprising heavy and light chain variable regions that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99% or 100% identical to the corresponding heavy and light chain variable regions of said mAb1 reference antibody as defined in SEQ ID NOs: 3 and 4, respectively, wherein the functional variant antibodies specifically bind to BTN3A and exhibit at least one of the following functional properties: (i) specificity for BTN3A1, in particular a K of 10 nM or less as measured by surface plasmon resonance (SPR), e.g., as described in the Examples; D, preferably 5 nM or less K D , or a K of 5 nM or less D Binding properties to human BTN3A1; (ii) an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 to bind to human PBMC; (iii) induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0080] In some embodiments, the functional variant exhibits all of the functional activities i)-iii). The present invention further relates to a functional variant antibody of a reference Fab fragment of mAb103.2 comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99% or 100% identical to the corresponding heavy chain and light chain variable regions of said Fab fragment of mAb103.2 as defined in SEQ ID NOs: 63 and 64, respectively, wherein the functional variant antibody specifically binds to BTN3A and exhibits at least one of the following functional properties: (i) specificity for BTN3A1, in particular a K of 10 nM or less as measured by surface plasmon resonance (SPR), e.g., as described in the Examples; D , preferably 5 nM or less K D , or a K of 5 nM or less D Binding properties to human BTN3A1; (ii) an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 to bind to human PBMC; (iii) induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0081] In some embodiments, the functional variant exhibits all of the functional activities i)-iii).

[0082] Typically, the functional properties according to points (i) to (iii) above of a functional variant of a Fab fragment of reference mAb3 or mAb1 or mAb103.2 are substantially equal to or better than the corresponding functional properties of the Fab fragment of the corresponding reference antibody mAb3 or mAb1 or mAb103.2 as described above. By substantially equal, it is intended herein that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding functional properties of the Fab fragment of reference mAb3 or mAb1 or mAb103.2.

[0083] The sequence of a CDR variant may differ from that of a CDR of a parent antibody sequence, mostly by conservative substitutions, for example at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

[0084] Functional variant antibodies having mutated amino acid sequences can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the encoding nucleic acid molecule, followed by testing the encoded altered antibody for retained functionality (i.e., the functions described above) using the functional assays described herein. Antibodies that cross-compete with the Fab fragments of reference mAb3 or mAb1 or mAb103.2. Additional antibodies having similar advantageous properties as the Fab fragment of reference mAb3 or reference mAb1 or mAb103.2 as disclosed herein can be identified based on their ability to cross-compete with the Fab fragment of said reference mAb3 or mAb1 or mAb103.2 (e.g., competitively inhibit binding of the Fab fragment of said reference mAb3 or mAb1 or mAb103.2) in a statistically significant manner in a standard BTN3A1 binding assay.

[0085] Test antibodies can be initially screened for their binding affinity to BTN3A1, for example, from human recombinant antibody libraries, e.g., using phage display technology, or from transgenic mice expressing human variable region antibodies immunized with BTN3A1 antigen as typically assessed in the Examples (see Materials and Methods section).

[0086] In another embodiment, an antibody for use in accordance with the present disclosure comprises an antibody that binds to at least the same epitope as the Fab fragment of reference mAb3 or reference mAb1 or mAb103.2 above.

[0087] The ability of the test antibody to cross-compete or inhibit the binding of an antibody of the present disclosure to human BTN3A1 demonstrates that the test antibody can compete with that antibody for binding to human BTN3A1. Such an antibody may, by non-limiting theory, bind to the same or a related (e.g., structurally similar or spatially proximal) epitope of human BTN3A1 as the antibody with which the test antibody competes.

[0088] For example, the following test can be used to screen BTN3A1 antibodies for their ability to cross-compete with the mAb3 reference antibody and / or to screen anti-BTN3A1 antibodies for their ability to bind to the same epitope as said reference antibody. BTN3KO cells (typically HEK293T) transfected with human BTN3A1 can be stained with a saturating concentration (e.g., 10 μg / mL) of the reference antibody mAb3. Different doses of the test BTN3A1 mAb can then be tested for their competitive potential with the mAb3 reference antibody. mAbs that compete with the reference antibody cannot recognize BTN3A1 in the presence of such reference antibody. Data can be expressed as mean fluorescence intensity. Alternatively, the competition assay can be performed in a binning assay as described in the Examples section. Typically, binning experiments can be performed by immobilizing recombinant human BTN3A1 on a biosensor and presenting the reference antibody followed by the competing antibody.

[0089] Selected antibodies can be further tested and selected for advantageous BTN3A activation properties, particularly as compared to Fab fragments of mAb3 or mAb1 or mAb103.2, as detailed above.

[0090] In some embodiments, the antibody for use in the present disclosure competes for binding to the BTN3A antibody described above, in particular the antibody for use in the present disclosure competes for binding with an antibody selected from mAb20.1 and mAb7.2, which can be obtained from one of the hybridomas accessible under CNCM deposit numbers I-4401 and I-4402 as described in WO 2012 / 080769 and WO 2012 / 080351, and from mAbs 1-6 described in WO 2020025703. In more specific embodiments, the antibody for use in the present disclosure competes for binding with an antibody selected from mAb20.1 produced by the hybridoma deposited at the CNCM under deposit number I-4401, and an antibody having a heavy chain of SEQ ID NO:23 and a light chain of SEQ ID NO:24. Thus, in one embodiment, the disclosure provides an isolated antibody for use in the treatment of an infectious disorder as disclosed herein, said isolated antibody competes with a Fab fragment of reference mAb3 or reference mAb1 or mAb103.2 for binding to BTN3A1, and said antibody has one or more of the following properties: (i) specificity for BTN3A1, in particular binding characteristics to human BTN3A1 as measured by a surface plasmon resonance (SPR) assay, e.g., as described in the Examples; (ii) in vitro induction of activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, as measured in a degranulation assay, e.g., as described in the Examples; (iii) reduction of C. burnetii bacterial burden in monocytes in the presence of Vγ9Vδ2 T cells; (iv) increased in vitro cytotoxic activity of Vγ9Vδ2 T cells against C. burnetii -infected monocytes after 4 h of coculture; (v) increased in vitro degranulation of Vγ9Vδ2 T cells against peripheral blood mononuclear cells from C. burnetii-infected patients; and / or (vi) An in vitro increase in the cytotoxic activity of Vγ9V52 T cells against cells infected with SARS-Cov2, for example, when measured in vitro in co-cultures of infected cells with Vγ9V52 T cells.

[0091] In certain embodiments, the functional properties according to points (i) to (vi) above of an antibody that competes with a Fab fragment of reference mAb3 or mAb1 or mAb103.2 for binding to BTN3A1 are substantially equal to or better than the corresponding functional properties of the Fab fragment of reference antibody mAb1 or mAb3 or mAb103.2, respectively, as described above. By substantially equal, it is intended herein that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95% or 100% of the corresponding functional properties of the Fab fragment of reference mAb1 or mAb3 or mAb103.2.

[0092] In certain embodiments, the cross-blocking antibody or the antibody that competes with the Fab fragment of reference mAb1 or mAb3 or mAb103.2 for binding to BTN3A1 is a chimeric, humanized or human recombinant antibody.

[0093] Framework or Fc Engineering BTN3A activating antibodies for use in the present disclosure may include modifications made to framework residues within VH and VL to reduce the immunogenicity of the activating antibody.

[0094] In some specific embodiments, the antibody for use in the present disclosure is a humanized monoclonal antibody of the parent murine antibody mAb20.1 and comprises at least the following amino acid mutations in the VH framework regions (compared to the VH parent framework regions): V5Q; V11L; K12V; V20L; R66K; M69L; T75S; M80I; E81Q; R83T; T87S; L108A; and at least the following amino acid mutations in the Vκ framework regions (compared to the Vκ framework regions): T5N; V15L; R18T; V19I; K39R; K42N; A43I; D70G; F73L; V104L.

[0095] In addition to modifications made within the framework regions, the antibodies of the disclosure may be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0096] Additionally, antibodies for use in the present disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter the glycosylation of the antibody, again altering one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[0097] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to designate the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody, with numbering according to the EU numbering system. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during antibody production or purification, or the codon corresponding to the lysine is deleted in a recombinant construct. Thus, the antibody composition of the present disclosure can include an antibody population with all K447 residues removed, an antibody population with no K447 residue removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0098] In other embodiments, the Fc region is modified to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to reduce the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. Such antibodies with reduced effector function, particularly reduced ADCC, include silent antibodies.

[0099] In certain embodiments, an Fc domain of IgG1 isotype is used. In some specific embodiments, a mutant variant of an IgG1 Fc fragment is used, such as a silent IgG1 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0100] In certain embodiments, an Fc domain of the IgG4 isotype is used. In some specific embodiments, a mutant variant of an IgG4 Fc fragment is used, such as a silent IgG4 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0101] Silenced effector function can be obtained by mutation of antibody Fc constant part, and has been described in the art (Baudino et al., 2008; Strohl, 2009). An example of silent IgG1 antibody is triple mutation variant IgG1 L247F L248E P350S. An example of silent IgG4 antibody is double mutation variant IgG4 S241P L248E.

[0102] In certain embodiments, the Fc domain is a silent Fc mutant that prevents glycosylation at position 314 of the Fc domain. For example, the Fc domain comprises an amino acid substitution of asparagine at position 314. An example of such an amino acid substitution is the substitution of N314 with glycine or alanine.

[0103] In yet other embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). The glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

[0104] Another modification of the antibodies herein contemplated for use according to the present disclosure is pegylation or hexylation or related techniques. Antibodies can be pegylated, for example, to extend the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods of pegylation of proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, EP 0154316 to Nishimura et al. and EP 0401384 to Ishikawa et al.

[0105] In certain embodiments, the C-terminal lysine commonly present in human IgG heavy chain constant domains is engineered to reduce the heterogeneity due to cleavage of this residue commonly observed during manufacturing or storage. Such modifications do not appreciably alter the desired function of these antibodies while providing stability benefits to these molecules. Nucleic acid molecules encoding antibodies of the present disclosure Also disclosed herein are nucleic acid molecules encoding BTN3A activating antibodies for use in accordance with the present disclosure. Exemplary variable light and heavy chain nucleotide sequences are those that encode the variable light and heavy chain amino acid sequences of any one of the exemplary BTN3A activating antibodies disclosed above, particularly mAb7.2, mAb20.1 and humanized forms of mAb7.2, mAb20.1, such as mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6, some of which are readily derived from Tables 1 and 2, using the genetic code and optionally taking into account codon bias depending on the host cell type.

[0106] The present disclosure also relates to nucleic acid molecules derived from the latter sequence that are optimized for protein expression in mammalian cells, such as CHO cell lines.

[0107] Further disclosed herein are nucleic acid molecules encoding the heavy chain of a humanized form of mAb20.1 of SEQ ID NO:23 and the light chain of a humanized form of mAb20.1 of SEQ ID NO:24, respectively.

[0108] In a specific embodiment, the antibody for use in the present disclosure is a humanized form of mAb20.1 having a VH encoded by SEQ ID NO:28 and a VL encoded by SEQ ID NO:29.

[0109] Further disclosed herein are nucleic acid molecules of SEQ ID NO:28 and SEQ ID NO:29, which encode the VH and VL, respectively, of a humanized form of mAb20.1.

[0110] The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others known in the art (Ausubel et al., 1988). The nucleic acid of the present disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. In one embodiment, the nucleic acid may be present in a vector, such as a phage display vector, or in a recombinant plasmid vector.

[0111] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For example, once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragments encoding VL or VH (for example, VL and VH as defined in Table 2) are operably linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are linked in a functional manner, for example, so that the amino acid sequences encoded by the two DNA fragments remain in frame, or so that the protein is expressed under the control of a desired promoter.

[0112] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (Kabat et al., 1992), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments, the heavy chain constant region is selected from among the IgG1 isotype, e.g., the human IgG1 isotype. In other embodiments, the heavy chain constant region is selected from among the IgG4 isotype, e.g., the human IgG4 isotype. In the case of a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0113] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (Kabat et al., 1992), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0114] To generate an scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a contiguous single-chain protein in which the VL and VH regions are linked by the flexible linker (Bird et al. 1988; Huston et al., 1988; McCafferty et al., 1990).

[0115] Methods for Producing Recombinant Antibodies for Use According to the Present Disclosure Antibodies of the present disclosure can be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, 1985).

[0116] For example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology or biochemistry techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting the light and heavy chain variable regions into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to the CH segment(s) in the vector and the VL segment is operably linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0117] In addition to the antibody chain genes, the recombinant expression vectors disclosed herein have regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Goeddel, 1990). Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the expression level of protein desired, and the like. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Furthermore, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and long terminal repeat of the human T-cell leukemia virus type 1 (Takebe et al., 1988).

[0118] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector of the present disclosure may have additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0119] For the expression of light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. It is theoretically possible to express the antibody of the present disclosure in either prokaryotic or eukaryotic host cells. It is believed that the expression of antibodies in eukaryotic cells, such as mammalian host cells, yeast or filamentous fungi, is because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0120] In one specific embodiment, a cloning or expression vector according to the present disclosure comprises one of the heavy and light chain coding sequences of either mAb1 or mAb3 operably linked to a suitable promoter sequence.

[0121] Mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese Hamster Ovary (CHO cells), including dhfr-CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker (described in Kaufman and Sharp, 1982), CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells and SP2 cells, such as GS CHO cell line with GS Xceed™ Gene Expression System (Lonza). When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for expression of the antibody in the host cell and, optionally, secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered and purified from the culture medium after secretion of the antibody, for example, using standard protein purification methods (Shukla et al., 2007).

[0122] In one specific embodiment, the host cell of the present disclosure is a host cell transfected with an expression vector having a coding sequence suitable for expression of a Fab fragment of mAb1 or mAb3 or mAb103.2, respectively, operably linked to a suitable promoter sequence.

[0123] For example, the disclosure relates to a host cell comprising at least the nucleic acid of SEQ ID NOs:28 and 29, which encode the VH and VL, respectively, of mAb3.

[0124] The latter host cells may then be further cultured under suitable conditions for the expression and production of an antibody of the present disclosure selected from the group consisting of mAb1, or mAb3, or the Fab fragment of mAb103.2, respectively.

[0125] Alternatively, cell-free expression systems can be used for the production of either mAb3 or mAb1. Typically, methods for cell-free expression of proteins or antibodies have been previously described (Stech et al., 2017).

[0126] Pharmaceutical Compositions In another aspect, the disclosure provides a composition, e.g., a pharmaceutical composition, for use in treating an infectious disorder as disclosed below comprising a BTN3A activating antibody, in particular a BTN3A activating antibody selected from the group consisting of mAb20.1, a BTN3A activating antibody having the six CDRs of mAb20.1 of SEQ ID NO:5-10, a BTN3A activating antibody having a VH of SEQ ID NO:1 and a VL of SEQ ID NO:2, a BTN3A activating antibody that is a humanized form of mAb20.1, and mAb3 having a heavy chain of SEQ ID NO:23 and a light chain of SEQ ID NO:24, and antigen-binding portions thereof, formulated together with a pharma- ceutically acceptable carrier.

[0127] Such compositions can include one or a combination (eg, two or more different) BTN3A activating antibodies, as described above.

[0128] The pharmaceutical compositions disclosed herein may also include additional active therapeutic agents. For example, the pharmaceutical compositions may include an antibody selected from the group consisting of an anti-BTN3A antibody of the present disclosure, such as mAb20.1, a BTN3A activating antibody having the six CDRs of mAb20.1 of SEQ ID NO:5-10, a BTN3A activating antibody having a VH of SEQ ID NO:1 and a VL of SEQ ID NO:2, a BTN3A activating antibody that is a humanized form of mAb20.1, and mAb3 having a heavy chain of SEQ ID NO:23 and a light chain of SEQ ID NO:24, or an antigen-binding portion thereof, in combination with at least one antiviral, anti-inflammatory, or antibacterial agent. Examples of such other active therapeutic agents that can be used are described in more detail below in the section on the use of the antibodies of the present disclosure.

[0129] As used herein, "pharmaceutical acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous or intravenous routes. Depending on the route of administration, the active compound, i.e., BTN3A-activating antibody, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0130] Sterile phosphate buffered saline is an example of a pharma- ceutically acceptable carrier. Other suitable carriers are known to those skilled in the art. (Remington and Gennaro, 1995) The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, and the like.

[0131] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will necessarily depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, and the like.

[0132] Pharmaceutical compositions for use in accordance with the present disclosure may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, and the like.

[0133] Preferably, the pharmaceutical composition comprises a pharma- ceutical acceptable vehicle for injectable preparations.The pharmaceutical composition may be in particular an isotonic sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or a mixture of their salts), or a dry composition, in particular a freeze-dried composition, that can be made into an injectable solution when adding sterile water or saline, as the case may be.

[0134] The dose used for administration may be adapted as a function of various parameters, in particular as a function of the mode of administration used, the pathology involved, or alternatively the duration of the treatment desired.

[0135] To prepare a pharmaceutical composition, an effective amount of a BTN3A activating antibody may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0136] Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders or lyophilisates for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy injectability exists. Pharmaceutical compositions must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.

[0137] The solution of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0138] The BTN3A activating antibody for use according to the present disclosure can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein) and are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0139] Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0140] Sterile injectable solution is prepared by incorporating the required amount of active compound into suitable solvent together with various other ingredients as listed above as necessary, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technique, which obtains powder of active ingredient + any additional desired ingredient from its previously sterile filtered solution.

[0141] Preparation of more or more concentrated solutions for direct injection is also contemplated, and it is envisioned that the use of DMSO as a solvent will result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0142] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although drug release capsules and the like may also be used.

[0143] For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0144] BTN3A activating antibodies for use according to the present disclosure may be formulated into a therapeutic mixture to contain about 0.001 to 1 gram / dose, or about 1 to 400 milligrams / dose, or about 1 to 200 milligrams / dose. Multiple doses may also be administered.

[0145] Suitable formulations for solutions for infusion or subcutaneous injection of antibodies have been described in the art and are reviewed, for example, in Cui et al. (Drug Dev Ind Pharm 2017, 43(4):519-530).

[0146] Preferred embodiments of BTN3A activating antibodies, their pharmaceutical compositions and methods for preparing them for use according to the present disclosure #1. An isolated BTN3A activating antibody comprising a variable heavy chain polypeptide VH of SEQ ID NO:1 and a variable light chain polypeptide VL of SEQ ID NO:2.

[0147] #2. An isolated BTN3A activating antibody comprising six CDRs of SEQ ID NO:5 to 10, a variable heavy chain polypeptide VH having at least 90% identity to SEQ ID NO:1, and a variable light chain polypeptide VL having at least 90% identity to SEQ ID NO:2.

[0148] #3. An isolated BTN3A activating antibody according to #1 or #2, optionally comprising a human IgG1 constant region that has been mutated or chemically modified such that said mutant or chemically modified IgG1 constant region confers no or reduced binding to Fcγ receptors when compared to a corresponding antibody having a constant region of the wild-type human IgG1 isotype. #4. An isolated BTN3A activating antibody according to any one of #1 to #3, wherein the antibody exhibits one or more of the following characteristics: (i) the antibody has a K of 10 nM or less, as measured by surface plasmon resonance (SPR), e.g., as described in the Examples. D , preferably 5 nM or less K D , or a K of 5 nM or less D binds to human BTN3A1; (ii) the antibody has an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 binds to human PBMCs; (iii) the antibody induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in vitro in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0149] #5. An isolated BTN3A activating antibody described in any of #1 to #4, comprising a heavy chain polypeptide of sequence number 23 and a light chain polypeptide of sequence number 24.

[0150] #6. A nucleic acid encoding the heavy and light chains of a BTN3A activating antibody described in any of #1 to #5, for example, comprising the nucleotide sequences of SEQ ID NO: 28 and SEQ ID NO: 29.

[0151] #7. An expression vector for recombinant production of a BTN3A activating antibody according to #1 in a host cell, the expression vector comprising at least one nucleic acid encoding said BTN3A activating antibody.

[0152] #8. A host cell comprising the expression vector of claim 7.

[0153] #9. A pharmaceutical composition comprising an anti-BTN3A antibody as defined in any one of #1 to #5 in combination with one or more pharma- ceutically acceptable excipients, diluents or carriers, and optionally other active ingredients, such as cytokines, such as IL-2 or IL-15, or functional derivatives and pegylated versions of said cytokines.

[0154] #10. The pharmaceutical composition of claim #9, which is a lyophilized formulation, a solution in a prefilled syringe or a solution in a vial.

[0155] #11. An isolated BTN3A activating antibody described in any one of #1 to #5, or a pharmaceutical composition described in claim #9 or #10, for use as a therapeutic agent.

[0156] #12. A method for producing a BTN3A antibody described in any one of #1 to #5, comprising: (i) culturing a host cell described in #8 for expression of the antibody by the host cell; optionally (ii) purifying the antibody; and (iii) recovering the antibody.

[0157] Use of BTN3A activating antibodies in the treatment of infectious disorders The present inventors have discovered BTN3A activating antibodies, such as mAb20.1, that have the following capabilities: (i) enhances monocyte reduction of C. burnetii bacterial burden in vitro in the presence of Vγ9Vδ2 T cells; (ii) increase the in vitro cytotoxic activity of Vγ9Vδ2 T cells against monocytes infected with C. burnetii, as measured in vitro using co-cultures of infected monocytes, typically after 4 hours of co-culture; and / or

[0158] The inventors also found that BTN3A activating antibodies such as mAb20.1 have the ability to enhance inhibition of SARS-Cov2 replication, for example, when measured in vitro in co-cultures of infected cells with Vy9V52 T cells.

[0159] Thus, the present disclosure relates to BTN3A activating antibodies, particularly certain BTN3A activating antibodies as described above, for use in the treatment of an infectious disorder, more particularly an infectious disorder selected from a viral or bacterial infectious disorder.

[0160] As used herein, the term "treat", "treating" or "treatment" refers to one or more of: (1) inhibiting a disease, e.g., inhibiting a disease, condition or disorder in an individual experiencing or exhibiting a pathology or symptom of the disease, condition or disorder (i.e., arresting further progression of the pathology and / or symptoms); and (2) alleviating a disease, e.g., alleviating a disease, condition or disorder in an individual experiencing or exhibiting a pathology or symptom of the disease, condition or disorder (i.e., reversing the pathology and / or symptoms), e.g., reducing the severity of a disease or alleviating or reducing one or more symptoms of a disease. In particular, with respect to the treatment of an infectious disorder, the term "treatment" can refer to preventing infection by an infectious agent, inhibiting replication of an infectious agent, reducing the severity of one or more symptoms associated with infection, or eradicating the infectious agent.

[0161] In a preferred embodiment, the subject is a human subject.

[0162] BTN3A activating antibodies for use as disclosed above may be administered as the sole active ingredient or, for example, in conjunction as an adjuvant or in combination with other drugs for the treatment or prevention of the above-mentioned diseases, such as cytokines, antiviral agents, anti-inflammatory agents.

[0163] For example, antibodies for use as disclosed above may be used in combination with cytokines, anti-viral agents, anti-bacterial agents, or anti-inflammatory agents.

[0164] Examples of cytokines include, but are not limited to, cytokines for expanding and / or activating Vy9V52 T cells in vivo, including interleukin 2 (IL-2) (Choudhry H et al, 2018, Biomed Res Int. 2018 May 6), interleukin 15 (IL-15) (Patidar M et al., Cytokine Growth Factor Rev. 2016 Oct;31:49-59), or derivatives thereof. The term derivative is used for any cytokine modification that may rely on pegylation (e.g., conjugation to polyethylene glycol (PEG) chains), mutations such as amino acid deletions, substitutions or insertions, or association with enhancers (e.g., an IL15 / IL15Ra complex fused to IgG1 Fc, where IL-15 is additionally mutated (asn72asp), further increasing biological activity and making this complex an IL-2 and IL-15Rβγ superagonist (Rhode PR et al, Cancer Immunol Res. 2016;4(1):49-60)) (Barroso-Sousa R et al, Curr Oncol Rep. 2018 Nov 15;21(1):1).

[0165] The term "IL-2" has the general meaning of IL-2 and refers to human interleukin-2. IL-2 is part of the body's innate immune response. IL-2 primarily regulates lymphocyte activity by binding to the IL-2 receptor.

[0166] The term "IL-15" has the general meaning of IL-15 and refers to human interleukin-15. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells.

[0167] Thus, the methods of use defined herein may comprise the co-administration, e.g., simultaneous or sequential administration, of a therapeutically effective amount of a BTN3A activating antibody with at least one second drug substance, said second drug substance being an antiviral or antibacterial, anti-inflammatory or cytokine, e.g., IL-2 or IL-15, or a cell therapy product, e.g., as set out above (such as γδ T cells).

[0168] Use of BTN3A activating antibodies in the treatment of disorders caused by SARS Cov2 infection In certain embodiments, the disclosure relates to BTN3A activating antibodies, particularly certain BTN3A activating antibodies as described above, for use in treating a subject in need of treatment for a disorder caused by SARS-Cov2 infection, typically Covid-19.

[0169] In certain embodiments, the present disclosure relates to a method of treating a disorder caused by SARS-Cov2 infection, typically Covid-19, in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an anti-BTN3A activating antibody.

[0170] In certain embodiments, the disclosure relates to the use of a BTN3A activating antibody in the preparation of a medicament for treating a disorder caused by a SARS-Cov2 infection, typically Covid-19, in a subject in need of such treatment.

[0171] In certain embodiments, subjects eligible for such treatment have been diagnosed with SARS-Cov2 infection.

[0172] In certain embodiments, the subject is selected from among those with mild or moderate COVID-19.

[0173] In certain embodiments, the subject is selected from those at high risk of progressing to severe COVID-19. Risk factors include, but are not limited to, (listed alphabetically) age (increasing risk with each decade after age 50), cancer, cardiovascular disease, chronic kidney disease, chronic pulmonary disease, diabetes, immunocompromised conditions or use of immunosuppressive medications, obesity (body mass index > 30), pregnancy, and sickle cell disease.

[0174] In certain embodiments, antiviral or anti-inflammatory drugs may be used in combination with BTN3A activating antibodies to treat the disorders caused by SARS-Cov2. Examples of such antiviral or anti-inflammatory drugs useful for treating SARS-Cov2 include, but are not limited to, remdesivir, baricitinib, bamlanivimab, bamlanivimab / etesevimab, casirivimab / imdevimab, dexamethasone, budesonide, and tocilizumab.

[0175] Use of BTN3A activating antibodies in the treatment of disorders caused by Coxiella burnetii In certain embodiments, the disclosure relates to BTN3A activating antibodies, particularly certain BTN3A activating antibodies as described above, for use in treating a subject in need of treatment for a disorder caused by Coxiella burnetii infection, typically Q fever.

[0176] In certain embodiments, the present disclosure relates to a method of treating a disorder caused by Coxiella burnetii infection, typically Q fever, in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a BTN3A activating antibody.

[0177] In certain embodiments, the disclosure relates to the use of a BTN3A activating antibody in the preparation of a medicament for treating a disorder caused by a Coxiella burnetii infection, typically Q fever, in a subject in need of such treatment.

[0178] In certain embodiments, subjects eligible for such treatment have been diagnosed with Coxiella burnetii infection.

[0179] In certain embodiments, antibacterial agents (such as antibiotics) or anti-inflammatory agents, including but not limited to doxycycline, tetracycline, chloramphenicol, ciprofloxacin, ofloxacin, and hydroxychloroquine, may be used in combination with a BTN3A activating antibody to treat the disorders caused by Coxiella burnetii.

[0180] Other specific embodiments for the use of BTN3A activating antibodies are disclosed below, but are not limited to these.

[0181] Specific Embodiments for Use of BTN3A Activating Antibodies #1. A BTN3A activating antibody for use in treating an infectious disorder in a human subject in need thereof.

[0182] #2. A BTN3A activating antibody for use according to #1, wherein the infectious disorder is a disorder caused by Coxiella burnetii infection, typically Q fever.

[0183] #3. A BTN3A activating antibody for use according to #1, wherein the infectious disorder is a disorder caused by SARS-Cov2, typically COVID-19.

[0184] #4. A K of 10 nM or less as measured by SPR, e.g., as described in the Examples. Dand preferably a K of 1 nM or less D A BTN3A activating antibody for use according to any one of #1 to #3, which binds to BTN3A at

[0185] #5. A BTN3A activating antibody for use according to any one of #1 to #4, having one or more of the following characteristics: (i) the antibody has a K of 10 nM or less, as measured by surface plasmon resonance (SPR), e.g., as described in the Examples. D and preferably a K of 5 nM or less D or K below 1 nM D binds to BTN3A1 at; (ii) the antibody has an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by a flow cytometry assay, e.g., as described in the Examples. 50 binds to human PBMCs at (iii) the antibody induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay, e.g. as described in the Examples.

[0186] #6. A BTN3A activating antibody for use according to any one of #1 to #5, having one or more of the following characteristics: (i) the antibodies enhance the reduction of C. burnetii bacterial burden in monocytes in the presence of Vγ9Vδ2 T cells in vitro; (ii) the antibodies increase the in vitro cytotoxic activity of Vγ9Vδ2 T cells against C. burnetii-infected monocytes, typically after 4 h of co-culture;

[0187] #7. A BTN3A activating antibody for use according to any one of #1 to #5, which increases the in vitro cytotoxic activity of Vγ9Vδ2 T cells against cells infected with SARS-Cov2, for example when measured in vitro in a co-culture of the infected cells with Vγ9Vδ2 T cells.

[0188] #8. A BTN3A activating antibody for use according to any one of #1 to #7, comprising HCDRs 1 to 3 of sequence numbers 5 to 7 and LCDRs 1 to 3 of sequence numbers 8 to 10.

[0189] #9. A BTN3A activating antibody for use according to any one of #1 to #7, comprising HCDRs 1 to 3 of SEQ ID NOs: 11 to 13 and LCDRs 1 to 3 of SEQ ID NOs: 14 to 16.

[0190] #10. A BTN3A activating antibody for use according to any one of #1 to #7, comprising HCDRs 1 to 3 of SEQ ID NOs: 17 to 19 and LCDRs 1 to 3 of SEQ ID NOs: 20 to 22.

[0191] #11. (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2; (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:3, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4; (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:63, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:64; - competes for binding to BTN3A1 with mAb20.1 produced by the hybridoma deposited at the CNCM under deposit number I-4401; or - competes for binding to BTN3A1 with mAb7.2 produced by the hybridoma deposited at the CNCM under the accession number I-4402 11. A BTN3A activating antibody for use according to any one of claims 1 to 10, which is any one of the following:

[0192] #12. A BTN3A activating antibody for use according to any one of #1 to #11, comprising a variable heavy chain VH of sequence number 1 and a light chain VL of sequence number 2.

[0193] #13. A BTN3A activating antibody for use according to any one of #1 to #12, wherein the BTN3A activating antibody comprises a mutant or chemically modified IgG1 constant region, which confers no or reduced binding to Fcγ receptors when compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0194] #14. A BTN3A activating antibody for use according to #13, wherein said mutated IgG1 constant region is the IgG1 triple mutation L247F L248E and P350S.

[0195] #15. A BTN3A activating antibody for use according to any one of #1 to #8 and #11 to #14, comprising a heavy chain of sequence number 23 and a light chain of sequence number 24.

[0196] #16. A BTN3A activating antibody for use according to any one of #1 to #15, administered simultaneously, sequentially or separately in combination with a cytokine for in vivo Vγ9Vδ2 T cell proliferation, such as IL2 or IL15 cytokines or pegylated derivatives thereof.

[0197] #17. A BTN3A activating antibody for use according to any one of #1 to #16 for treating viral infectious disorders of SARS-Cov2, wherein the anti-BTN3A antibody is administered simultaneously or separately, preferably in combination with an antiviral or anti-inflammatory treatment selected from the group consisting of remdesivir, baricitinib, bamlanivimab, bamlanivimab / etesevimab, casirivimab / imdevimab, dexamethasone, budesonide and tocilizumab.

[0198] #18. A BTN3A activating antibody for use according to any one of #1 to #17 for treating a SARS-Cov2 viral infectious disorder, wherein the subject is a human subject diagnosed as being SARS-Cov2 positive.

[0199] #19. A BTN3A activating antibody for use according to any one of #1 to #18 for treating SARS-Cov2 viral infectious disorders, wherein the subject is a human subject with mild or moderate COVID-19.

[0200] #20. A BTN3A activating antibody for use according to any one of #1 to #18 for treating SARS-Cov2 viral infectious disorder, wherein the subject is at high risk of progressing to severe COVID-19.

[0201] #21. A BTN3A activating antibody for use according to any one of #1 to #18 for treating a SARS-Cov2 viral infectious disorder, wherein the subject has severe COVID-19.

[0202] #22. A BTN3A activating antibody for use according to any one of #1 to #15 for treating a disorder caused by Coxiella Burnetii infection, administered simultaneously or separately, preferably in combination with an antibacterial treatment selected from an antibiotic selected from doxycycline, tetracycline, chloramphenicol, ciprofloxacin, ofloxacin, and hydroxychloroquine.

[0203] #23. A BTN3A activating antibody for use according to any one of #1 to #15 and #22 for treating a disorder caused by Coxiella Burnetii infection, wherein the subject has been diagnosed as positive for Coxiella Burnetii infection.

[0204] #24. A BTN3A activating antibody for use according to any one of #1 to #15 and #22 to #23 for treating a disorder caused by Coxiella Burnetii infection, wherein the subject has Q fever.

[0205] #25. A BTN3A activating antibody for use according to any one of #1 to #24, administered to a subject in need thereof by intravenous infusion, for example at a dose of 1 mg to 1 g, for example 1 mg to 200 mg. EXAMPLES

[0206] Assays for testing the functional properties of anti-BTN3A activating antibodies 1. Assay to determine binding affinity of BTN3A-activating antibodies by SPR Multi-cycle kinetic analysis may be performed on any BTN3A antibody candidate using a Biacore T200 (serial number 1909913) instrument running Biacore T200 evaluation software V2.0.1 (Uppsala, Sweden).

[0207] Purified antibodies are diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the beginning of each cycle, each antibody is captured onto Protein A at a density (RL) of approximately 146.5 RU (theoretical value to obtain an RMax of approximately 50 RU). After capture, the surface is stabilized before injection of BTN3A1 antigen (Sino Biological catalogue no. 15973-H08H). BTN3A1 is titrated in 0.1% BSA / HBS-P+ (running buffer) in a two-fold dilution range from 25 to 0.78 nM. The association phase is monitored for 400 s and the dissociation phase for 35 min (2100 s). Kinetic data are obtained using a flow rate of 50 μl / min to minimize potential mass transfer effects. Regeneration of the Protein A surface is performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle. Two blanks (without BTN3A1) and single concentration analyte replicates were performed for each test antibody to confirm surface and analyte stability over the kinetic cycle. The signal from reference channel Fc1 is subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface. In addition, a blank run is subtracted for each Fc to correct for any antigen-independent signal fluctuations such as drift. Sensorgrams were fitted using a one-to-one binding mathematical model with global RMax parameters and no bulk signal (constant RI=0 RU).

[0208] 2. Flow cytometric assay for binding of BTN3A activating antibodies to human PBMCs BTN3A activating antibodies for use according to the present disclosure can be characterized for their binding to BTN3A expressed in human PBMCs isolated from the blood of healthy donors.PBMCs are isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation.PBMCs are then frozen and stored at -80°C or in liquid nitrogen until required.

[0209] 1×10 6100 μl of cells / ml are transferred to each well of a fresh U-bottom 96-well plate, the plate is then centrifuged and the supernatant discarded.

[0210] Serial dilutions of antibody from 0.001 μg / ml to 150 μg / ml are prepared in PBS 2 mM EDTA. Human PBMC are resuspended in 50 μl of the prepared diluted test antibody titration series.

[0211] After 30 min incubation at 4°C in the dark, plates are centrifuged and washed twice with 150 μl / well PBS 2 mM EDTA, after which wells are resuspended in a mixture consisting of 50 μl of goat anti-human antibody (PE-labeled) diluted 1 / 100 in PBS 2 mM EDTA and live / dead neat IR diluted 1 / 500.

[0212] After 15 min incubation at 4° C. in the dark, plates are centrifuged and washed once with 150 μl / well PBS 2 mM EDTA, then wells are resuspended in 200 μl PBS 2 mM EDTA. Cells are analyzed on a BD LSR Fortessa Cytometer. Data are analyzed using FlowJo software (version 10, FlowJo, LLC, Ashland, USA) (data not shown).

[0213] The same protocol can be performed to test binding to cynomolgus BTN3A expressed from cynomolgus PBMCs and the Daudi Burkitt's lymphoma cell line.

[0214] 3. In vitro functional efficacy: γδ T cell degranulation assay The assay consists of measuring the activating effect of the BTN3A antibody on the degranulation of γδ-T cells against the Daudi Burkitt's lymphoma cell line (Harly et al., 2012). γδ-T cells from PBMCs of healthy donors are expanded by culturing for 11-13 days with zoledronic acid (1 μM) and IL2 (200 Ui / ml). IL2 is added on days 5, 8 and every 2 days thereafter. The percentage of γδ-T cells is determined at the beginning of the culture and assessed with respect to the culture time by flow cytometry until it reaches at least 80%. Frozen or fresh γδ-T cells are then used in a degranulation assay against the Daudi cell line (E:T ratio 1:1), whereby the cells are co-cultured for 4 h at 37 °C in the presence of 10 μg / ml of humanized variants of 7.2 and 20.1, as well as chimeric versions of 7.2 and 20.1. Activation with PMA (20 ng / ml) plus ionomycin (1 μg / ml) served as a positive control for γδ-T cell degranulation, and medium alone served as a negative control. At the end of the 4-h co-incubation, cells were analyzed by flow cytometry to assess the percentage of γδ-T cells positive for CD107a (LAMP-1, lysosomal-associated membrane protein-1) + CD107b (LAMP-2). As CD107 is mobilized to the cell surface following activation-induced granule exocytosis, measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytolytic T cells.

[0215] 4. Assay to determine the cytotoxic activity of Vγ9Vδ2 T cells against SARS-Cov2-infected cells in the presence of BTN3A-activating antibodies Monocytes, MDM, BEAS-2B and MRC-5 are labeled with 10 μM cell proliferation dye eFluor® 670 (Invitrogen) and then stimulated with virus. Target cells are co-cultured with Vγ9Vδ2 T cells (effector) at an effector to target (E:T) ratio of 1:1 in the presence of candidate anti-BTN3A activating antibodies or control mAb20.1 (0, 0.1, 1 or 10 μg / ml). After 24 hours, cells are stained with CellEvent caspase-3 / 7 green (Invitrogen) to identify dead cells. Cytotoxicity is assessed by flow cytometry as the percentage of caspase 3 / 7+ cells in the target cell population. Data are collected on a BD Canto II instrument (BD Biosciences) and analyzed using FlowJo software (FlowJo v10.6.2).

[0216] 5. Assay for IFN-γ secretion by Vγ9Vδ2 T cells against SARS-Cov2-infected cells in the presence of BTN3A-activating antibodies Monocytes, MDM, BEAS-2B and MRC-5 are stimulated with virus and co-cultured with Vγ9Vδ2 T cells (effector) at an effector to target (E:T) ratio of 1:1 in the presence of candidate anti-BTN3A activating antibodies or control mAb20.1 (0, 0.1, 1 or 10 μg / ml). After 24 h, culture supernatants are collected from the co-cultures and IFN-γ secretion is detected using a human IFN-γ immunoassay kit (R&D Systems) according to the manufacturer's instructions.

[0217] 6. Assay to determine degranulation of Vγ9Vδ2 T cells against Coxiella burnetii-infected cells in the presence of BTN3A-activating antibodies Monocytes infected with C. burnetii are co-cultured with Vγ9Vδ2 T cells at an effector to target (E:T) ratio of 1:1 in the presence of candidate anti-BTN3A activating antibodies or control mAb 20.1 and fluorochrome-labeled CD107a and CD107b (BD Biosciences). After 4 hours, cells are harvested and stained with fluorochrome-labeled TCR-specific mAbs (Miltenyi Biotec) and viability markers (Live / Dead Near IR, Invitrogen). Degranulation is observed by upregulation of CD107a / b in the γδ T cell population. + Cell percentages are assessed by flow cytometry. Data are collected on a Navios instrument (Beckman Coulter) and analyzed using FlowJo software (FlowJo v10.6.2).

[0218] 7. Assay to determine the cytotoxic activity of Vγ9Vδ2 T cells against Coxiella burnetii-infected cells in the presence of BTN3A-activating antibodies Monocytes infected with C. burnetii are labeled with 10 μM cell proliferation dye eFluor® 670 (Invitrogen) and then co-cultured with Vy9V52 T cells at an E:T ratio of 1:1 in the presence of candidate anti-BTN3A activating antibodies or control mAb20.1. After 4 hours, cells are stained with CellEvent Caspase-3 / 7 Green (Invitrogen) to identify dead cells. Cytotoxicity is assessed by the expression of caspase 3 / 7 in the target cell population. + Percentages of cells were assessed by flow cytometry. Data were collected on a BD Canto II instrument (BD Biosciences) and analyzed using FlowJo software (FlowJo v10.6.2).

[0219] Example 1: Humanization and characterization of mAb20.1 1. Description of humanization strategy a. Design of Composite Human Antibody™ variable region sequences Structural models of the V-regions of the murine 7.2 and 20.1 antibodies were generated using the Swiss PDB and analyzed to identify key "constraining" amino acids in the V-regions that are likely to be essential for the binding properties of the antibodies. Most residues contained within the CDRs (using both Kabat and Chothia definitions) were considered important along with several framework residues. From the above analysis, Composite Human sequences of the 7.2 and 20.1 antibodies were generated.

[0220] b. Avoidance of CD4+ T cell epitopes Based on the structural analysis, a large preliminary set of sequence segments that could be used to create humanized variants of 7.2 and 20.1 were selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al., 2008) and TCED™ of known antibody sequence-associated T cell epitopes (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II or scored significant hits to TCED™ were discarded. This resulted in a reduced set of segments, and these combinations were analyzed again as above to ensure that the junctions between segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V region sequences predicted to lack significant T cell epitopes. Several heavy and light chain sequences were then selected for gene synthesis and expression in mammalian cells for mAbs 7.2 and 20.1.

[0221] 2. Generation and preliminary characterization of humanized variants a. Construction of humanized variant plasmids Humanized variants of 7.2 and 20.1 were synthesized for human IgG4 (S241P, L248E) heavy chain and kappa light chain with flanking restriction enzyme sites for cloning into an expression vector system. All constructs were verified by sequencing.

[0222] b. Antibody expression Chimeras 7.2 and 20.1 (VH0 / Vκ0), two control combinations (VH0 / Vκ1, VH1 / Vκ0) and humanized heavy and light chain combinations were transiently transfected into FreeStyle™ CHO-S cells (ThermoFisher, Loughborough, UK) using the MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA) from the corresponding endotoxin-free DNA. Transfections were performed for each antibody using an OC-400 processing assembly. After cell harvesting, cells were diluted 3x10 in CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-glutamine (ThermoFisher, Loughborough, UK) and 1x hypoxanthine-thymidine (ThermoFisher, Loughborough, UK). 6 The cells were diluted to 1000 cells / mL. 24 hours after transfection, the culture temperature was reduced to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. Cultures were fed daily with 3.6% (of starting volume) feed (2.5% CHO CD Efficient Feed A (ThermoFisher, Loughborough, UK), 0.5% Yeastolate (BD Biosciences, Oxford, UK), 0.25 mM Glutamax (ThermoFisher, Loughborough, UK) and 2 g / L glucose (Sigma, Dorset, UK)). IgG supernatant titers were monitored by IgG ELISA and transfections were cultured for up to 14 days before supernatants were harvested.

[0223] c. Selection of mAb3 (humanized form of mAb20.1) Two humanized variants of mAb20.1 were selected from 20 humanized candidates for further characterization.

[0224] Table 4 below summarizes comparative data between the murine parent antibody mAb7.2, mAb20.1, and a humanized version of mAb20.1 having a VH of SEQ ID NO:1 and a VL of SEQ ID NO:2.

[0225] [Table 4]

[0226] Example 2: Evidence for the use of activating BTN3A antibodies to treat SARS-Cov2 infection material and method Cell isolation and cell line culture Blood samples from 15 healthy volunteers were obtained from a local blood bank (agreement N° 7828, "Etablissement Francais du Sang", Marseille, France). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from buffy coats (using Ficoll (Eurobio, Les Ulis, France).

[0227] Monocytes were purified from PBMCs by CD14 selection using MACS magnetic beads (Miltenyi Biotec, Bergisch Glabach, Germany) and cultured in Roswell Park Memorial Institute-1640 medium (RPMI, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, Life Technologies), 2 mM L-glutamine, 100 U / mL penicillin, and 50 μg / mL streptomycin (Life Technologies). For monocyte-derived macrophages (MDMs), cells were cultured in RPMI-1640 containing 10% inactivated human AB serum (MP Biomedicals, Solon, OH), 2 mM glutamine, 100 U / mL penicillin, and 50 μg / mL streptomycin. After 3 days, the medium was replaced with RPMI-1640 containing 10% FBS and 2 mM glutamine, and the cells were allowed to differentiate into macrophages for an additional 4 days.

[0228] Vy9V52 T cells were expanded from fresh PBMCs. Briefly, PBMCs were expanded in RPMI-1640 medium supplemented with 10% FBS, interleukin-2 (IL-2, to a final concentration of 200UI / ml) and zoledronic acid monohydrate (final concentration 1 μM). IL-2 was added every 2 days starting on day 5. After 12 days of culture, the purity of Vy9V52 T cells was assessed by flow cytometry analysis and then frozen. Vy9V52 T cells were further purified and enriched using a TCR γ / δ+ T cell isolation kit (Miltenyi Biotec) to a purity of up to 98%.

[0229] Normal human bronchial epithelial cells (BEAS-2B cells, ATCC® CRL-9609™) were cultured in LHC-9 medium (Life Technologies) and normal human lung fibroblast cells (MRC-5 cells, ATCC® CCL-171™) were cultured in Minimum Essential Medium (MEM, Life Technologies) supplemented with 4% FBS and 2 mM L-glutamine at 37° C. in a 5% CO2 atmosphere.

[0230] Virus production and cell infection SARS-CoV-2 strain IHU-MI6 was obtained after infection of Vero E6 cells (ATCC® CRL-1586™) in MEM supplemented with 4% FBS as previously described (Boumaza et al., 2020). Monocytes and MDMs isolated from PBMCs of healthy donors, as well as BEAS-2B and MRC-5 cells, were infected with the virus suspension at a multiplicity of infection (MOI) of 1 for 24 h at 37°C in the presence of 5% CO2 and 95% air in a humidified incubator.

[0231] RNA isolation and qRT-PCR Total RNA was purified from cells (2.10 μg / mL) using the RNeasy mini kit (Qiagen, Courtaboeuf, France) with DNase I treatment to eliminate DNA contamination, as previously described ( Mezouar et al., 2019c ). 6The RNA was extracted from 1000 cells / well of 10000 ng / ml of 10 ... Results were normalized using ACTB or GAPDH housekeeping genes as previously described (Mezouar et al., 2019a) and expressed as the relative expression of the investigated genes, ΔCt = Ct target - Ct housekeeping gene. The threshold cycle (Ct) was defined as the number of cycles required to detect a fluorescent signal.

[0232] [Table 5]

[0233] Flow cytometry staining and data acquisition and analysis For analysis of cells infected with SARS-CoV-2 in vitro, cells were suspended in phosphate-buffered saline (Life Technologies) containing 1% FBS and 2 mM EDTA (Sigma-Aldrich). Cells were labeled with a viability dye (LIVE / Dead Near IR, Invitrogen) using anti-BTN3A (103.2) or anti-BTN2A (7.48) mAb or with an isotype control (Miltenyi Biotech). After 30 min of incubation, binding of the primary antibody was detected with Alexa Fluor 488 anti-mouse (Invitrogen). Data were collected on a BD Canto II instrument (BD Biosciences) and analyzed using FlowJo software (FlowJo v10.6.2, Ashland, OR).

[0234] Viral RNA extraction and qRT-PCR Viral RNA was extracted from infected cells using the NucleoSpin® Viral RNA Isolation Kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer's recommendations. Viral detection was performed using the One-Step RT-PCR SuperScript™ III Platinum™ Kit (Life Technologies). Thermal cycling was achieved using a LightCycler 480 real-time PCR system (Roche, Rotkreuz, Switzerland) at 55°C for 10 min for reverse transcription, followed by 95°C for 3 min, then 45 cycles of 95°C for 15 s and 58°C for 30 s. Primers and probes were designed against the E gene (Boumaza et al., 2020).

[0235] Cytotoxicity assay Monocytes, MDM, BEAS-2B and MRC-5 were labeled with 10 μM cell proliferation dye eFluor® 670 (Invitrogen) and then stimulated with virus. Target cells were co-cultured with Vγ9Vδ2 T cells (effector) at an effector to target (E:T) ratio of 1:1 in the presence of anti-BTN3A 20.1 mAb (0, 0.1, 1 or 10 μg / ml). After 24 hours, cells were stained with CellEvent caspase-3 / 7 green (Invitrogen) to identify dead cells. Cytotoxicity was assessed by flow cytometry as the percentage of caspase 3 / 7+ cells in the target cell population. Data were collected on a BD Canto II instrument (BD Biosciences) and analyzed using FlowJo software (FlowJo v10.6.2).

[0236] IFN-γ secretion assay Monocytes, MDM, BEAS-2B and MRC-5 were stimulated with virus and co-cultured with Vγ9Vδ2 T cells (effector) at an effector to target (E:T) ratio of 1:1 in the presence of anti-BTN3A 20.1 mAb (0, 0.1, 1 or 10 μg / ml). After 24 h, culture supernatants were collected from the co-cultures and IFN-γ secretion was detected using a human IFN-γ immunoassay kit (R&D Systems) according to the manufacturer's instructions.

[0237] statistical analysis Statistical analyses were performed with GraphPad Prism (version 8.0, La Jolla, CA) for transcriptional analysis using Student's t test or nonparametric Mann-Whitney U test, and for spectral cytometry using the Kruskal-Wallis test, followed by Dunn's multiple comparisons and Mann-Whitney tests (significance boundary: p < 0.05).

[0238] result Effect of SARS-CoV-2 infection on the expression of BTN3A and BTN2A First, we evaluated whether SARS-CoV-2 infection affects the expression of BTN3A and BTN2A in cells of bone marrow origin (primary cell cultures of monocytes or monocyte-derived macrophages (MDM)) or cells of lung origin (normal epithelial lung cell lines: BEAS-2B or MRC-5). No effect on BTN2A transcription (isoform genes 2A1, 2A2) or protein expression was observed (Figure 1A). In contrast, gene expression of the three isoforms of BTN3A (3A1, 3A2, 3A3) was significantly increased in the presence of SARS-CoV-2 in MDM cultures compared to cultures without virus. Indeed, the relative expression of the three isoforms of BTN3A was increased approximately 5-fold in SARS-CoV-2-stimulated MDMs compared to non-stimulated MDMs (3A1 p=0.012, 3A2 p=0.006, 3A3 p=0.028) (Figure 1B). No statistically significant differences in BTN3A transcripts were observed in monocyte or lung cell cultures. However, we observed a significant increase in BTN3A protein expression in MDM, BEAS-2B, and MRC-5 upon SARS-CoV-2 stimulation (Figure 1B). Protein expression of BTN3A increased 3.5-fold in MDM and approximately 2-fold in BEAS-2B and MRC-5 cells after stimulation with SARS-CoV-2 (p=0.048, p=0.012, and p=0.002, respectively).

[0239] Characterization of Vγ9Vδ2 T cell responses to SARS-CoV-2-infected cells in vitro We then studied the ability of Vγ9Vδ2 T cells to inhibit SARS-CoV-2 replication in vitro when activated with the reference anti-BTN3A monoclonal antibody 20.1. After confirming that viral infection did not affect the viability of Vγ9Vδ2 T cells (Figure 2), we co-cultured Vγ9Vδ2 T cells with bone marrow or lung cells in the presence of the SARS-CoV-2 IHU-MI6 strain and increasing concentrations of the reference 20.1 mAb. Reference anti-BTN3A 20.1 resulted in a dose-dependent inhibition of viral replication (28.4% in monocyte cultures, 42.4% in MDM, 33.7% in MRC-5 and 53.0% in BEAS-2B cultures at 10 μg / ml) (Figure 3A). Cytotoxicity (caspase 3 / 7) of Vγ9Vδ2 T lymphocytes appeared to be higher against SARS-CoV-2 infected lung cells than against bone marrow cells and increased with the concentration of the reference anti-BTN3A 20.1 antibody. The differences were statistically significant between doses of 0.1 and 10 μg / ml in each of the four target cell cultures (Table 6).

[0240] Finally, we investigated whether Vγ9Vδ2 T cells activated with reference anti-BTN3A 20.1 exerted IFN-γ-mediated non-cytolytic anti-SARS-CoV-2 activity. Increasing concentrations of anti-BTN3A were associated with a significant and dose-dependent increase in IFN-γ in the supernatants of the four cell cultures (Figure 3B and Table 6).

[0241] [Table 6]

[0242] Example 3: Evidence for the use of activating anti-BTN3A antibodies to treat Coxiella burnetii infection material and method Cell isolation The blood samples (leukopacks) used in our study came from the French Blood Agency (Etablissement Francais du sang, EFS), which handles donor reception, informed consent, and sample collection. According to an agreement established between our laboratory and the EFS (N°7828), buffy coats were obtained and peripheral blood mononuclear cells (PBMCs) were isolated as previously described.

[0243] Monocytes were purified from PBMCs by CD14 selection using MACS magnetic beads (Miltenyi Biotec, Bergisch Glabach, Germany) and cultured in Roswell Park Memorial Institute-1640 medium (RPMI, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, Life technologies), 2 mM L-glutamine, 100 U / mL penicillin and 50 μg / mL streptomycin (Life Technologies).

[0244] Vy9V52 T cells were expanded from fresh PBMCs as previously described. Briefly, PBMCs were expanded in RPMI-1640 medium supplemented with 10% FBS, interleukin-2 (IL-2, to a final concentration of 200UI / ml) and zoledronic acid monohydrate (Zometa, to a final concentration of 1 μM). IL-2 was added every 2 days starting on day 5 for 12 days, and the purity of Vy9V52 T cells was assessed by flow cytometry analysis (>85%) and then frozen at -80°C in 10% dimethylsulfoxide (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and 90% FBS.

[0245] Bacterial production Coxiella burnetii bacterial phase I (Nine Mile strain, RSA493 and Guiana strain, MST17) were cultured in L929 cells for 10 days. Briefly, infected cells were sonicated and centrifuged at 10,000×g for 10 min, then washed and stored at −80° C. Bacterial concentration was determined using Jimenez staining, and bacterial viability was assessed using the Live / Dead BacLight bacterial viability kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer's instructions.

[0246] Bacteria detection DNA was extracted from cells infected with C. burnetii using a DNA mini kit (Qiagen, Courtaboeuf, France). Infection was quantified by real-time quantitative PCR (qPCR) performed with specific primers F (5'-GCACTATTTTTAGCCG-GAACCTT-3' [SEQ ID NO: 43]) and R (5'-TTGAGGAGAAAAACTGGATTGAGA-3' [SEQ ID NO: 44]) targeting the C. burnetii COM-1 gene, as previously described.

[0247] The presence of C. burnetii in cells was also assessed by flow cytometry. Briefly, infected cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). After washing, cells were incubated for 30 min with anti-rabbit directed against C. burnetii, followed by incubation with Alexa647 anti-rabbit antibody (Invitrogen). Data were collected on a BD Canto II instrument (BD Biosciences, Le Pont-de-Claix, France) and analyzed using FlowJo software (FlowJo v10.6.2, Ashland, OR).

[0248] RNA isolation and qRT-PCR Total RNA was extracted from cells (2.10) using the RNeasy mini kit (Qiagen) with Dnase I treatment to eliminate DNA contamination, as previously described. 6 Cells / well) were extracted from the cells. The quality and quantity of extracted RNA was assessed using a NanoDrop spectrophotometer (Nanodrop Technologies, Wilmington, USA). Reverse transcription of isolated RNA was performed using the Moloney Murine Leukemia Virus Reverse Transcriptase Kit (Life Technologies) and oligo(dT) primers. Expression levels of genes involved in M1 / M2 response, as well as BTN3A isoform genes, were assessed using real-time qPCR, Smart SYBR Green fast Master kit (Roche Diagnostics, Meylan, France) and specific primers (Table 7). Expression levels of BTN2A were assessed using real-time qPCR, TaqMan® Fast Advanced Master Mix (Applied Biosystems, Life Technologies) and specific probes (Table 7). All qPCR was performed using a CFX Touch real-time PCR detection system (Bio-Rad, Marnes-la-Coquette, France). Results were normalized using ACTB or GAPDH housekeeping genes as previously described, ΔCt = Ct target -Ct housekeeping gene 2 -ΔCt The expression is expressed as the relative expression of the investigated genes using the threshold cycle (Ct) was defined as the cycle number required to detect a fluorescent signal.

[0249] [Table 7]

[0250] Protein expression of BTN3A and BTN2A Cells were harvested in PBS (Life Technologies) containing 1% FBS and 2 mM EDTA (Sigma-Aldrich) and labeled with a viability dye (Live / Dead Near IR, Invitrogen), anti-BTN3A (clone 103.2) or anti-BTN2A (clone 7.48) mAb or appropriate isotype controls (Miltenyi Biotech). After 30 min incubation, binding of primary antibodies was detected with PE anti-mouse antibody (Invitrogen) and data were collected on a Navios instrument (Beckman Coulter) and analyzed using FlowJo software (FlowJo v10.6.2).

[0251] Degranulation assay Monocytes were co-cultured with Vγ9Vδ2 T cells at an effector to target (E:T) ratio of 1:1 in the presence of anti-BTN2A mAb (clone 7.48) or anti-BTN3A mAb (clone 20.1 or 103.2) and fluorochrome-labeled CD107a and CD107b (BD Biosciences). After 4 hours, cells were harvested and stained with fluorochrome-labeled TCR-specific mAbs (Miltenyi Biotec) and viability markers (Live / Dead Near IR, Invitrogen). Degranulation was observed by upregulation of CD107a / b in the γδ T cell population. + Cell percentages were assessed by flow cytometry. Data were collected on a Navios instrument (Beckman Coulter) and analyzed using FlowJo software (FlowJo v10.6.2).

[0252] Cytotoxicity assay Monocytes were labeled with 10 μM cell proliferation dye eFluor® 670 (Invitrogen) and then co-cultured with Vy9V52 T cells at an E:T ratio of 1:1 in the presence of anti-BTN3A mAb (clone 20.1). After 4 hours, cells are stained with CellEvent Caspase-3 / 7 Green (Invitrogen) to identify dead cells. Cytotoxicity was assessed by the expression of caspase 3 / 7 in the target cell population. +Percentages of cells were assessed by flow cytometry. Data were collected on a BD Canto II instrument (BD Biosciences) and analyzed using FlowJo software (FlowJo v10.6.2).

[0253] Immunoassays Tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D Systems), granzyme B, perforin and granulysin (Abcam) levels were quantified in supernatants of monocyte / Vγ9Vδ2 T cell co-cultures using specific immunoassay kits. Levels of TNF-α, IFN-γ, interleukin (IL)-1β, IL-6, IL-10 and transforming growth factor beta (TGF-β) (R&D Systems) were quantified in supernatants of BTNKO cells following C. burnetii infection. The sensitivities of the assays were 6.2 pg / mL for TNF-α, 5.7 pg / mL for IFN-γ, 1.0 pg / mL for IL-1β, 0.7 pg / mL for IL-6, 3.9 pg / mL for IL-10, 15.4 pg / mL for TGF-β, 3.0 pg / mL for GM-CSF, 20 pg / mL for granzyme B, 40 pg / mL for perforin, and 10 pg / mL for granulysin.

[0254] statistical analysis Statistical analysis was performed with GraphPad Prism (8.0, La Jolla, CA) using Mann-Whitney U tests for transcriptional analysis and t tests for flow cytometry and ELISA results. Hierarchical clustering of gene expression was analyzed using the ClustVis web tool. The boundary of significance was set at p<0.05.

[0255] result C. burnetii infection regulates the expression of BTN3A and BTN2A To evaluate whether C. burnetii infection affects the expression of BTN, monocytes from healthy donors were isolated and infected with the reference strain NM1 or the Guiana strain, described as more virulent. 24 h after C. burnetii NM1 strain infection, an increase in the transcriptional expression of both BTN3A1 and BTN3A2 isoforms, but not BTN3A3, was found. Guiana strain infection induced the three isoforms (Figure 4A). Interestingly, a significant difference was observed between C. burnetii NM1 infection and the heat-inactivated form of this strain (p=0.0374), suggesting that virulence factors are involved in the expression of BTN3A1. A significant increase in BTN3A protein expression was found in monocytes infected with C. burnetii NM1 and the Guiana strain (p=0.0021 and p=0.0096, respectively) (Figure 4B). Finally, expression of BTN3A was assessed in PBMCs from Q fever patients with C. burnetii infection, either in active or persistent forms of BTN3A. Protein expression of BTN3A was significantly higher in patients with Q fever compared to healthy donors (p=0.0185), with similar expression according to disease form (Figure 4C).

[0256] Since BTN2A is involved in the activation of Vγ9Vδ2 T cells, we also investigated whether C. burnetii infection affects the expression of BTN2A. After 24 h of infection, BTN2A transcriptional expression of both isoforms (2A1 and 2A2) was significantly increased after C. burnetii NM1 and Guiana infection compared to non-infected cells (2A1, p=0.0170 and p=0.0021, respectively; and 2A2, p=0.0054 and p=0.0463, respectively), with no significant regulation compared to heat-inactivated forms (Figure 4D). Regarding the protein expression of BTN2A, a significant increase (NM1 strain, p=0.0160, and Guiana strain, p=0.0018) was observed in monocytes infected with C. burnetii compared to non-infected cells (Figure 4E). Similar to BTN3A, PBMCs from Q fever patients showed significantly higher expression of BTN2A compared to healthy donors (p=0.0125), without modulation by the clinical form of the disease (Figure 4F).

[0257] Overall, C. burnetii infection results in increased expression of BTN3A and BTN2A in the host following in vitro infection, as well as in samples from infected patients.

[0258] Involvement of BTN3A and BTN2A in C. burnetii infection Next, we investigated whether BTN could be involved in the uptake or replication of C. burnetii. To this end, we performed CRISPR-Cas9 knockout of three BTN3A genes or two BTN2A genes in THP-1 cell line. Cells were transduced with guides targeting either BTN2A1 and BTN2A2 (BTN2AKO) or BTN3A1, BTN3A2 and BTN3A3 (BTN3AKO) isoforms, or with unrelated CRISPR guides (mock). BTN3A- or BTN2A-neutralized (KO) cells were infected with C. burnetii NM1 and bacterial load was assessed by qRT-PCR. No differences were observed in bacterial uptake ( Fig. 5A ) or replication ( Fig. 5B ) between BTN3AKO or BTN2AKO and mock cells ( Fig. 5A ), suggesting that BTN3A and BTN2A are not directly involved in the process of C. burnetii cell infection.

[0259] Involvement of BTN3A and BTN2A in the inflammatory response to C. burnetii infection We then investigated the involvement of BTN in the host immune response following C. burnetii infection. As observed in Figure 3, C. burnetii infection leads to the regulation of both pro- and anti-inflammatory genes in THP-1 cells. When infected with C. burnetii, hierarchical clustering revealed cell type-dependent clustering. BTN3AKO and BTN2KO cells clustered away from mock cells (Figure 6A). BTN3AKO and BTN2KO cells showed suppressed inflammatory responses following C. burnetii infection. Indeed, the expression of inflammatory genes TNF and IL1B was significantly reduced by 3- and 2-fold in BTN3AKO and BTN2AKO cells, respectively, compared to mock cells (Figure 6B). Moreover, a reduced immune regulatory response was observed following infection only in BTN3AKO cells. Indeed, transcriptional expression of the IL10 gene was significantly reduced by two-fold compared to mock cells (p=0.0435) (Figure 6B). With regard to protein levels, BTN3AKO and BTN2AKO cells showed a significant reduction in TNF-a (50% and 30%, respectively) and IL-1β (up to 20%) release after C. burnetii infection compared to mock cells (Figure 6C). No significant differences were observed in the levels of anti-inflammatory cytokines such as IL-10.

[0260] Taken together, these data demonstrate that both BTN3A and BTN2A are involved in the inflammatory response to C. burnetii infection. The fact that the inflammatory response was suppressed in the presence of only BTN3A or only BTN2A suggests that BTN3A and BTN2A may act independently.

[0261] C. burnetii infection leads to activation of Vγ9Vδ2 cells in a BTN3A- and BTN2A-dependent manner Since we showed that BTN is overexpressed in monocytes after C. burnetii infection, we hypothesized that this process may enhance Vγ9Vδ2 T cell activation. After 4 h of co-culture, C. burnetii infection of monocytes leads to increased degranulation (up to 5-fold) as shown by increased plasma membrane expression of CD107, in a dose-response depending on the bacteria:target cell ratio (Figure 7A). We then investigated whether Vγ9Vδ2 T cell activation on cells infected with C. burnetii is dependent on BTN using specific antagonist antibodies, including anti-BTN3A antagonist (clone 103.2) and anti-BTN2A antagonist (clone 7.48). Both antibodies led to the abrogation of Vγ9Vδ2 T cell degranulation after C. burnetii infection, indicating that both BTNs are involved in Vγ9Vδ T cell activation upon C. burnetii infection (Figure 7B and Figure 7C). Interestingly, a higher tendency for degranulation of Vγ9Vδ2 T cells was found in co-cultures of PBMCs from patients with Q fever compared to healthy donors without antibody treatment (10% vs. 3%, p=0.173). This degranulation was also inhibited in the presence of anti-BTN3A 103.2 and anti-BTN2A 7.48 antagonist antibodies (Figures 7D and 7E). Taken together, C. burnetii infection leads to the activation of Vγ9Vδ2 T cells in a BTN3A- and BTN2A-dependent manner.

[0262] Next, we hypothesized that Vγ9V52 T cell activation against C. burnetii-infected cells could be enhanced by a BTN3A-activating antibody (clone 20.1), mimicking pAg-induced Vγ9V52 T cell activation. As shown in Figure 7F and Table 8, we observed that BTN3A-activating antibodies led to an increase in the expression of CD107 (Figure 7F) and the cytotoxic activity of Vγ9V52 T cells against C. burnetii-infected monocytes (Figure 7G) after 4 h of co-culture. A similar effect was observed against all C. burnetii strains, suggesting that the reference 20.1 antibody can induce Vγ9V52 T cell activation even against more virulent bacteria. Finally, the use of the reference 20.1 antibody was also able to increase degranulation of Vγ9Vδ2 T cells from PBMCs derived from C. burnetii patients and healthy donors (mean 24.13% and 3.243%, respectively, p=0.0002, n=3 each) (Figure 7H). These data indicate that targeting Vγ9Vδ2 T cells with a BTN3A activating antibody leads to activation of Vγ9Vδ2 T cell effector function.

[0263] [Table 8]

[0264] BTN3A 20.1 activating antibodies increase the antibacterial activity of Vγ9Vδ2 T cells Since the reference BTN3A-activating 20.1 antibody increases Vy9V52 T cell activation, we wondered whether the reference BTN3A-activating 20.1 antibody could boost the antibacterial activity of Vy9V52 T cells. To this end, monocytes were infected with C. burnetii NM1 for 24 h and then co-cultured with Vy9V52 T cells in the presence of 20.1 antibody (0, 0.1, 1 or 10 μg / ml) for 4 h and the bacterial load was measured by flow cytometry and qPCR. Co-incubation of Vy9V52 T cells resulted in a significant reduction in the MFI associated with C. burnetti staining in co-cultures of infected monocytes and Vy9V52 T cells (Figure 8A) and a 5.10 increase in the MFI associated with C. burnetti staining in co-cultures of infected monocytes and Vy9V52 T lymphocytes (Figure 8B). 7 From 6.10 6 The reference BTN3A-activating 20.1 antibody caused a dose-dependent reduction in C. burnetii load in monocytes, with a 6.10% reduction in C. burnetii load, as shown by a reduction in the number of copies (p=0.0021) (FIG. 8B). 6 From 4.2.10 6 copies (0 vs. 10 μg / ml, respectively, p=0.0501) (Figure 8B). Overall, the reference BTN3A 20.1 activating antibody increases the antibacterial activity of Vγ9Vδ2 T lymphocytes against monocytes infected with Coxiella burnetii.

[0265] The reference BTN3A 20.1 antibody increases secretion of cytokines and cytotoxic molecules by Vγ9Vδ2 T cells Since BTN3A activating antibodies allow the inhibition of bacterial load, we investigated whether this could be related to the secretion of cytokines and cytotoxic molecules strongly produced by activated Vγ9V52 T cells. Indeed, treatment of Vγ9V52 T cells / C. burnetii-infected monocytes co-cultures with reference 20.1 antibody increased the secretion of TFN-α, IFN-γ and GM-CSF in a dose-dependent manner (Figure 9A, left panel). Moreover, in the case of infection with C. burnetii guiana, significant differences were observed between the 0.1 μg / ml and 10 μg / ml doses for IFN-γ, TFN-α and GM-CSF secretion (p=0.0260, p=0.0443 and p=0.0265, respectively). Concerning cytotoxic molecules, the secretion of granzyme B and perforin was significantly higher in the presence of 10 μg / ml of 20.1 antibody in monocytes infected with C. burnetii NM1 and Gina, and also in non-infected monocytes (Figure 9B, right panel). Overall, the presence of the reference 20.1 antibody allows an increased secretion of cytokines and cytotoxic molecules, both produced by activated Vγ9Vδ2 T cells (Table 9).

[0266] [Table 9]

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Claims

1. A BTN3A activating antibody comprising a variable heavy chain polypeptide VH that is at least 95% identical to SEQ ID NO: 1 and a variable light chain polypeptide VL that is at least 95% identical to SEQ ID NO: 2, and comprising HCDR1-3 of SEQ ID NOs: 5-7 and LCDR1-3 of SEQ ID NOs: 8-10.

2. A BTN3A activating antibody as described in claim 1, comprising a variable heavy chain polypeptide VH of sequence number 1 and a variable light chain polypeptide VL of sequence number 2.

3. A BTN3A activating antibody described in claim 1, comprising a heavy chain polypeptide of sequence number 23 and a light chain polypeptide of sequence number 24.

4. A nucleic acid molecule encoding the heavy and light chains of the BTN3A activating antibody described in claim 1.

5. An expression vector comprising at least one nucleic acid molecule described in claim 4.

6. A host cell comprising the expression vector described in claim 5.

7. 10. A pharmaceutical composition comprising the BTN3A activating antibody of claim 1 for treating an infectious disorder in a human subject in need thereof.

8. The pharmaceutical composition of claim 7, wherein the infectious disorder is selected from the group consisting of (i) a disorder caused by SARS-Cov2 infection, and (ii) a disorder caused by Coxiella burnetii infection.

9. The pharmaceutical composition of claim 7, wherein the BTN3A-activating antibody has one or more of the following properties: (i) the BTN3A activating antibody has an EC of 50 μg / ml or less, as measured by a flow cytometry assay; ５０ binds to human PBMCs with (ii) the BTN3A activating antibody has an EC50 activity of less than 5 μg / ml, as measured by a degranulation assay. ５０ to induce γδ-T cell activation in vitro in co-culture with cells expressing BTN3; (iii) the BTN3A activating antibody enhances the reduction of C. burnetii bacterial burden in vitro, as measured in vitro using co-cultures of infected monocytes in the presence of Vγ9Vδ2 T cells; (iv) the BTN3A activating antibody increases the in vitro cytotoxic activity of Vγ9Vδ2 T cells against C. burnetii-infected cells, as measured in vitro using co-cultures of infected monocytes; and / or (v) the BTN3A activating antibody increases the in vitro cytotoxic activity of Vγ9Vδ2 T cells against cells infected with SARS-Cov2, as measured in vitro in co-cultures of infected cells with Vγ9Vδ2 T cells.

10. 8. The pharmaceutical composition of claim 7, wherein the BTN3A activating antibody comprises a mutant or chemically modified IgG1 constant region, which confers no or reduced binding to Fcγ receptors when compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

11. The pharmaceutical composition of claim 7 for treating disorders caused by SARS-Cov2 infection, wherein the BTN3A-activating antibody is used in combination with a second drug substance selected from antiviral treatments, anti-inflammatory treatments and cell therapy products, administered simultaneously or separately.

12. The pharmaceutical composition of claim 11, wherein the second drug substance is selected from the group consisting of remdesivir, baricitinib, bamlanivimab, bamlanivimab / etesevimab, casirivimab / imdevimab, dexamethasone, budesonide and tocilizumab.

13. 12. The pharmaceutical composition of claim 7 or 11, wherein the subject is (i) a human subject who has been diagnosed as SARS-Cov2 positive, (ii) a human subject with mild or moderate COVID-19, (iii) a human subject at high risk of progressing to severe COVID-19, and / or (iv) a subject with severe COVID-19.

14. The pharmaceutical composition of claim 7 for treating disorders caused by Coxiella burnetii infection, wherein the BTN3A-activating antibody is used in combination with a second drug selected from antibacterial and cell therapy products, administered simultaneously or separately.

15. The pharmaceutical composition of claim 14, wherein the second drug is selected from doxycycline, tetracycline, chloramphenicol, ciprofloxacin, ofloxacin, and hydroxychloroquine.

16. 15. The pharmaceutical composition of claim 7 or 14 for treating a disorder caused by a Coxiella burnetii infection, wherein the subject has been diagnosed as positive for a Coxiella burnetii infection or the subject has Q fever.

17. The pharmaceutical composition of claim 7, wherein the BTN3A-activating antibody is administered to a subject in need thereof by topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration.

18. The pharmaceutical composition described in claim 7, wherein the BTN3A-activating antibody is administered at a dose of 1 mg to 1 g.

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