Methods for treating multidrug resistant bacterial infections

Abstract

The present disclosure relates to methods for treating multidrug resistant bacterial infectious conditions. In particular, the disclosure provides BTN3A-activating antibodies, and their use in the treatment of multidrug resistant bacterial infectious disorders, such as disorders caused by multidrug resistant Mycobacterium tuberculosis infection, in human subjects in need thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to methods for treating a multi-drug resistant bacterial infectious disorder. In particular, the present disclosure provides BTN3A-activating antibodies, and their use in treating a multi-drug resistant bacterial infectious disorder, e.g., a disorder caused by a multi-drug resistant Mycobacterium tuberculosis infection, in a human subject in need thereof. BACKGROUND

[0002] Antimicrobial resistance is the ability of a microorganism to resist the action of different antimicrobial agents. In this type of resistance, microorganisms can resist drugs that were once able to successfully combat them (Colson, et al. 2021). When this resistance occurs to multiple drugs, it is called multi-drug resistance (MDR). Different types of resistance mechanisms are observed in microorganisms, such as natural resistance to specific antimicrobial agents in some microorganisms, genetic mutations, or acquired resistance from other species (Catalano, et al. 2022). Initially, MDR bacteria were associated with hospital-acquired infections. MDR bacteria have spread and are now the main cause of community-acquired infections. The spread of MDR bacteria in society has led to an increase in morbidity, mortality, healthcare spending, and antibiotic use. This overdevelopment is the cause of a higher incidence of MDR and leads to the development of a vicious cycle. Resistant microorganisms are difficult to treat, requiring alternative or higher doses of antimicrobial agents or a lack / shortage of effective antimicrobial agents, thus adversely affecting countries at all levels of development. According to the World Health Organization (WHO) and the Infectious Diseases Society of America (IDSA) statements, MDR pathogens, known as “superbugs,” are one of the main public threats that cause millions of deaths worldwide each year (Bloom, et al. 2018; Infectious Diseases Society of, et al. 2011). In 2021, the WHO published a list of antibiotic-resistant pathogens (priority pathogens), highlighting the greatest threat to human health, drug-resistant Gram-negative bacteria (https: / / www.who.int / medicines / publications / WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf). It is estimated that, without the discovery or formulation of new drugs, there will be no effective antibiotics available to treat these deadly resistant pathogens by 2050 (Bharadwaj, et al. 2022). Among these MDR strains, Staphylococcus aureus (S. aureus) Staphylococcus aureus ), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) Pseudomonas aeruginosa ), Klebsiella pneumoniae (K. pneumoniae) Klebsiella pneumoniae ), and Streptococcus pneumoniae (S. pneumoniae) are the most common causes of infections in humans (Bharadwaj, et al. 2022). The WHO has declared that the spread of MDR bacteria is a global threat and has called for urgent action to combat this problem (https: / / www.who.int / news-room / q-a-detail / who-global-action-plan-on-antimicrobial-resistance).Streptococcus pneumoniae ), Enterococcus faecalis (E. faecalis) Enterococci ) (E. faecium (E. faecalis Entorococcus faecium and E. faecalis (E. faecalis Enterococcus Faecalis ) and Mycobacterium tuberculosis (M. tuberculosis) Mycobacterium tuberculosis ) are the most frequently cited strains.

[0003] Tuberculosis (TB) caused by M. tuberculosis remains one of the leading causes of death worldwide, with increasing incidence and mortality over the past two years. Globally, the treatment success rate for patients with active pulmonary TB is 86-56%, depending mainly on the level of Mtb drug resistance to treatment (https: / / www.who.int / teams / global-tuberculosis-programme / tb-reports / global-tuberculosis-report-2022). In 2021, 166991 people were diagnosed with pulmonary TB with resistance to at least one antimicrobial, resulting in survival rates of less than 50% of these patients receiving WHO-recommended treatment. Even new regimens effective against MDR-TB such as BPaL (Pretomanid, Bedaquiline and Linezolid) are now associated with new bacterial resistance (Peloquin and Davies 2021). To overcome antimicrobial resistance, reduce treatment duration and achieve complete remission, new therapeutic strategies and agents are urgently needed. Host-directed therapies represent an innovative approach to amplify host immune responses and increase Mtb killing (Tiberi, et al. 2018). Immune checkpoint inhibitors evaluated in different preclinical models of TB can have the potential to restore effective T cell responses against Mtb. However, treatment with PD-1 blockade worsened tuberculosis in infected rhesus macaques and was associated with reactivation of tuberculosis in cancer patients (Kauffman, et al. 2021; Picchi, et al. 2018). Strategies to activate and target more relevant immune cell populations can provide an alternative to immunotherapy of tuberculosis. In particular, gammadelta T cells are unconventional T cells that recognize phosphoantigens (pAg), metabolites of the mevalonate pathway produced by pathogens such as Mtb (Chen, et al. 2013) and in response to stress-like infections in eukaryotic cells (Harly, et al. 2012) and appear to be an attractive target for immunotherapy in infectious diseases (Gay, et al. 2022).

[0004] Gamma delta T cells, particularly gamma9delta2 T cells, have been studied as an anti-tuberculosis component in vitro and in vivo using non-human primate (NHP) models. The in vitro anti-mycobacterial effector properties (cytotoxicity through perforin and granulysin, granzymes and pro-inflammatory molecules…) and recognition of Mtb infected cells of Vdelta2 T cells have been demonstrated using cells from healthy donors (Das, et al. 2001; Dieli, et al. 2000; Dieli, et al. 2001; Spencer, et al. 2013; Yang, et al. 2019). However, a loss of gamma9delta2 T cells has been observed in the blood and BAL (bronchoalveolar lavage) of active TB patients with severe disease, with reduced T EM and T EMRA frequencies and reduced function (Dieli, et al. 2002; El Daker, et al. 2013; Gioia, et al. 2002; Meraviglia, et al. 2010; Szereday, et al. 2003), suggesting an inhibitory local environment in the lung. Reduced frequencies of Vgamma9Vdelta2 T cell receptor (TCR) sequences have also been described in lung resections of human TB infections (Kulicke, et al. 2020). In the NHP model of tuberculosis, pAg-induced expansion, transendothelial migration and anti-bacterial function of Vgamma9Vdelta2 T cells in Mtb infected cynomolgus monkeys were associated with reduced TB burden (Huang, et al. 2008; Shen, et al. 2002). Moreover, such studies allowed the evaluation of the impact of gamma9delta2 T cell expansion on TB pathology, which was often proven to be beneficial (Chen, et al. 2013; Chen 2016). In particular, using IL-2 and pAg HMBPP, it was shown that Vgamma9Vdelta2 T cells were able to inhibit IL2-mediated expansion of Tregs and restore the suppression of Mtb-specific T cell responses upon pAg stimulation in BCG infected cynomolgus monkeys (Gong, et al. 2009). Adoptive transfer of Vdelta2 T cells was proven to be therapeutically effective in the cynomolgus monkey model of tuberculosis (Qaqish, et al. 2017), which led to a clinical pilot study in MDR tuberculosis patients to evaluate allogeneic Vgamma9Vdelta2 T cell therapy in a clinical trial (Liang, et al. 2021).

[0005] WO2012 / 080351 reports BTN3A-activating antibodies, such as murine mAb 7.2 or mAb 20.1, which have the ability to induce proliferation and cytokine secretion of Vγ9Vδ2 T cells.

[0006] WO2020 / 025703 further reports specific humanized BTN3A-activating antibodies, in particular their use for treating cancer disorders.

[0007] WO2020 / 136218 also discloses fragments derived from the Fab fragment of the anti- BTN3A antibody mAb 103.2 and their use as BTN3A-activating antibodies for inducing proliferation and cytokine secretion of Vγ9Vδ2 T cells.

[0008] However, to the best of the inventors' knowledge, there is no evidence of a reasonable use of an activating compound of Vγ9Vδ2 T cells, in particular a BTN3A-activating antibody, for treating MDR bacterial infectious disorders, in particular MDR bacterial infectious disorders caused by Mtb infection. SUMMARY

[0009] A first aspect of the present disclosure relates to a BTN3A-activating antibody for use in the treatment of a multi-drug resistant bacterial infectious disorder, preferably selected from a multi-drug resistant Mycobacterium tuberculosis infection, in a human subject in need thereof.

[0010] Another aspect relates to a method for treating a multi-drug resistant bacterial infectious disorder in a human subject in need thereof, said method comprising administering a therapeutically effective amount of a BTN3A-activating antibody as disclosed herein, such as mAb3, optionally in combination with a standard of care therapy such as rifampicin, isoniazid, ethambutol and pyrazinamide (HRZE).

[0011] In particular embodiments, the BTN3A-activating antibody has one or more of the following properties: (i) an EC50of 50 pg / ml or less, preferably 10 pg / ml or less, as measured in a flow cytometry assay; 50 binds human PBMCs; (ii) an EC50of less than 5 pg / ml, preferably 1 pg / ml or less, as measured in a degranulation assay in co-culture with cells expressing BTN3A; 50 induces activation of gd T cells, typically Vγ9Vδ2 T cells, in vitro; (iii) induces activation of gd T cells, typically Vγ9Vδ2 T cells, in vitro in co-culture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by a CFU (colony forming unit) assay; or (iv) induces in vivo activation of γδ T cells, typically Vγ9Vδ2 T cells.

[0012] In vivo activation of γδ T cells, typically Vγ9Vδ2 T cells, can be reflected by one or more of: (i) a decrease in the number of circulating γδ T cells, typically Vγ9Vδ2 T cells, following injection of an anti-BTN3A antibody; (ii) an increase in the expression of an activation marker, in particular CD69, on circulating γδ T cells following injection of an anti-BTN3A antibody; or (iii) an increase in the amount of pro-inflammatory cytokines, in particular IL-6, IFNg, IL-15, MIP-1b, MCP-1, G-CSF, in the circulation following injection of an anti-BTN3A antibody.

[0013] The induction of in vivo activation of γδ T cells is typically measured in a subject, for example following injection of an anti-BTN3A antibody at a unit dose of 0.1 mg to 1 g, in particular 1 mg to 200 mg.

[0014] In particular embodiments, the BTN3A-activating antibody comprises HCDR1-3 of SEQ ID NO: 5-7 and LCDR1-3 of SEQ ID NO: 8-10. In particular embodiments, the BTN3A-activating antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6 or 35-38, and HCDR3 of SEQ ID NO: 7, and LCDR1 of SEQ ID NO: 8 or 39 or 40, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10, or a variant of said HCDRs and LCDRs having 1, 2, or 3 amino acid substitutions, deletions, or insertions in the CDR sequences.

[0015] In particular embodiments, the BTN3A-activating antibody: (i) comprises HCDR1-3 of SEQ ID NO: 11-13 and LCDR1-3 of SEQ ID NO: 14-16; (ii) comprises a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, and a variable light chain (VL) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2; (iii) comprises a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3, and a variable light chain (VL) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4; (iv) competes for binding with the mAb3 antibody having a heavy chain of SEQ ID NO: 23 and a light chain of SEQ ID NO: 24.

[0016] In preferred embodiments, the BTN3A-activating antibody comprises a variable heavy chain VH of SEQ ID NO: 1 and a light chain VL of SEQ ID NO: 2, more preferably a heavy chain of SEQ ID NO: 23 and a light chain of SEQ ID NO: 24.

[0017] In specific embodiments, the infectious condition is caused by a multi-drug resistant strain, preferably Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Enterococcus (Enterococcus faecium and Enterococcus faecalis).

[0018] In preferred embodiments, the infectious condition is tuberculosis caused by a multi-drug resistant Mycobacterium tuberculosis. In specific embodiments, the method of treatment increases the efficacy of standard-of-care treatment and shortens the duration of standard-of-care treatment. In specific embodiments, the method of treatment reduces the risk of relapse after treatment interruption. In specific embodiments, the method of treatment prevents progression from latent tuberculosis to active tuberculosis.

[0019] In specific embodiments, the BTN3A-activating antibody is administered to a subject in need thereof by intravenous infusion, preferably at a unit dose of 0.1 mg to 1 g or 0.5 mg to 1 g, for example at a dose of 0.1 mg to 1 g or 5 mg to 200 mg, for example 2 to 6 times every 2 to 4 weeks.

[0020] Other specific aspects and specific embodiments are disclosed infra. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 PK-PD study design. Pharmacokinetic and pharmacodynamic parameters were evaluated in 4 cynomolgus monkeys. In particular, lung distribution and proper activation of g9d2 T cells were analyzed.

[0022] Figure 2PK-PD study. Frequency and absolute counts of circulating Vγ9Vδ2 T cells in blood of treated animals decrease after mAb3 treatment. Dashed lines indicate mAb3 administration. (A) Frequency of total T cells, CD4+ and CD8+ αβ T cells, Vδ1 and Vδ2 T cells, and NK cells in the CD45+ leukocyte population. (B) Absolute counts of each population calculated with WBC counts measured in the hematology parameters and percentage of each subpopulation within the CD45+ leukocyte population.

[0023] Figure 3 PK-PD study. In vivo activation of circulating Vγ9Vδ2 T cells by mAb3, e.g. increase in frequency of CD69+ cells in the Vγ9Vδ2 T cell population. mAb3 administration at dO and d14 (dashed lines).

[0024] Figure 4 PK-PD study. In vivo activation of circulating Vγ9Vδ2 T cells by mAb3, e.g. production of inflammatory cytokines and chemokines in vivo. Dashed lines indicate mAb3 administration.

[0025] Figure 5 PK-PD study. Serum concentration of mAb3 in treated healthy animals. mAb3 administration at dO and d14.

[0026] Figure 6 Study design and readouts to evaluate the therapeutic efficacy of mAb3 as a single agent.

[0027] Figure 7 Administration of mAb3 reduces lung lesions in Mtb-infected cynomolgus monkeys. The study was performed as described in Figure 6 (A) PET-CT images of lung granulomas (white spots) measured at baseline (before infection), 4 WPI (before mAb3 treatment), 8 WPI (after 1st and before 2nd mAb3 administration) and 12 WPI (after 3rd and before 4th mAb3 administration). (B) Quantification of lung granuloma volume measured in (A). Dashed lines indicate mAb3 administration.

[0028] Figure 8 Frequency and absolute counts of circulating VγVδ2 T cells in blood of Mtb-infected animals decrease after mAb3 treatment. Vertical interrupted lines indicate infection, vertical dashed lines indicate mAb3 administration. (Upper panel) Frequency of Vδ2 T cells in the total T cell population. (Lower panel) Frequency of CD69+ cells in the Vγ9Vδ2 T cell population.

[0029] Figure 9Study design to evaluate the therapeutic efficacy of mAb3 in combination with current standard of care in a NHP model of tuberculosis. Treatment efficacy of mAb3 and current standard of care (HZRE) will be evaluated in MTB infected NHPs. If the first round is successful, a second group of animals will be included to strengthen the results. Finally, relapse studies can be considered in this particular clinical setting. DETAILED DESCRIPTION

[0030] Definitions For the purposes of the present disclosure, certain terms are defined first. Additional definitions are set forth throughout the detailed description of the application.

[0031] The terms "polypeptide," "protein," or "peptide" as used herein refer to any chain of amino acid residues, regardless of length or post-translational modification (e.g., glycosylation).

[0032] The term "BTN3A" as used herein has its general meaning in the art. In particular embodiments, it refers to a human BTN3A polypeptide, including BTN3A1 of SEQ ID NO: 32, BTN3A2 of SEQ ID NO: 33, or BTN3A3 of SEQ ID NO: 34.

[0033] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen- binding site that immunospecifically binds an antigen. The terms "antibody" or "immunoglobulin" have the same meaning and are used equivalently in the present 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., a ligand for another antibody or receptor) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, an antibody can be derivatized or linked to more than one other functional molecule to produce a multispecific molecule that binds to more than two different binding sites and / or target molecules; the term "bispecific molecule" as used herein also includes such multispecific molecules. To produce a bispecific molecule, an antibody of the present disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, an antibody fragment, a peptide or a binding mimetic, such that a bispecific molecule results. In addition, for embodiments in which the bispecific molecule is multispecific, such a molecule can include a third binding specificity in addition to the first and second target epitopes.

[0034] In natural antibodies of both 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 and kappa. There are five major heavy chain types (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for an antigen. The constant regions of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR).

[0035] The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and is composed of the variable portions of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining site is composed of residues from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) can participate in the antibody combining site, or influence the overall domain structure and thus the combining site. Complementarity determining regions or CDRs refer to the amino acid sequences of the variable region of a native Fv that together define the binding affinity and specificity of a native immunoglobulin combining site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically includes six CDRs, comprising the CDR sets from each of the heavy and light chain V regions. Framework regions (FRs) refer to the amino acid sequences that are interposed between the CDRs. Thus, the variable region of the light and heavy chains typically comprises 4 framework regions and 3 CDRs having the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0036] Residues in the variable domain of an antibody are typically numbered according to a system devised by Kabat et al. This system was described in Kabat et al., 1987, in *Sequences of Proteins of immunological interest, US Department of Health and Human Services, NIH, USA* (Kabat et al., 1992, hereinafter referred to as "Kabat et al."). This numbering system is used in this specification. Kabat residue nomenclature does not always directly correspond to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or more amino acids than the strict Kabat number, corresponding to truncation or insertion of structural components (whether framework or complementarity-determining regions (CDRs)) of the basic variable domain structure. For a given antibody, the correct Kabat number of the residues can be determined by aligning homologous residues in the antibody sequence to a "standard" Kabat numbered sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable region 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 variable region of the light chain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3).

[0037] Non-CDR regions of mammalian antibodies can be replaced by similar regions of allogeneic or xenoantibodies, while typically retaining the epitope specificity of the original antibody. This is most clearly demonstrated in the development and use of “humanized” antibodies, where non-human CDRs are covalently linked to human FR and / or Fc / pFc' regions to produce functional antibodies.

[0038] As used herein, “humanization” describes an antibody in which some, most, or all of the amino acids outside the CDR region are replaced by 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. Patents 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. U.S. Patents 5,585,089 and 5,693,761, as well as WO90 / 07861, also propose four possible criteria for designing humanized antibodies. The first proposal is, for the receptor, to use a framework from a specific human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or to use a common framework from many human antibodies. The second proposal is that if the amino acids in the human immunoglobulin framework are unusual, and the donor amino acid at that position is typical for human sequences, then the donor amino acid can be chosen instead of the receptor. The third proposal is to select donor amino acids instead of acceptor amino acids at positions immediately adjacent to the three CDRs in the humanized immunoglobulin chain. The fourth proposal is to use donor amino acid residues at frame positions, where the amino acid is predicted to have side-chain atoms within 3 Å of the CDR in the antibody's three-dimensional model and is predicted to interact with the CDR. The above methods are merely examples of some methods that can be used by those skilled in the art to prepare humanized antibodies. Other methods of antibody humanization are familiar to those skilled in the art. In some humanized forms of antibodies, some, most, or all of the amino acids outside the CDR region can be replaced by amino acids from human immunoglobulin molecules, but some, most, or all of the amino acids within one or more of the CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permitted, as long as they do not eliminate the antibody's ability to bind a given antigen. Suitable human immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. "Humanized" antibodies typically retain antigen specificity similar to the original antibody. However, certain humanization methods can be used to increase the affinity and / or specificity of antibody binding through “directed evolution” approaches, as described in Wuet al., Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

[0039] Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice with most 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 the references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified to have a functional loss in the production of endogenous (e.g., mouse) antibodies. The animals are further modified to contain all or part of the human germline immunoglobulin loci, such that immunization of these animals will result in the production of fully human antibodies against the target antigen. After immunizing these mice (e.g., Xeno mice (Abgenix), HuMAB mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma techniques. These monoclonal antibodies have the amino acid sequence of human immunoglobulins and therefore do not elicit a human anti-mouse antibody (HAMA) response when administered to humans.

[0040] There are also in vitro methods for selecting human antibodies from human antibody libraries. These include phage display technology (US Patent Nos. 5,565,332 and 5,573,905) or in vitro stimulation of human B cells (US Patent Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0041] As used herein, the term "antigen-binding fragment" (or simply "antibody fragment") of an antibody refers to the full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as the BTN3A protein as defined above) and its BTN3A activation properties. In specific embodiments, the BTN3A-activated antibody for treating infectious diseases disclosed herein is an antibody fragment, and more specifically includes any protein of the antigen-binding domain of the BTN3A-activated antibody disclosed herein. Antibody fragments well known in the art include: Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in a hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments (Ward et al., 1989 Nature 341:544-546), which consist of a VH domain, or any fusion protein containing such an antigen-binding fragment; and bivalent antibodies, which are small antibody fragments having two antigen-binding sites, the fragments comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between two domains on the same chain, the domain is forced to pair with a complementary domain of another chain, resulting in two antigen-binding sites. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked using a recombination approach via a synthetic adapter that allows them to be made into a single-chain protein, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" (also referred to herein as antibody fragment). More generally, the antibody fragments intended herein also encompass single-domain antibodies, which are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US Patent No. 6,248,516 B1). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for intact antibodies. Suitable antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and biantibodies.Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and production via recombinant host cells as described herein.

[0042] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody molecule with single-specificity. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity, possessing variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from fully synthetic sequences. The method of preparing monoclonal antibodies is independent of binding specificity.

[0043] "Recombinant antibody" is an antibody produced, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells; antibodies isolated from a recombinant antibody library; antibodies isolated from animals (e.g., mice) transgenic due to a human immunoglobulin gene; or antibodies produced, expressed, generated, or isolated in any other manner, wherein a specific immunoglobulin gene sequence (e.g., a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies. In some embodiments, the recombinant human antibody used according to this disclosure has the same amino acid sequence as the corresponding naturally occurring human antibody, but is structurally different from the said naturally occurring human antibody. For example, in some embodiments, the glycosylation pattern is different due to the recombinant production of the recombinant human antibody. In some embodiments, the recombinant human antibody is chemically modified by adding or subtracting at least one covalent chemical bond relative to the structure of a naturally occurring human antibody.

[0044] As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of antibodies that specifically bind to antigens other than BTN3A). However, isolated antibodies that specifically bind to BTN3A may be cross-reactive with other antigens, such as related BTN3A molecules from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0045] The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” in this document. The terms “anti-BTN3A antibody” or “BTN3A antibody” are used briefly in this document to mean “antibody that recognizes BTN3A”.

[0046] As used herein, the term "activating antibody" refers to an antibody capable of directly or indirectly inducing the immune function of effector cells. Specifically, as used herein, a BTN3A activating antibody possesses at least the ability to induce activation of γδ T cells (typically Vγ9Vδ2 T cells) co-cultured with cells expressing BTN3A, wherein EC 50 The concentration is below 5 μg / ml, preferably 1 μg / ml or lower, as measured in a degranulation assay (described in the examples below). Typically, the degranulation assay measures the activation effect of BTN3A antibody against the degranulation of γδ T cells in the Daudi Burkitt lymphoma cell line, for example by co-culturing such cells with a selected concentration of antibody at 37°C for 4 hours.

[0047] As used herein, the term "binding" in the context of antibody binding to a predetermined antigen or epitope (particularly BTN3A) generally refers to the binding of an antibody to a predetermined antigen or epitope (particularly BTN3A) when measured in a BIAcore instrument using, for example, surface plasmon resonance (SPR) technology, with an antigen in a typically soluble form as a ligand and an antibody as an analyte, corresponding to approximately 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller, approximately 10 -10 M or smaller, or about 10 -11 M or even smaller K D The binding affinity. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of many SPR assays routinely used for epitope sites of monoclonal antibodies. Typically, antibodies bind with K... D The K has an affinity for binding to the predetermined antigen. D K, compared to its binding to non-specific antigens that are not the same as or closely related to the intended antigen (e.g., BSA, casein), D At least 10 times lower, for example, at least 100 times lower, for example, at least 1,000 times lower, for example, at least 10,000 times lower, for example, at least 100,000 times lower. When the antibody's K... D When the antigen is very low (i.e., the antibody has high affinity), then its Kb for binding the antigen is... D Typically, it is more effective than K against non-specific antigens. D The binding is at least 10,000 times lower. If such binding is undetectable (e.g., using SPR technology in a BIAcore 3000 instrument, with a soluble form of antigen as a ligand and an antibody as an analyte), or is 100, 500, 1000, or greater than 1000 times lower than the binding detected by such antibody with an antigen or epitope having a different chemical structure or amino acid sequence, then the antibody is said to essentially not bind to the antigen or epitope.

[0048] In the context of antibodies, the term "affinity" refers to the strength of antibody-epitope binding.

[0049] The terms “Kon” or “Kass” (Ka) used in this article refer to the binding rate of a specific antibody-antigen interaction, while the terms “Kdis” (Kd) or “Koff” (Koff) refer to the dissociation rate of a specific antibody-antigen interaction.

[0050] The term "K" used in this article D "" refers to the equilibrium dissociation constant, which is determined by k off With k on The ratio (i.e., koff / kon) is obtained and expressed as molar concentration (M). D The value relates to the antibody concentration (the amount of antibody required for a specific experiment), therefore K D The lower the K value (lower concentration), the higher the antibody affinity. D The value can be determined using methods known in the art. K is used to determine mAb. D Preferred methods for determining the K value can be found in Harlow, et al., Antibodies: A Laboratory Manual, ColdSpring 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), all of which are incorporated herein by reference. Determination of the K value of the antibody DThe method involves using SPR, or using biosensor systems such as Biacore® (see also for details on affinity assessment Rich RL, DayYS, Morton TA, Myszka DG. High-resolution and high-throughput protocols formeasuring drug / human serum albumin interactions using BIACORE®. AnalBiochem. 2001 Sep 15;296(2):197-207) or Octet® systems. The Octet® platform is based on biolayer interferometry (BLI) technology. The principle of BLI technology is based on an optical interference pattern of white light reflected from two surfaces—an immobilized protein layer and an internal reference layer. The binding between the ligand immobilized on the surface of the biosensor tip and the analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of the biolayer, and when this shift is measured over a period of time and its magnitude is plotted as a function of time, a classic binding / dissociation curve is obtained. Real-time measurement of this interaction allows for monitoring of binding specificity, binding rate, dissociation rate, and concentration (see Abdiche et al. 2008). Affinity measurements are typically performed at 25 °C.

[0051] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope present 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 mononuclear cells (PBMCs), preferably having an EC50 concentration of less than 50 μg / ml, more preferably less than 10 μg / ml, as determined by flow cytometry as described in the examples. 50 In other embodiments, its K-type antigen binds to the recombinant polypeptide. D The value is 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less, as measured by the SPR measurement described in the examples.

[0052] Antibodies that "cross-react with antigens other than BTN3A" refer to those with a K+ concentration of 10 nM or less, 1 nM or less, or 100 pM or less. D Antibodies that bind to antigens other than BTN3A. Antibodies that "do not cross-react with a specific antigen" are intended to indicate K+ levels of 1 μM or higher, or 10 μM or higher. DAntibodies that bind to the antigen. In some embodiments, these antibodies, which do not cross-react with the antigen, exhibit substantially undetectable binding to these proteins in standard binding assays.

[0053] Specificity can also be expressed by, for example, a ratio of approximately 10:1, approximately 20:1, approximately 50:1, approximately 100:1, 10.000:1, or greater, of the affinity / affinity for binding to a specific antigen to the affinity / affinity for binding to other unrelated molecules (in this case, the specific antigen is the BTN3A peptide).

[0054] As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc.

[0055] As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence using preferred codons from a production cell or organism, typically a eukaryotic cell such as a Chinese hamster ovary (CHO) cell or a human cell. Optimized nucleotide sequences are engineered to fully or as much as possible preserve the amino acid sequence originally encoded by the starting nucleotide sequence. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as optimized.

[0056] The term "identity" used in this article regarding polypeptide sequences refers to the amino acid sequence identity between two molecules. When the same amino acid occupies a position in two molecules, the molecules are identical at that position. The identity between two polypeptides is a direct function of the number of identical positions. Typically, sequences are aligned to obtain the highest possible sequence match (including gaps if necessary). The percentage of identity between two sequences is a function of the number of shared identical positions (i.e., identity % = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap; the length of the gaps needs to be introduced for optimal alignment of the two sequences. As described below, sequence comparison and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms.

[0057] The percentage 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), which has been incorporated into the ALIGN program (version 2.0) using the PAM120 weighted residue table with a gap length penalty of 12 and a gap penalty of 4. Alternatively, publicly available techniques and widely available computer programs can be used, such as BLASTP, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990), or the algorithm of Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970), which has been incorporated into the GAP program in the GCG software package (Devereux et al., Nucleic Acids Res. 12:387, 1984, typically available at http: / / www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0058] Normally, but not necessarily, the amino acid substitutions relative to the reference peptide, such as the CDR region, are conserved amino acid substitutions.

[0059] As used herein, “conservative amino acid substitution” means that a given amino acid can be substituted with residues having similar physiological and chemical characteristics, such as replacing one aliphatic residue with another (e.g., replacing another aliphatic residue with Ile, Val, Leu, or Ala), or replacing one polar residue with another polar residue (e.g., between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conserved substitutions, such as substitutions of an entire region having similar hydrophobic characteristics, are known. Peptides containing conserved amino acid substitutions can be tested in any of the assays described herein to confirm retention of the desired activities, such as antigen-binding activity and specificity, of the native or reference peptide. Amino acids can be grouped according to the similarity of their side chain properties (AL Lehninger, in Biochemistry, second ed., pp. 73-75, WorthPublishers, New York (1975)): (1) Nonpolar: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Nonpolar: 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 grouped based on common side-chain properties: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitution replaces members of one of these categories with members of another. Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr is Trp; and / or Phe to Val, to Ile or to Leu.

[0060] The percentage of identity between two nucleotide amino acid sequences can also be determined using algorithms such as the BLASTN program for nucleic acid sequences, with a word length (W) of 11, an expected value (E) of 10, M=5, and N=4 as default values, and by comparing the two strands.

[0061] Other antibodies can be identified based on their ability to cross-compete with other antibodies of this disclosure in standard antigen binding assays such as ELISA binding assays (e.g., competitively inhibiting binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of the antibodies of this disclosure to a target demonstrates that the test antibody can compete with that antibody for binding to the target; according to a non-limiting theory, such an antibody can bind to the same or related (e.g., structurally similar or spatially proximate) epitopes on the target as the competing antibody. Therefore, another aspect of this disclosure provides antibodies that bind to and compete with the antibodies disclosed herein for the same antigen. As used herein, the antibody “competitively” binds when, in the presence of an equimolar concentration of the competitive antibody, it inhibits the target binding of the disclosed antibody or antigen-binding fragment by more than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0062] "Combination therapy," "co-administration," "combination administration," or "concomitant administration" refers to the combined administration of at least two therapeutic agents, wherein a first agent (typically a BTN3A-activating compound) and a second agent (e.g., one or more antibacterial agents) are administered simultaneously or separately at time intervals in the same subject in need, where these time intervals allow the combination partners to exhibit cooperative or synergistic effects for treating conditions such as multidrug-resistant tuberculosis or other conditions caused by multidrug-resistant bacterial agents. While these delivery methods are within the scope described herein, they are not intended to imply that the therapeutic agents must be administered simultaneously and / or formulated for joint delivery. BTN3A-activating antibodies disclosed herein, such as mAb3, can be administered simultaneously, before, or after one or more other additional therapies or therapeutic agents. The term also covers treatment regimens in which the agents do not necessarily take place via the same route of administration.

[0063] Activated BTN3A antibodies for use according to the present disclosure This disclosure relates to the use of BTN3A-activated antibodies in a method for treating an infectious condition in a subject in need, more specifically a condition caused by a multidrug-resistant bacterial infection (e.g., Mycobacterium tuberculosis).

[0064] In specific embodiments, the BTN3A activating antibody according to the present invention for use exhibits one or more of the following properties: (i) As measured by SPR, it has a K value of 10 nM or lower. D K is preferred to be 1 nM or lower. D Combined with BTN3A, for example as described in the embodiments; (ii) EC50 μg / ml or lower, preferably 10 μg / ml or lower, as measured in a flow cytometry assay. 50 Combined with human PBMCs, such as those described in the embodiments; and, (iii) As measured by a granulation assay, its concentration in co-culture with cells expressing BTN3A is below 5 μg / ml, preferably 1 μg / ml or lower at EC50 concentrations. 50 In vitro induction of γδ T cells, typically Vγ9Vδ2 T cells, is performed, for example, as described in the examples.

[0065] In some embodiments, the antibodies disclosed herein for use are chimeric, humanized, or human antibodies.

[0066] In a preferred embodiment of this disclosure, the BTN3A activating antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while having at least the same (or higher) affinity as the parent non-human antibody. More specifically, the BTN3A activating antibody is a humanized form of mouse antibody 20.1 or 7.2, disclosed in WO2012080351. In a specific embodiment, the humanized antibody comprises all six CDRs of the non-human antibody, such as the CDRs of mouse mAb 20.1 or 7.2, and a FR (or a portion thereof) derived from the mouse antibody sequence having one or more mutations to reduce immunogenicity.

[0067] The humanized antibody optionally further comprises at least a portion of the human constant region. Preferably, the recombinant antibody according to this disclosure is a humanized silencing antibody, typically a humanized silencing IgG1 or IgG4 antibody.

[0068] As used herein, the term "silent" antibody refers to an antibody that shows no or low FcγR binding and / or C1q binding when measured in a binding assay (such as that described in WO2020 / 025703). In one embodiment, the term "no or low FcγR and / or C1q binding" means that the silent antibody exhibits FcγR and / or C1q binding that is at least 50% lower, for example, less than 80%, than that observed with a corresponding antibody having a wild-type human IgG1 or IgG4 isotype.

[0069] Examples of BTN3A activating antibodies are described in the following paragraphs. In some embodiments, the BTN3A activating antibodies are selected from, for example, those described in International Patent Applications WO2012 / 080769; WO2012 / 080351 and WO2020 / 025703, WO2020 / 136218, WO2020 / 033923, WO2020 / 033926, WO2023 / 161457 and Dai et al, 2024, the contents of which are incorporated herein by reference in their entirety.

[0070] In some specific embodiments, the BTN3A activating antibody is selected from the humanized antibody described in WO2020 / 025703, or the humanized form of the BTN3A activating antibody described in WO2012 / 080769, WO2012 / 080351, WO2020 / 136218, WO2020 / 033923, WO2020 / 033926, WO2023 / 161457 and Dai et al, 2024.

[0071] In some implementations, the BTN3A antibody may be selected from the mouse mAb20.1 and mAb7.2 described in WO2012 / 080769 and WO2012 / 080351, or their humanized forms.

[0072] In some embodiments, the BTN3A activating antibody comprises any one of the six CDRs (VH CDR1 (also known as HCDR1), VH CDR2 (also known as HCDR2), VH CDR3 (also known as HCDR3), VL CDR1 (also known as LCDR1), VL CDR2 (also known as LCDR2), VL CDR3 (also known as LCDR3)) of antibody 20.1 or 7.2 as described in WO2012 / 080769 and WO2012 / 080351, or any one of mAb1-5 as described in WO2020 / 025703, or any one of the antibodies as described in WO2020 / 033923, WO2020 / 033926, WO2023 / 161457 and Dai et al, 2024.

[0073] In the specific implementation scheme, the BTN3A activating antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in Table 1 below: Table 1: CDR areas of mAb 20.1, mAb7.2 or mAb1-mAb6, or variants defined in WO2020 / 025703 or WO2023 / 161457, according to Kabat number.

[0074] In some embodiments, the antibodies disclosed herein for use contain six CDR regions that are 100% identical to the six CDR regions of mAb20.1 or mAb7.2 as described in Table 1, particularly mAb20.1.

[0075] Other antibodies disclosed herein include those with amino acids that have been mutated through amino acid deletion, insertion, or substitution, but which have at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% identity in the CDR regions compared to the six CDR regions of antibodies 20.1 or 7.2 described herein, particularly compared to the six CDR regions defined in Table 1.

[0076] The BTN3A activating antibodies disclosed herein for use 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, the BTN3A activating antibodies disclosed herein for use 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 described in Table 2 below: Table 2: Variable heavy and light chain amino acid sequences of mAb1-mAb6

[0077] The corresponding amino acid and nucleotide coding sequences for the constant isotype regions of IgG1 and IgG4 used to generate mAb1 to mAb6, and their mutant forms IgG1 L247F / L248E / P350S and IgG4 S241P / L248E, are well known in the art (Oganesyan et al., 2008; Reddy et al., 2000).

[0078] The C-terminal lysine found in IgG can be naturally cleaved, and this modification does not affect the properties of the antibody; therefore, this residue can be additionally omitted in the constructs of mAb1 to mAb6.

[0079] The full-length light and heavy chains and their coding sequences for the preparation of preferred humanized antibodies mAb 1 and mAb 3 for use in this disclosure are shown in Table 3 below.

[0080] Table 3: DNA coding sequences of mAb1 and mAb3

[0081] Functional variants of mAb20.1 and mAb7.2 for use according to the present disclosure Epitope mapping analysis indicated that reference mAb 20.1 binds to residues at positions 79, 83, and 88 of human BTN3A1 of SEQ ID NO: 32. Therefore, this disclosure covers the use of BTN3A antibodies that bind to epitopes containing amino acid residues located at positions 79 to 88 of SEQ ID NO: 32 and have one or more functional properties as previously defined and further mentioned below, particularly having one or more functional properties of reference mAb 20.1 or its humanized form mAb3.

[0082] As shown in the epitope mapping analysis, reference mAb 7.2 binds to residues at positions 73, 79, 83, 88, 90, and 93 of human BTN3A1 in SEQ ID NO: 32. Therefore, this disclosure covers epitopes that bind to amino acid residues located at positions 73 to 93 of SEQ ID NO: 32, and most specifically, epitopes that bind to amino acid residues located at positions 73, 79, 83, 88, 90, and 93 of SEQ ID NO: 32, and have one or more functional properties as previously defined and further mentioned below, particularly having one or more functional properties of reference mAb 7.2 or its humanized form mAb1.

[0083] In other embodiments, the functional variant antibody of this disclosure has a full-length heavy chain and light chain amino acid sequence; or a variable region heavy chain and light chain amino acid sequence; or all six CDR region amino acid sequences that are homologous to or more specifically identical to the corresponding amino acid sequence of any of the above-described reference antibody mAb 20.1 or mAb 7.2 or their humanized forms (mAb3 or mAb1, respectively), and wherein the functional variant antibody retains the desired functional properties of the reference antibody.

[0084] A functional variant of the reference mAb20.1 antibody, or its humanized form mAb3, or the reference antibody mAb7.2, or its humanized form mAb1, particularly having VH and VL, or any of the six CDR functional variants used in the case of the monoclonal antibodies of this disclosure, still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of its affinity (typically measured by K, as indicated by the SPR of the parent antibody (e.g., mAb3 or mAb1). D (Evaluation) and in some cases, this functional variant can be associated with higher affinity, selectivity and / or specificity compared to a reference antibody (e.g., mAb3 or mAb1).

[0085] Typically, when the reference antibody is mAb3 or mAb1, the desired functional properties of the reference antibody, or any exemplary reference antibody as disclosed herein, can be selected from the following group: (i) The specificity of BTN3A1, particularly its binding property to human BTN3A1, as measured by SPR assay; for example, as described in the examples; (ii) Activation of γδ T cells, typically Vγ9Vδ2 T cells, in vitro in co-culture with cells expressing BTN3A, as measured in a granulation assay, for example as described in the examples; (iii) Activation of γδ T cells, typically Vγ9Vδ2 T cells, in vitro in a co-culture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by CFU (colony forming unit) assay.

[0086] For example, the functional properties of a functional variant of reference mAb3 or 1 at points (i) to (iii) above are substantially equal to or superior to the corresponding functional properties of the corresponding reference antibody mAb3 or mAb1 as described above. In this document, substantially equal to means that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding functional properties of reference mAb3 or mAb1.

[0087] In a specific embodiment, the BTN3A activating antibody for use according to this disclosure is a functional variant of mAb20.1 or its humanized form mAb3, or mAb7.2 or its humanized form mAb1, having no more than 1, 2, 3 or 4 amino acid changes (including deletions, insertions or substitutions) in one or more CDRs compared with the CDR sequence of antibody 20.1 or 7.2, or more particularly with the CDR sequence of mAb20.1.

[0088] For example, this disclosure relates to a functional variant antibody of reference mAb20.1 or its humanized form mAb3, which comprises a variable heavy chain (V H ) and variable light chains (V L The sequence, wherein the CDR sequence, i.e., the 6 CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 and the corresponding CDR sequences of mAb20.1 or its humanized form mAb3 reference antibody as defined in SEQ ID NO: 5-10, share at least 60%, 70%, 90%, 95% or 100% sequence identity with the corresponding CDR sequences of the reference antibody, wherein the 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, as measured by SPR, particularly at 10 nM or lower K+. D Preferably with K 5 nM or lower D Or with 5nM or lower K D The property of binding to human BTN3A1, for example as described in the embodiments; (ii) EC50 μg / ml or lower, preferably 10 μg / ml or lower, as measured in flow cytometry analysis. 50 Integrating with human PBMCs, for example as described in the embodiments; (iii) As measured in the threshing assay, in co-culture with cells expressing BTN3A, at EC50 concentrations of less than 5 μg / ml, preferably 1 μg / ml or lower. 50 In vitro induction of γδ T cells, typically Vγ9Vδ2 T cells, is performed, for example, as described in the examples.

[0089] Most preferably, it exhibits properties i) to iii).

[0090] This disclosure also relates to functional variant antibodies of reference mAb7.2 or its humanized form mAb1, which contain a variable heavy chain (V H ) and variable light chains (V L The sequence, wherein the CDR sequence, i.e., the 6 CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 share at least 60%, 70%, 90%, 95%, or 100% sequence identity with the corresponding CDR sequences of the mAb7.2 or mAb1 reference antibody as defined in SEQ ID NO: 11-16, wherein the functional variant antibody specifically binds to BTN3A1, and the antibody exhibits at least one of the following functional properties: (i) Specificity for BTN3A1, as measured by SPR, particularly at 10 nM or lower K+. DPreferably with K 5 nM or lower D Or with 5nM or lower K D The property of binding to human BTN3A1, for example as described in the embodiments; (ii) EC50 μg / ml or lower, preferably 10 μg / ml or lower, as measured in flow cytometry analysis. 50 Integrating with human PBMCs, for example as described in the embodiments; (iii) As measured in the threshing assay, in co-culture with cells expressing BTN3A, at EC50 concentrations of less than 5 μg / ml, preferably 1 μg / ml or lower. 50 In vitro induction of γδ T cells, typically Vγ9Vδ2 T cells, is performed, for example, as described in the examples.

[0091] Preferably, the functional variant exhibits all functional activities from i) to iii).

[0092] It further relates to a functional variant antibody of the mAb3 reference antibody, comprising at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding heavy and light chain variable regions of the mAb3 reference antibody as defined in SEQ ID NO: 1 and 2, respectively; the functional variant antibody specifically binds to BTN3A and exhibits at least one of the following functional properties: (i) Specificity for BTN3A1, as measured by SPR, particularly at 10 nM or lower K+. D Preferably with K 5 nM or lower D Or with 5nM or lower K D The property of binding to human BTN3A1, for example as described in the embodiments; (ii) EC50 μg / ml or lower, preferably 10 μg / ml or lower, as measured in flow cytometry analysis. 50 Integrating with human PBMCs, for example as described in the embodiments; (iii) As measured in the threshing assay, in co-culture with cells expressing BTN3A, at EC50 concentrations of less than 5 μg / ml, preferably 1 μg / ml or lower. 50 In vitro induction of γδ T cells, typically Vγ9Vδ2 T cells, is performed, for example, as described in the examples.

[0093] Preferably, it exhibits properties i) to iii).

[0094] It further relates to a functional variant antibody of the mAb1 reference antibody, comprising at least 80%, 90%, or at least 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding heavy and light chain variable regions of the mAb1 reference antibody as defined in SEQ ID NO: 3 and 4, respectively; the functional variant antibody specifically binds to BTN3A and exhibits at least one of the following functional properties: (i) Specificity for BTN3A1, as measured by SPR, particularly at 10 nM or lower K+. D Preferably with K 5 nM or lower D Or with 5nM or lower K D The property of binding to human BTN3A1, for example as described in the embodiments; (ii) EC50 μg / ml or lower, preferably 10 μg / ml or lower, as measured in flow cytometry analysis. 50 Integrating with human PBMCs, for example as described in the embodiments; (iii) As measured in the threshing assay, in co-culture with cells expressing BTN3A, at EC50 concentrations of less than 5 μg / ml, preferably 1 μg / ml or lower. 50 In vitro induction of γδ T cells, typically Vγ9Vδ2 T cells, is performed, for example, as described in the examples.

[0095] In some implementations, the functional variants exhibit all the functional activities of i) to iii).

[0096] Generally, the functional properties of the reference mAb3 or mAb1 at points (i) to (iii) above are substantially equal to or better than the corresponding functional properties of the corresponding reference antibody mAb3 or mAb1 as described above. In this document, substantially equal to means that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding functional properties of the reference mAb3 or mAb1.

[0097] The sequence of a CDR variant may differ from that of the CDR of the parent antibody sequence by a majority of conserved substitutions; for example, at least 10 substitutions, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1, in the variant are conserved amino acid residue substitutions.

[0098] Functional variant antibodies with mutated amino acid sequences can be obtained by mutagenesis of the encoded nucleic acid molecule (e.g., site-directed mutagenesis or PCR-mediated mutagenesis), followed by testing the retained function of the encoded altered antibody (i.e., the function described above) using the functional assays described herein.

[0099] Antibodies that cross-compete with reference mAb3 or mAb1 Additional antibodies possessing similar advantageous properties to the reference mAb3 or reference mAb1 disclosed herein can be identified based on their ability to cross-compete with the reference mAb3 or mAb1 in a statistically significant manner (e.g., competitively inhibit their binding) in a standard BTN3A1 binding assay.

[0100] Antibodies can be screened first to test their binding affinity to BTN3A1, for example from a human recombinant antibody library using, for example, phage display technology or from transgenic mice immunized with BTN3A1 antigen that express antibodies expressing human variable regions, as typically evaluated in the examples (see Materials and Methods section).

[0101] In another embodiment, the antibody for use according to this disclosure comprises an antibody that binds to the same epitope as at least reference mAb3 or reference mAb1.

[0102] The ability of the test antibody to cross-compete with or inhibit the binding of the antibody of this disclosure to human BTN3A1 demonstrates that the test antibody can compete with the antibody for binding to human BTN3A1; according to the non-limiting theory, such an antibody can bind to the same or related (e.g., structurally similar or spatially adjacent) epitopes on human BTN3A1 with the competing antibody.

[0103] For example, the following test can be used to screen BTN3A1 antibodies for their ability to cross-compete with a mAb3 reference antibody and / or for their ability to bind to the same epitope as the reference antibody: BTN3A-KO cells transfected with human BTN3A1 (typically HEK293T) can be stained with a saturated concentration (e.g., 10 μg / mL) of the reference antibody mAb3. The competitive potential of different doses of the test BTN3A1 mAb against the mAb3 reference antibody can then be tested. The mAb that does indeed compete with the reference antibody will not recognize BTN3A1 in the presence of this reference antibody. The 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 then providing a reference antibody followed by a competing antibody.

[0104] Further testing and selection of antibodies that have favorable BTN3A activation properties compared to mAb3 or mAb1 can be conducted, particularly as detailed above.

[0105] In some embodiments, the antibody of this disclosure for use competitively binds to the aforementioned BTN3A antibody, particularly to antibodies selected from mAb 20.1 and mAb 7.2, which are available from, for example, one of the hybridomas obtainable with CNCM accessions I-4401 and I-4402 as described in WO2012 / 080769 and WO2012 / 080351, and mAb1-6 as described in WO 2020025 / 703. In a more specific embodiment, the antibody of this disclosure for use competitively binds to an antibody selected from mAb 20.1 produced from a hybridoma with CNCM accession number I-4401 and an antibody having the heavy chain of SEQ ID NO: 23 and the light chain of SEQ ID NO: 24.

[0106] Therefore, in one embodiment, this disclosure provides isolated antibodies for treating infectious conditions as disclosed herein, wherein said isolated antibody competitively binds to BTN3A1 with reference mAb3 or reference mAb1, and wherein said antibody has one or more of the following properties: (i) The specificity of BTN3A1, particularly its binding property to human BTN3A1, as measured by SPR assay; for example, as described in the examples; (ii) Activation of γδ T cells, typically Vγ9Vδ2 T cells, in vitro in co-culture with cells expressing BTN3A, as measured in a granulation assay, for example as described in the examples; (iii) Activation of γδ T cells, typically Vγ9Vδ2 T cells, in vitro in a co-culture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by CFU (colony forming unit) assay.

[0107] In specific implementations, the functional properties of the antibodies that compete with reference mAb3 or mAb1 for binding to BTN3A1, according to points (i) to (iii) above, are substantially equal to or superior to the corresponding functional properties of the reference antibody mAb1 or mAb3 as described above. In this document, "substantially equal" means that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the corresponding functional properties of reference mAb1 or mAb3.

[0108] In some implementations, the cross-blocking antibody or the antibody that competes with reference mAb1 or mAb3 for binding to BTN3A1 is a chimeric, humanized, or recombinant human antibody.

[0109] Framework or Fc engineering The BTN3A-activated antibody disclosed herein for use may include modifications to framework residues within VH and VL to reduce their immunogenicity.

[0110] In some specific embodiments, the antibody disclosed herein for use is a humanized monoclonal antibody of parental mouse antibody mAb 20.1, which includes at least the following amino acid mutations in the VH frame region (compared to the parental VH frame region): V5Q; V11L; K12V; V20L; R66K; M69L; T75S; M80I; E81Q; R83T; T87S; L108A; and includes at least the following amino acid mutations in the Vκ frame region (compared to the Vκ frame region): T5N; V15L; R18T; V19I; K39R; K42N; A43I; D70G; F73L; V104L.

[0111] In addition to modifications performed within the frame region, the antibodies disclosed herein can be engineered to include modifications within the Fc region, typically altering one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity.

[0112] Furthermore, the antibodies disclosed herein for use may be chemically modified (e.g., one or more chemical moieties may be linked to the antibody) or modified to alter their glycosylation to further modify one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[0113] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as the amino acid residue from position C226 or P230 to the carboxyl terminus of the IgG antibody, numbered according to the EU numbering system. The C-terminal lysine (K447 residue) of the Fc region may be removed, for example, during antibody production or purification, or its corresponding codon may be omitted in the recombinant construct. Therefore, antibody compositions of this disclosure may comprise antibody populations with all K447 residues removed, antibody populations with K447 residues not removed, and antibody populations consisting of mixtures of antibodies containing and without K447 residues.

[0114] In other embodiments, the Fc region is modified by modifying one or more amino acids to reduce the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or reduce the antibody's affinity for the Fcγ receptor. Antibodies with reduced effector function, particularly reduced ADCC, include silencing antibodies.

[0115] In some embodiments, the Fc domain of the IgG1 isotype is used. In some specific embodiments, mutant variants of the IgG1 Fc fragment are used, such as silent IgG1 Fc, which reduce or eliminate the ability of the fusion peptide to mediate ADCC and / or bind to the Fcγ receptor.

[0116] In some embodiments, the Fc domain of the IgG4 isotype is used. In some specific embodiments, mutant variants of the IgG4 Fc fragment are used, such as silent IgG4 Fc, which reduce or eliminate the ability of the fusion peptide to mediate ADCC and / or bind to the Fcγ receptor.

[0117] Silent effector functions can be acquired through mutations in the constant Fc region of an antibody and have been described in the art (Baudino et al., 2008; Strohl, 2009). Examples of silencing IgG1 antibodies include the triple mutant variant IgG1L247F L248E P350S. Examples of silencing IgG4 antibodies include the double mutant variant IgG4 S241P L248E.

[0118] In some implementations, the Fc domain is a silent Fc mutant that prevents glycosylation at position 314 of the Fc domain. For example, the Fc domain contains an amino acid substitution of asparagine at position 314. An example of such amino acid substitution is that N314 is replaced by glycine or alanine.

[0119] In other embodiments, the glycosylation of the antibody is modified. For example, non-glycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. This carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This glycosylation-free process can increase the antibody's affinity for the antigen. This method is described in more detail in U.S. Patents 5,714,350 and 6,350,861 to Co et al.

[0120] Another modification of the antibody intended for use according to this disclosure is PEGylation or HES hydration or related techniques. Antibodies can be PEGylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become linked to the antibody or antibody fragment. PEGylation can be carried out by acylation or alkylation reactions with reactive polyethylene glycol molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-PEG or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a non-glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this disclosure. See, for example, Nishimura et al. EP 0154316 and Ishikawa et al. EP 0401384.

[0121] In some embodiments, a C-terminal lysine residue typically present in the constant domain of the human IgG heavy chain is engineered to reduce heterogeneity due to cleavage of this residue, which is commonly observed during production or storage. This modification does not significantly alter the desired function of these antibodies while conferring stability benefits to these molecules.

[0122] Nucleic acid molecules encoding antibodies of the present disclosure This document also discloses nucleic acid molecules encoding BTN3A activating antibodies for use according to this disclosure. Examples of variable light and heavy chain nucleotide sequences are those encoding the variable light and heavy chain amino acid sequences of any of the exemplary BTN3A activating antibodies disclosed above, particularly mAb 7.2, mAb 20.1 and their humanized forms, such as mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6, some of which are readily derived from Tables 1 and 2 and use the genetic code and optionally take into account codon preferences depending on the host cell species.

[0123] This disclosure also relates to nucleic acid molecules derived from the latter sequence, which have been optimized for protein expression in mammalian cells such as the CHO cell line.

[0124] This article also discloses nucleic acid molecules that encode the humanized heavy chain of mAb 20.1 of SEQ ID NO: 23 and the humanized light chain of mAb 20.1 of SEQ ID NO: 24, respectively.

[0125] This article also discloses nucleic acid molecules SEQ ID NO: 28 and SEQ ID NO: 29, which encode humanized forms of mAb 20.1, VH and VL, respectively.

[0126] Nucleic acids can be present in intact cells, cell lysates, or in partially purified or substantially pure forms. When purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, using standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (Ausubel et al., 1988), nucleic acids are “isolated” or “made substantially pure.” The nucleic acids of this disclosure can 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.

[0127] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. Once DNA fragments encoding, for example, VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting 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 (e.g., VL and VH as defined in Table 2) are operatively linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked” as used in this context means that the two DNA fragments are functionally linked, for example, such that the amino acid sequences encoded by the two DNA fragments are kept within a frame, or that the protein is expressed under the control of a desired promoter.

[0128] By operatively ligating DNA encoding the VH region to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (Kabat et al., 1992), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions. In some embodiments, the heavy chain constant regions are selected from the IgG1 isotype, such as the human IgG1 isotype. In other embodiments, the heavy chain constant regions are selected from the IgG4 isotype, such as the human IgG4 isotype. For Fab fragment heavy chain genes, the DNA encoding the VH region can be operatively ligated to another DNA molecule encoding only the heavy chain CH1 constant region.

[0129] By operatively linking DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (Kabat et al., 1992), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be either a κ or λ constant region.

[0130] To generate the scFv gene, DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible adapter (e.g., the amino acid sequence (Gly4-Ser)3), so that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by a flexible adapter (Bird et al., 1988; Huston et al., 1988; McCafferty et al., 1990).

[0131] Methods for producing recombinant antibodies for use according to the present disclosure The antibodies disclosed herein can be generated in host cell transfected tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, 1985).

[0132] For example, to express an antibody or an antibody fragment thereof, DNA encoding a partial or full-length light and heavy chain can be obtained using standard molecular biology or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the target antibody), and the DNA can be inserted into an expression vector such that the gene is operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" refers to linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating antibody gene transcription and translation. An expression vector and expression control sequences compatible with the host cell used for expression are selected. The antibody light chain gene and 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 using standard methods (e.g., linking the antibody gene fragment to a complementary restriction site on the vector, or blunt-end ligation if no restriction site is present). By inserting the light and heavy chain variable regions of the antibodies described herein into expression vectors encoding the heavy and light chain constant regions of the desired isotype, they can be used to generate full-length antibody genes of any antibody isotype, such that the VH fragment is operatively linked to the CH fragment within the vector, and the VL fragment is operatively linked to the CL fragment within the vector. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that promotes the 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 within the N-terminal frame of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0133] In addition to the antibody chain gene, the recombinant expression vectors disclosed herein carry regulatory sequences that control the expression of the antibody chain gene in host cells. 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 gene. Such regulatory sequences are described, for example, in publications by Goeddel (Goeddel, 1990). Those skilled in the art will understand that the design of expression vectors, including the selection of regulatory sequences, can depend on factors such as the choice of host cells to be transformed and the desired protein expression level. Regulatory sequences for mammalian host cell expression include viral elements that guide high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP)), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or P-globin promoters, may be used. In addition, regulatory elements consist of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and long terminal repeats from human T-cell leukemia virus type 1 (Takebe et al., 1988).

[0134] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of this disclosure can carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selective marker genes. Selective marker genes facilitate the selection of host cells to which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017 to Axel et al.). For example, selective marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells to which the vector has been introduced. Selective marker genes include the dihydrofolate reductase (DHFR) gene (for dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0135] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. It is theoretically possible to express the antibodies of this disclosure in prokaryotic or eukaryotic host cells. The expression of the antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, is discussed because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0136] In one specific implementation, the cloning or expression vector according to this disclosure comprises one of the coding sequences of the heavy and light chains of either mAb 1 or mAb 3, which are operatively linked to a suitable promoter sequence.

[0137] Mammalian host cells used to express the recombinant antibodies of this disclosure include Chinese hamster ovaries (CHO cells), including dhfr-CHO cells (as described in Kaufman and Sharp, 1982) used in conjunction with DHFR selective labeling (as described in Urlaub and Chasin, 1980), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells, and SP2 cells, such as the GSCHO cell line along with GS Xceed TM 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 antibody expression within the host cell, and optionally by secreting the antibody into the culture medium in which the host cell is grown. The antibody can be recovered and purified from the culture medium, for example, using standard protein purification methods after its secretion (Shukla et al., 2007).

[0138] In one specific embodiment, the host cell of this disclosure is a host cell transfected with an expression vector having coding sequences suitable for expression of mAb1 or mAb3, respectively, which are operatively linked to a suitable promoter sequence.

[0139] For example, this disclosure relates to a host cell containing at least nucleic acids encoding VH and VL, respectively, of SEQ ID NO: 28 and 29.

[0140] The host cells can then be further cultured under suitable conditions for expressing and producing the antibodies of this disclosure, wherein the antibodies are selected from mAb1 or mAb3, respectively.

[0141] Alternatively, cell-free expression systems can be used to produce any mAb3 or mAb1. Methods for cell-free expression of proteins or antibodies have been generally described (Stech et al., 2017).

[0142] Pharmaceutical compositions On the other hand, this disclosure provides compositions for treating infections caused by multidrug-resistant bacteria as disclosed below, such as pharmaceutical compositions, comprising a BTN3A activating antibody formulated with a pharmaceutically acceptable carrier, particularly selected from the group consisting of: mAb 20.1, a BTN3A activating antibody having six CDRs of mAb 20.1 having SEQ ID NO: 5-10, a BTN3A activating antibody having VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, a BTN3A activating antibody as a humanized form of mAb 20.1, and mAb 3 having a heavy chain of SEQ ID NO: 23 and a light chain of SEQ ID NO: 24, and their antigen-binding moieties.

[0143] Such compositions may include one BTN3A activating antibody as described above or (e.g., a combination of two or more different) BTN3A activating antibodies.

[0144] Pharmaceutical compositions containing BTN3A activating antibodies can be formulated at various concentrations. For example, formulations may contain activated BTN3A activating antibodies at concentrations of 0.1 μM to 1 mM, more preferably 1 μM to 500 μM, 500 μM to 1 mM, 300 μM to 700 μM, 1 μM to 200 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 800 μM to 900 μM, or 900 μM to 1 mM. Typically, formulations contain BTN3A activating antibodies at concentrations of 300 μM to 700 μM.

[0145] The pharmaceutical composition can be formulated in a single unit dose of 0.1 mg to 1 g. For example, the formulation may contain an activated BTN3A antibody in doses of 0.5 mg to 1 g, more preferably 0.1 mg to 1 mg, 1 mg to 1 g, 1 mg to 5 mg, 5 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 500 mg, or 500 mg to 1 g. The activated BTN3A antibody may also be formulated in doses of 1 mg to 1 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 100 mg.

[0146] The pharmaceutical compositions disclosed herein may also include additional active therapeutic agents. For example, the pharmaceutical compositions may comprise the anti-BTN3A antibody of this disclosure, such as an antibody selected from: mAb 20.1, a BTN3A activating antibody having six CDRs of mAb20.1 having SEQ ID NO: 5-10, a BTN3A activating antibody having VH of SEQ ID NO: 1 and VL of SEQ ID NO: 2, a BTN3A activating antibody as a humanized form of mAb 20.1, and mAb3 having the heavy chain of SEQ ID NO: 23 and the light chain of SEQ ID NO: 24, or the antigen-binding portion thereof. Examples of such other active therapeutic agents that may be used are described in more detail below in the section concerning the use of the antibodies of this disclosure.

[0147] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, epidermal, oral, or intranasal administration (e.g., by injection, infusion, or nebulization). In one embodiment, the carrier should be suitable for subcutaneous or intravenous routes. Depending on the route of administration, the active compound, i.e., the BTN3A-activated antibody, can be coated with material to protect the compound from acids and other natural conditions that may inactivate it.

[0148] Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well 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 from the vial surface, etc.

[0149] The dosage form, route of administration, dosage, and regimen of a drug composition naturally depend on the condition to be treated, the severity of the disease, the patient's age, weight, sex, and inflammatory status.

[0150] The pharmaceutical compositions according to this disclosure can be formulated for application topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, or intraocularly, for example by injection, infusion, or nebulization.

[0151] Suitable formulations of solutions for antibody infusion or subcutaneous injection have been described in the art and are reviewed, for example, in Cui et al. (Drug Dev Ind Pharm 2017, 43(4): 519-530). In a preferred embodiment, the anti-BTN3A antibody is formulated for intravenous infusion as defined above.

[0152] To prepare a pharmaceutical composition, an effective amount of BTN3A-activated antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0153] Use of BTN3A-activating antibodies in the treatment of infectious conditions This disclosure relates to BTN3A-activating antibodies as described above, particularly specific BTN3A-activating antibodies, for the treatment of infectious diseases caused by multidrug-resistant bacteria, more specifically by multidrug-resistant Mycobacterium tuberculosis.

[0154] As used herein, the term “treat, treating, or treatment” means one or more of the following: (1) suppressing disease; for example, suppressing disease, condition, or disorder in an individual experiencing or exhibiting a pathology or symptomology of disease, condition, or disorder (i.e., preventing further development of the pathology and / or symptomology); and (2) improving disease; for example, improving disease, condition, or disorder in an individual experiencing or exhibiting a pathology or symptomology of disease, condition, or disorder (i.e., reversing the pathology and / or symptomology), such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with respect to the treatment of infectious conditions, the term “treatment” may mean preventing infection with an infectious agent, suppressing the replication of an infectious agent, reducing the severity of one or more symptoms associated with the infection, or eradicating the infectious agent. In the specific implementation plan, particularly for subjects with tuberculosis who are further treated with a combination of rifampin, isoniazid, ethambutol and pyrazinamide (HZRE treatment), the term "treatment" refers to shortening the duration of combination chemotherapy, reducing the risk of relapse after treatment interruption, and preventing the progression from latent tuberculosis to active tuberculosis.

[0155] Latent tuberculosis infection involves a state of sustained immune response to stimulation by Mycobacterium tuberculosis antigens, without clinical evidence of active tuberculosis.

[0156] Active tuberculosis refers to the disease occurring in people infected with Mycobacterium tuberculosis, characterized by signs or symptoms of active disease or both, and differs from latent tuberculosis infection that occurs without signs or symptoms of active disease. The active status of tuberculosis can be determined by, for example, clinical, imaging, or laboratory evidence.

[0157] In a preferred embodiment, the subject is a human subject.

[0158] The BTN3A activating antibody described above can be administered as the sole active ingredient, or in combination with, for example, an adjuvant, or in combination with other drugs such as cytokines, antiviral agents, antimicrobial agents, or anti-inflammatory agents, for the treatment or prevention of the aforementioned diseases.

[0159] For example, antibodies used for purposes as described above can be used in combination with cytokines, antiviral agents, antimicrobial agents, anti-inflammatory agents, therapeutic cells, and / or immune checkpoint inhibitors.

[0160] Examples of cytokines include those used to expand and / or activate Vγ9Vδ2 T cells in vivo, including but not limited to 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, mutation (e.g., amino acid deletion, substitution, or insertion) that may depend on polyethylene glycolation (e.g., conjugation with polyethylene glycol (PEG) chains), or combination with an activator (e.g., an IL15 / IL15Ra complex fused with IgG1Fc, wherein IL-15 is further mutated (asn72asp), which further increases biological activity, making such a 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).

[0161] The term "IL-2" has its general meaning 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.

[0162] The term "IL-15" has its general meaning and refers to human interleukin-15. Like IL-2, IL-15 binds to a complex consisting of the IL-2 / IL-15 receptor β chain (CD122) and a common γ chain (γ-C, CD132) and signals through it. IL-15 regulates the activation and proliferation of T and natural killer (NK) cells.

[0163] Therefore, the method of use as defined herein may include co-administration, such as simultaneous or sequential administration of a therapeutically effective amount of a BTN3A activating antibody and at least one second pharmaceutical substance, the second pharmaceutical substance being an antiviral or antibacterial, anti-inflammatory agent or cytokine, such as IL-2 or IL-15, or a cell therapy product (e.g., γδ T cells), as described above.

[0164] In specific implementation plans, antimicrobial agents are standard care treatments for infections caused by multidrug-resistant bacteria, such as combinations of rifampin, isoniazid, ethambutol, and pyrazinamide (HZRE).

[0165] In a specific implementation plan, the infectious disease is caused by MDR Staphylococcus aureus, MDR Escherichia coli, MDR Pseudomonas aeruginosa, MDR Klebsiella pneumoniae, MDR Streptococcus pneumoniae, or MDR Enterococcus (Enterococcus faecalis and Enterococcus faecium).

[0166] Magiorakos et al. (2012) described antimicrobial classes, including antimicrobial agents associated with the antimicrobial susceptibility of bacterial organisms, including Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae, and Pseudomonas aeruginosa. In some embodiments, multidrug-resistant bacterial infections are resistant to at least one agent from three or more antimicrobial classes.

[0167] In a preferred embodiment, the infectious disease is caused by multidrug-resistant Mycobacterium tuberculosis, particularly resistant to at least rifampin or isoniazid.

[0168] In a specific implementation, the BTN3A antibody treatment reduces Vγ9Vδ2 T cells in the blood of the treated subject, particularly by at least 25%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0169] Use of BTN3A-activating antibodies in the treatment of conditions caused by infection with multi-drug resistant Mycobacterium tuberculosis In specific implementations, this disclosure relates to BTN3A activating antibodies, particularly specific BTN3A activating antibodies as described above, for the treatment of conditions caused by multidrug-resistant Mycobacterium tuberculosis infection in subjects of need.

[0170] In a specific implementation, this disclosure relates to a method for treating a condition caused by MDR Mycobacterium tuberculosis infection in a subject in need, the method comprising administering a therapeutically effective amount of BTN3A activated antibody to the subject.

[0171] In a specific implementation, this disclosure relates to the use of BTN3A-activated antibody in the preparation of a medicament for treating conditions caused by MDR Mycobacterium tuberculosis infection in subjects of need.

[0172] In the specific implementation plan, subjects who meet this treatment criteria have been diagnosed with MDR Mycobacterium tuberculosis infection.

[0173] In a specific implementation scheme, the subjects are selected from those with active tuberculosis. Specifically, the BTN3A activating antibody delays or prevents the progression of tuberculosis in subjects with active tuberculosis. In particular, the BTN3A activating antibody can reduce the Mtb infection burden in subjects with active tuberculosis. The BTN3A activating antibody can also prevent the progression of active tuberculosis or reduce the clinical symptoms of active tuberculosis.

[0174] In particular, Mycobacterium tuberculosis infection is resistant to at least isoniazid and rifampin.

[0175] In the specific implementation plan, the subjects were selected from those at high risk of developing severe tuberculosis.

[0176] For example, people at high risk of developing tuberculosis are divided into two categories: (i) People who have recently been infected with Mycobacterium tuberculosis and people with medical conditions that weaken the immune system. People who have recently been infected with Mycobacterium tuberculosis include: those who have had close contact with people who have infectious tuberculosis; those who have moved to areas with a high incidence of tuberculosis in the world; children under the age of 5 who have tested positive for tuberculosis; and groups with high rates of tuberculosis transmission, such as the homeless, injection drug users, and people living with HIV; (ii) People who work or live with people at high risk of tuberculosis in facilities or institutions such as hospitals, homeless shelters, reformatories, nursing homes, and residences of people living with human immunodeficiency virus (HIV). People with medical conditions that weaken the immune system include infants and young children, people with HIV infection, substance abuse, silicosis, diabetes, severe kidney disease, low body weight, people who have received organ transplants, people with cancer, people who are receiving medical treatment such as corticosteroids or for autoimmune conditions such as rheumatoid arthritis or Crohn's disease (https: / / www.cdc.gov / tb / default.htm).

[0177] In a specific implementation, the antimicrobial agent can be used in combination with BTN3A-activated antibodies to treat the condition caused by multidrug-resistant Mycobacterium tuberculosis infection.

[0178] Examples of such antimicrobial agents include, but are not limited to, rifampin, isoniazid, ethambutol, pyrazinamide, levofloxacin, chloroquine, premani, linezolid, and moxifloxacin (https: / / apps.who.int / iris / rest / bitstreams / 1211676 / retrieve).

[0179] In a specific implementation, the antibacterial drug is HZRE treatment, a combination of rifampin, isoniazid, ethambutol, and pyrazinamide, such as as described at https: / / www.who.int / publications / i / item / 9789240065116.

[0180] The disclosure, which has been fully described, will now be further illustrated by the following examples, which are merely illustrative and do not imply further limitation.

[0181] Example Determination of the functional properties of anti-BTN3A activated antibodies 1. Determination of the binding affinity of BTN3A-activated antibody by SPR. Multicycle kinetic analysis of any BTN3A antibody candidate can be performed using a Biacore T200 instrument (serial number 1909913) running Biacore T200 evaluation software V2.0.1 (Uppsala, Sweden).

[0182] The purified antibodies were diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, each antibody was captured onto protein A at a density (RL) of ~146.5 RU (to obtain the theoretical value of RMax of ~50 RU). After capture, the surface was stabilized and then injected with BTN3A1 antigen (Sino Biological catalog number 15973-H098H). BTN3A1 was titrated at a two-fold dilution of 25 to 0.78 nM in 0.1% BSA / HBS-P+ (flow buffer). The binding phase was monitored for 400 seconds and the dissociation phase for 35 minutes (2100 seconds). Kinetic data were obtained using a flow rate of 50 μl / min to minimize any potential mass transfer effects. At the end of each cycle, the protein A surface was regenerated by two injections of 10 mM glycine-HCl pH 1.5. Two replicates were performed for each antibody test, one blank (without BTN3A1) and one analyte at a single concentration, to examine the stability of the surface and the analyte during kinetic cycling. The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. Additionally, a blank run was subtracted for each Fc to correct for any antigen-independent signal variations, such as drift. The sensor map was fitted using a one-to-one binding mathematical model with a global RMax parameter and no large signal (constant RI = 0RU).

[0183] 2. Determination of the binding of BTN3A-activated antibody to human PBMCs by flow cytometry. The BTN3A activating antibody according to this disclosure for use can be characterized for its binding to BTN3A expressed in human PBMCs isolated from healthy donor blood. PBMCs were isolated from white membrane cells using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs can be used fresh or frozen and stored at -80°C or in liquid nitrogen until needed.

[0184] 100 μl 1 x 10 6 Cells were transferred at a rate of 1 cell / ml into each well of a fresh U-bottom 96-well plate, and then the plate was centrifuged and the supernatant was discarded.

[0185] Prepare serially diluted antibody solutions in PBS and 2 mM EDTA, from 0.001 μg / ml to 150 μg / ml. Resuspend human PBMCs in 50 μl of the prepared diluted test antibody titration system.

[0186] After incubating in the dark at 4°C for 30 minutes, the plate was centrifuged and washed twice with 150 μl / well of PBS 2mM EDTA. The wells were then resuspended in 50 μl of a mixture consisting of goat anti-human antibody (PE-labeled) diluted 1 / 100 in PBS 2mM EDTA and live / dead pure IR diluted 1 / 500 in PBS 2mM EDTA.

[0187] After incubating in the dark at 4°C for 15 minutes, the plates were centrifuged and washed once with 150 μl / well PBS 2mM EDTA, followed by resuspending in 200 μl PBS 2mM EDTA. Cells were analyzed on a BD LSR Fortessa cytometer. Data were analyzed using Flowjo software (Version 10, Flowjo, LLC, Ashland, USA) (data not shown).

[0188] The same protocol can be used to test the binding of cynomolgus BTN3A expressed on cynomolgus monkey PBMCs to DaudiBurkitt lymphoma cell lines.

[0189] 3. In vitro functional efficacy: γδ T cell degranulation assay This assay involved measuring the activation effect of the BTN3A antibody on the degranulation of γδ-T cells against the Daudi Burkitt lymphoma cell line (Harly et al., 2012). γδ-T cells were expanded from healthy donor PBMCs by culturing them for 11–13 days with zoledronic acid (1 μM) and IL2 (200 IU / ml). IL2 was added on days 5, 8, and every 2 days thereafter. The percentage of γδ-T cells was determined at the start of culture, and culture time was evaluated by flow cytometry until at least 80% was reached. Frozen or fresh γδ-T cells were then used for a degranulation assay against the Daudi cell line (E:T ratio 1:1), wherein the cells were co-cultured at 37°C for 4 hours in the presence of 10 μg / ml mAb1 and mAb3 and their chimeric forms. Activation with PMA (20 ng / ml) plus lonomycin (1 μg / ml) served as a positive control for γδ-T cell degranulation, and culture medium alone served as a negative control. At the end of the 4-hour co-incubation, cells were analyzed by flow cytometry to evaluate the percentage of CD107a (LAMP-1, lysosome-associated membrane protein-1) + CD107b (LAMP-2) positive γδ-T cells. CD107 is mobilized to the cell surface after activation-induced granule exocytosis, therefore, the measurement of surface CD107 is a sensitive marker recently used to identify degranulated cytolytic T cells.

[0190] 4. In vitro functional activity: γδ T cells kill extracellular or intracellular Mtb bacteria (from Dieli et al, J Infect Dis, 2001 and Meraviglia S et al, I Immunol, 2010). As described above, expanded γ9δ2 T cells were co-cultured with Mtb-infected macrophages at different ratios at 37°C in the presence of different concentrations of mAb1 or mAb3 for 24 hours. Mtb-infected macrophages could be obtained by infecting primary monocyte-derived macrophages or THP-1 cells with Mtb H37Rv bacteria at an MOI of 10. The cells were then washed to remove bacilli released from the macrophages, lysed with saponins, and sonicated for 20 seconds to release intracellular bacteria, followed by mycobacterial viability measurement. Alternatively, expanded γ9δ2 T cells were co-cultured at 37°C with 2 × 10⁻⁶ Mtb H37Rv bacteria. 4 Bacteria (in 7H9 medium supplemented with albumin-glucose catalase) were cultured at 37°C for 24 hours with or without incremental concentrations of mAb1 or mAb3. For mycobacterial viability assessment, serial 10-fold dilutions of cell lysates were prepared in 7H9 broth and plated onto 7H10 agar plates. The plates were sealed in plastic and maintained at 37°C, and colony counts (CFU) were performed after 14–21 days.

[0191] Example 1: Humanization and Characterization of mAb 20.1 1. Description of the humanization strategy a. Composite human antibody TM Design of variable region sequences Structural models of the V region of mouse 7.2 and 20.1 antibodies were generated using Swiss PDB and analyzed to identify important "restriction" amino acids in the V region that may be essential for antibody binding properties. Most residues contained within the CDR (defined using Kabat and Chothia), along with many framework residues, were considered important. From the above analysis, composite human sequences of the 7.2 and 20.1 antibodies were generated.

[0192] b. CD4+ T cell epitope avoidance Based on structural analysis, iTope was used for simulation analysis of peptides binding to human MHC class II alleles. TM The technology (Perry et al., 2008) and TCED using known antibody sequence-associated T cell epitopes. TM (Bryson et al., 2010) Selection and analysis yielded a large set of preliminary sequence segments that could be used to generate humanized variants 7.2 and 20.1. Significantly non-human germline conjugates or TCEDs identified as human MHC class II were discarded. TM The sequence fragments with significant hit scores were selected. This produced a reduced set of fragments, which were then analyzed again as described above to ensure that the connections between the fragments did not contain potential T-cell epitopes. The selected sequence fragments were assembled into complete V-region sequences predicted to have no significant T-cell epitopes. Several heavy and light chain sequences were then selected for gene synthesis and expression in mammalian cells for mAb 7.2 and 20.1.

[0193] 2. Generation and preliminary characterization of humanized variants a. Construction of humanized variant plasmids Humanized variants 7.2 and 20.1 were synthesized and cloned into expression vector systems for human IgG4 (S241P, L248E) heavy and κ light chains using flanking restriction enzyme sites. All constructs were confirmed by sequencing.

[0194] b. Antibody expression Chimeric 7.2 and 20.1 (VH0 / Vκ0), two control combinations (VH0 / Vκ1, VH1 / Vκ0), and combinations of humanized heavy and light chains were transiently transfected into FreeStyle DNA using the MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA) derived from the corresponding endotoxin-free DNA. TMIn CHO-S (ThermoFisher, Loughborough, UK). Each antibody was transfected using an OC-400 processing unit. After cell recovery, cells were loaded at 3 × 10⁻⁶ cells / year. 6 Cells / mL were diluted in CD Opti-Cho medium (ThermoFisher, Loughborough, UK) containing 8 mM glutamine (ThermoFisher, Loughborough, UK) and 1× xanthine-thymidine (ThermoFisher, Loughborough, UK). 24 hours post-transfection, the culture temperature was lowered to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. The culture was fed daily with 3.6% (starting volume) of diet (2.5% CHO CD High-Efficiency Diet 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)). The titer of IgG supernatant was monitored by IgG ELISA, and the transfected material was cultured for up to 14 days before the supernatant was collected.

[0195] c. Select the humanized form of mAb3, mAb 20.1. Two humanized variants of mAb 20.1 were selected from the 20 humanized candidates for further characterization.

[0196] Table 4 below summarizes the comparative data between humanized forms of mouse parental antibody mAb 7.2, mAb 20.1 and mAb 20.1 with SEQ ID NO: 1 (VH) and SEQ ID NO: 2 (VL).

[0197] Table 4: Functional properties of humanized mAb 20.1 Example 2: Evidence for the use of activated BTN3A antibodies in the treatment of active multidrug-induced bacterial infections The inventors hypothesize that Vγ9Vδ2 T cells are important effectors against Mtb and potentially other MDR bacterial strains, and that they can be safely activated by mAb3 to control bacterial development.

[0199] For tuberculosis, it is hypothesized that the local lung environment during active tuberculosis is associated with reduced responsiveness and γ9δ2 T cell infiltration. Restoring and enhancing γ9δ2 T cell responses in the lungs using mAb3 host-directed therapy could help control tuberculosis pathology and infection. Indeed, mAb3 can be expected to trigger Mtb clearance by directly killing infected cells and through the local production of pro-inflammatory cytokines by γ9δ2 T cells, which in turn could reverse immunosuppressive mechanisms within tuberculous granulomas, enhance local recruitment of anti-mycobacterial effector cells, and strengthen their anti-mycobacterial activity.

[0200] As a single agent or in combination with standard care (HZRE: rifampin, isoniazid, ethambutol, and pyrazinamide), mAb3 can shorten chemotherapy duration, reduce the risk of relapse after treatment interruption, and prevent latent tuberculosis from progressing to active tuberculosis. Finally, mAb3 treatment should increase the cure rate, especially for rifampin-resistant and extensively resistant tuberculosis. This also applies to other MDR bacterial infections.

[0201] In vitro and in vivo demonstration of the concept of mAb3 therapeutic activity as a single agent or in combination with current standard of care in the TB capuchin monkey model Evaluation of mAb3 monotherapy activity in the capuchin monkey model of active tuberculosis · PK-PD study of mAb3: Cynomolgus monkey models of tuberculosis infection have been developed using bronchoalveolar lavage or nebulization, allowing for the exploration of disease heterogeneity observed in humans (Capuano, et al. 2003; Lin, et al. 2009; White et al., 2015; Sharpe et al., PLoS One, 2017). Pulmonary tuberculosis is a chronic lung infection characterized by granulomatous formation, in which the dynamics of the immune response are complex and heterogeneous.

[0202] Previous studies using pAg to stimulate Vγ9Vδ2 T cells prior to Mtb infection have shown significant reductions in clinical signs, disease burden, and CFU counts in cynomolgus monkey lesions (Gong, et al. 2009; Huang, et al. 2008; Shen, et al. 2002). However, in these studies, activation of Vγ9Vδ2 T cells alone did not appear to be sufficient to achieve a cure. The experiments described in the following paragraphs aim to demonstrate the therapeutic benefit of activating Vγ9Vδ2 T cells in infected cynomolgus monkeys.

[0203] Figure 1 Since mAb3 has never been injected into animals, PK-PD studies are needed to verify the dosimetry, half-life, and in vivo pharmacodynamic activity (i.e., activation of Vγ9Vδ2 T cells) of mAb3 in cynomolgus monkeys.

[0204] In this study, two doses of 0.3 and 10 mg / kg were evaluated in parallel. Two cynomolgus monkeys in each group received two intravenous (iv) infusions at a two-week interval between weeks 0 and 2. mAb3 concentrations in circulation and bronchoalveolar lavage (BAL) were measured using a universal ELISA, and Vγ9Vδ2 T cell activation was measured by mass cytometry at different time points after the IV infusion. Figure 2 ).

[0205] Thaw the treatment items at 4°C. Once thawed, gently shake the animal items to homogenize the solution before use. Dilute the stock solution of mAb3 (20 mg / mL) with Vanguard buffer (PBS 1X, 125 mM NaCl, 0.02% Tween 80, 25 mM L-His, and distilled water) to 0.2 mg / mL for 0.3 mg / kg treatment and 5 mg / mL for 10 mg / kg treatment. Fill syringes with the appropriate volume of treatment preparation based on the final data of animal body weight. Observe the animals 7 days a week. At each bleeding episode, perform a clinical examination and record body weight and rectal temperature. Collect whole blood in 1 mL EDTA tubes for complete blood count (CBC) and immunophenotypic analysis.

[0206] Serum was obtained by centrifuging dried test tubes at 1800g for 10 minutes at room temperature. The following cytokines in the serum were quantified according to the manufacturer's instructions using an NHP Milliplex (PCYTMG-40K-PX23, Millipore) and a Bioplex 200 analyzer (Bio-Rad): granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor (GM-CSF), IFN-γ, IL-10, IL-12 / 23 (p40), IL-13, IL-15, IL-17a, IL-18, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, MCP-1, MIP1α, MIP1β, TGF-α, TNF-α, VEGF, and soluble CD40 ligand. mAb3 was quantified in the serum samples by ELISA.

[0207] The following charts were used to analyze whole blood collected on EDTA tubes by flow cytometry:

[0208] Cells were obtained using a ZE5 cell counter (Biorad) after staining and erythrocyte lysis.

[0209] In both experimental groups, the first mAb3 injection induced a rapid and transient decrease in Vδ2 T cells on day 1 post-injection, returning to baseline by day 7 in the 10 mg / kg group, and in one animal by day 14. However, no effect on Vγ9Vδ2 T cell counts was observed after a second mAb3 treatment at 10 mg / kg, and a less significant effect was observed at 0.3 mg / kg. The reduction after injection suggests activation of these T cell subsets (…). Figure 3 ).

[0210] A rapid decrease in circulating Vδ1 γδ T cells was also observed after treatment with mAb3 in only the 0.3 mg / kg group. Subsequently, the number of cells increased in both groups on days 7 and 21 after treatment (7 days after each treatment), and returned to baseline on days 14 after the first injection and days 28 after the second injection.

[0211] NK cell expansion was observed after each injection, peaking at days 7 and 21 in both groups. A decrease in CD4+ T cell count was observed 24 hours after the first injection in the 0.3 mg / kg group, followed by stable expansion until the second injection, where a decrease was observed again. Following the second injection on day 15, a decrease in CD4+ T cell count was observed in both groups, subsequently returning to baseline counts, which remained stable until the end of the study. An increase in CD8+ T cell count was observed in all animals 7 days after the first injection. CD8+ T cell levels decreased in all animals 24 hours after the second injection and returned to day 15 levels. These data suggest that mAb3 activation of Vγ9Vδ2 T cells also induces bystander activation in all other circulating immune populations.

[0212] This was confirmed by evaluating the expression of activating markers, particularly CD69. Figure 4 24 hours after the first injection of two doses of mAb3, CD69 was significantly increased. This was less pronounced after the second injection. Other immune subsets were not significantly affected.

[0213] An increase in circulating pro-inflammatory IL-6, IFNg, IL-15, MIP-1b, and MCP-1 was observed in all animals following the first injection of both doses of mAb3. Figure 5 This indicates that Vγ9Vδ2 T cells are activated. G-CSF and IL1-RA were also induced.

[0214] The p-value of the antibodies in the treated animals was then determined by ELISA. After the first treatment, mAb3 was no longer detectable from day 7 in the 0.3 mg / kg group, while it was still detectable on day 14 in the 3 mg / kg group. Therapeutic activity of mAb3 as a single agent (study 1):Following the second treatment, mAb3 clearance was faster in the 0.3 mg / kg group than after the first injection, as mAb3 was undetectable 24 hours post-injection. The kinetics in the 3 mg / kg group were similar between the first and second injections. Cmax values ​​were comparable for both groups after the first and second injections.

[0215] This study is important for determining the appropriate treatment range and timing of mAb3 administration for efficacy studies.

[0216] Figure 6 The objective was to evaluate the ability of mAb3 to reduce the infection burden of Mtb in cynomolgus monkeys. 3 × 10⁻⁶ 5 Erdman strain CFU (colony-forming units) / mL was used to infect Mauritian cynomolgus macaques via aerosol nebulizer using CH technology (Sharpe, et al. 2017; White, et al. 2015). mAb3 treatment began 6 weeks post-infection, at which time the first pulmonary granuloma was visible via PET-CT imaging of the lungs. mAb3 was administered every two weeks. Two different doses, 0.3 and 3 mg / kg, were tested based on the PK / PD properties of the antibody. A group of untreated infected animals was also included. Figure 6 ).

[0217] The stock solution was diluted to obtain 6.5 × 10⁻⁶ in PBS-Tween 80 (0.05%). 5 Mtb inoculum was prepared using a bacterial suspension of CFU / mL, with a total volume of 7 mL per animal. Cynomolgus monkeys were individually exposed to Mtb for 10 minutes using a CH-technology sprayer. All imaging acquisitions were performed on a digital photon counting (DPC) PET-CT system (Vereos-Ingenuity, Philips) implemented in the BSL3 laboratory. For the imaging phase, animals were anesthetized via the IM route with a mixture of 5 mg / kg ketamine and 0.05 mg / kg metoprine, and placed in a supine position on a bear hugger (3M). Anesthesia was monitored using heart rate, oxygen saturation, carbon dioxide levels, and temperature. During imaging, anesthesia was maintained by intratracheal administration of 1–5% isoflurane and 100% oxygen. CT was performed 5 minutes prior to the PET scan for attenuation correction and anatomical localization. The CT detector used was collimated to 64 × 0.6 mm, with a tube voltage of 120 kV and an intensity of approximately 150 mA. Whole-body PET scans were performed 40 minutes after administration of 3 MBq / kg of [18F]-FDG via the saphenous vein (4-5 bed positions, 3 minutes / bed position).

[0218] Considering the number of granulomas measured by PET-CT, animals were randomized into three different groups at 4 weeks post-infection (WPI):

[0219] The stock solution of mAb3 (20 mg / mL) was diluted to 0.2 mg / mL with Vanguard buffer (PBS 1X, 125 mM NaCl, 0.02% Tween 80, 25 mM L-His, and distilled water) for treatment at a dose of 0.3 mg / kg and 2 mg / mL for treatment at a dose of 3 mg / kg. Starting from week 6, anti-BTN3A mAb3 antibody was administered via IV every two weeks at an infusion rate of 2 mL / min. Two animals were injected with Vanguard buffer.

[0220] Disease progression monitoring is based on clinical scores, PET-CT imaging, immune surveillance, and Vγ9Vδ2 T cell phenotype ( Figure 7 ).

[0221] The following cynomolgus monkeys were monitored for infection: - By analyzing lesion volume and PET / CT images taken at baseline and 4, 8, and 12 weeks post-infection (WPI) [ 18 F-FDG signal is used to evaluate the progression of lung lesions and inflammation.

[0222] Immunophenotypic analysis of whole blood collected on EDTA tubes at baseline and at 2, 4, 6, 8, 10 and 12 WPIs was performed by flow cytometry.

[0223] - After centrifuging the dried tubes (at 1800 rpm for 10 minutes at room temperature), serum was collected, aliquoted, and stored at -80°C until administration of anti-BTN3A (mAb3). Serum was collected at baseline and at 4, 6, 8, and 12 weeks post-treatment (WPI). Samples were taken at weeks 4, 6, 8, 10, and 12, before mAb3 administration and at tmax (30 minutes), 24 hours, 96 hours, and 168 hours after treatment.

[0224] - For bronchoalveolar lavage (BAL) collection at baseline and at 4, 6, 8, 10, and 12 WPI, animals were anesthetized with ketamine hydrochloride (Imalgene 1000, 10 mg / kg, intramuscular route). Animals were placed in the left lateral decubitus position. The epiglottis and larynx were examined using a laryngoscopy, and a 5% lidocaine spray was applied to the larynx. Two minutes after spray application, the animals were intubated using an endotracheal tube (COVETO ref: 706188). Pressure was applied inside the balloon using a 5 mL syringe. Intubation was confirmed by controlling respiration at the distal end of the tube. 30 mL of 0.9% NaCl was injected into the endotracheal tube using a syringe. A 50 mL conical tube (Falcon®) was quickly placed in front of the endotracheal tube. The cynomolgus monkey septum was compressed to collect the first fraction of the lavage fluid. The remaining saline was then very rapidly recovered directly from the endotracheal tube. BAL was maintained at 4°C until centrifuged at 500 g for 10 min at 4°C. BAL cells are used for immunophenotyping.

[0225] like Figure 8 As shown, infected animals treated with the vector developed pulmonary granulomas indicative of active Mtb infection. Two treated animals died during or shortly after the experiment. It is suspected that the disease progression stage in these two animals was too late when the first dose was administered. As measured by the volume occupied by the pulmonary granulomas, no active TB infection developed in the six animals treated with mAb3 (two in the 0.3 mg / kg group and two in the 3 mg / kg group). Of these four animals, three did develop very few small lesions, and one began to develop some granulomas at the end of the study. This suggests that mAb3 treatment can prevent the progression of active Mtb infection. The extent to which Mtb infection was eradicated in most treated animals in the MDR-Tb infection animal model was unexpected and unprecedented.

[0226] Phenotypic analysis of circulating cells showed that, following the first mAb3 injection, the frequency and count of Vγ9Vδ2 T cells were reduced in the blood of both groups of treated animals as early as 24 hours post-injection, indicating the activation of these cells. Evaluation of mAb3 in combination with standard of care chemotherapy (study 2): This was confirmed by the increased frequency of CD69+ Vγ9Vδ2 T cells remaining in the circulation. Following this decrease, the Vγ9Vδ2 T cells did not return to the circulation, indicating that these cells are constantly transported out of the circulation.

[0227] · Figure 9 The evaluation of mAb3 combined with HZRE chemotherapy is also relevant to clinical translation. Standard care HZRE chemotherapy in a tuberculosis NHP model has been shown to reproduce therapeutic efficacy and reduce relapse risk in patients with treatment interruption (Lin, et al. 2013; Sibley, et al. 2022). Administered via aerosol at a dose of 3 × 10⁻⁶.5 Cynomolgus monkeys were infected with CFU / mL of *Erdman* strain, and the same efficacy criteria (PET-CT imaging, clinical monitoring, immune monitoring, and Vγ9Vδ2 T cell phenotype) were used as in Study 1, which evaluated mAb3 as a single agent. Treatment strategies and protocols were defined based on the results obtained from studies evaluating the therapeutic efficacy of mAb3 as a single agent. As an example ( Characterization of NHP Vγ9Vδ2 T cell anti-tuberculosis activity mAb3 + HZRE treatment was initiated 8 weeks after infection and continued for 8 weeks, mimicking the treatment of active tuberculosis. NHPs were euthanized upon completion of treatment to evaluate lesion sterilization and drug penetration, as well as the immune response in the tissue. In cases of protocol success, a small number of NHPs were followed after treatment interruption until the human endpoint was reached to assess the ability of mAb3 to delay or prevent relapse (relapse study). Untreated infected animals were included as controls in this study. The dynamics and function of γ9δ2 T cells were longitudinally tracked as described in Study 1.

[0228] · ​ Due to the low frequency of Vγ9Vδ2 T cells in cynomolgus monkey blood, NHP PBMCs or cells from BAL were used. Classical intracellular cytokine staining (IFN-γ, TNF-α, IL17A) and surface staining for CD69 activation and degranulation assessment were performed using freshly isolated somatic cells and ex vivo expansion of γ9δ2 T cells. Essentially, prior to staining and collection, PBMCs were incubated for 6 hours with intact Mtb lysates, IPP, HMB-PP, or other pAg of interest in the presence or absence of mAb3.

[0229] Co-culture assays using expanded or purified Vγ9Vδ2 T cells and Mtb-infected macrophages were used to evaluate the intracellular killing effect of Mtb bacteria. Briefly, monocyte-derived macrophages were infected with Mtb for 4 hours and then co-cultured with autologous δ2+ T cells for 3 days. CFU counts were measured on 7H11 plates or a BD BACTEC MGIT (automated mycobacterial detection system). These assays were first established on Vγ9Vδ2 T cells expanded from healthy NHP using different pAg combinations with rIL2. The in vitro potency of mAb3 in increasing Vγ9Vδ2 T cell activation, cytokine production, degranulation, and intracellular mycobacterial killing was then evaluated in cells from uninfected and infected cynomolgus monkeys. These data support the fact that Vγ9Vδ2 T cells from infected animals still respond to mAb3 treatment.

[0230] The effects of HZRE on expanded or purified Vγ9Vδ2 T cells derived from human or cynomolgus monkey blood can also be measured. Viability, activation phenotype, and function (cytokine production and degranulation) are measured at different time points following in vitro treatment. Once the in vitro effects of HZRE are established, in vitro treatment with HZRE and mAb3 can be envisioned, and the same readouts as those used to assess the in vitro effects of mAb3 can be performed.

[0231] In vitro transformation of mAb3 from tuberculosis patient samples was supported. Vγ9Vδ2 T cells from healthy donors can be activated by different bacterial strains (Escherichia coli, Mtb, Staphylococcus aureus, Clostridium difficile) and further enhanced by BTN3A activation antibodies, as exemplified by increased expression of CD154, CD137, and IFNγ (unpublished data). The differences in Vδ2 T cell response to Mtb lysates, HMBPP or zoledronic acid, mAb3, BCG, and bacterial antigens (PPD, ESAT6 / CFP-10) were evaluated in healthy donors, patients with latent or disease-free tuberculosis, tuberculosis patients, and tuberculosis patients with antibiotic-resistant Mtb strains. Diagnosis of tuberculosis was based on IFNγ-release assays, clinical symptoms, positive cultures, and positive PCR. Blood from confirmed tuberculosis patients was obtained as treatment-naïve blood and reanalyzed after initiation of antimycobacterial therapy.

[0232] This longitudinal study assessed the stability of the reactivity patterns of Vγ9Vδ2 T cells in patients with tuberculosis (TB) over time and post-treatment. The ability of Vδ2 T cells from four groups to be activated by mAb3 in short-term and antigen-responsive γδ T cells differed in phenotype (differentiation markers, co-stimulatory / inhibitory receptors) or function (IFNγ, TNFα, granulolysin / perforin). Finally, Vδ2 T cells from the four groups were activated by mAb3 and classified according to CD154 / CD137 induction to observe whether their functional / specific profiles differed after in vitro cloning / expansion.

[0233] For short-term activation of Vδ2 T cells, PBMCs were activated over a period of 16–24 h using Mtb lysate, HMBPP or zoledronic acid, mAb3, BCG, and bacterial antigens (PPD, ESAT-6 / CFP-10). Subsequently, extensive immunophenotyping was performed using mass cytometry to assess the following markers:

[0234] Functional analysis of mAb3-activated and expanded γδ T cells involved short-term activation (o / n) of PBMCs from tuberculosis-infected (latent) and tuberculosis patients in the presence of mAb3. Activated (i.e., CD154+ and / or CD137+) Vδ2 T cells were FACS-sorted, cloned into 96-well plates (100 cells / well), and expanded with IL-2 for 2–3 weeks. Reactivity patterns of the expanded clones were tested after restimulation with Mtb lysate, HMBPP, mAb3, BCG, bacterial antigens (PPD, ESAT-6), and selected additional bacteria (Staphylococcus aureus, Clostridium difficile, Escherichia coli). The re-induction of CD137 / CD154, the proliferation and intracellular expression of IFN-γ, TNFα, and granulosin / perforin were analyzed.

[0235] BAL can be obtained from selected patients, and the reactivity patterns of BAL with blood γδ T cells can be compared, especially in cases already described in the literature.

[0236] Table 5 (mAb sequences and, in particular, CDR sequences according to Kabat):

[0237] References Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, and Struhl, K. (1988). Current Protocols in Molecular Biology (John Wiley&Sons). Baudino, L., Shinohara, Y., Nimmerjahn, F., Furukawa, J.-I., Nakata, M., Martínez-Soria, E., Petry, F., Ravetch, JV, Nishimura, S.-I., and Izui, S. (2008). J. Immunol. 181, 6664–6669. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman,B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S., and Whitlow, M. (1988).Science 242, 423–426. Goeddel, D.V. (1990). [1] Systems for heterologous gene expression.In Methods in Enzymology, (Academic Press), pp. 3–7. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotný,J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., and Crea, R.(1988). Proc. Natl. Acad. Sci. 85, 5879–5883. Kabat, E.A., Wu, T.T., Foeller, C., Perry, H.M., and Gottesman, K.S.(1992). Sequences of Proteins of Immunological Interest (DIANE Publishing). Kaufman, R.J., and Sharp, P.A. (1982). Mol. Cell. Biol. 2, 1304–1319. McCafferty, J., Griffiths, A.D., Winter, G., and Chiswell, D.J.(1990). Nature 348, 552–554. Morrison, S.L. (1985). Science 229, 1202–1207. Oganesyan, V., Gao, C., Shirinian, L., Wu, H., and Dall’Acqua, W.F.(2008). Acta Crystallogr. D Biol. Crystallogr. 64, 700–704. Perry, L.C.A., Jones, T.D., and Baker, M.P. (2008). Drugs RD 9, 385–396. Reddy, M.P., Kinney, C.A.S., Chaikin, M.A., Payne, A., Fishman-Lobell, J., Tsui, P., Monte, P.R.D., Doyle, M.L., Brigham-Burke, M.R.,Anderson, D., et al. (2000). J. Immunol. 164, 1925–1933. Remington, J.P., and Gennaro, A.R. (1995). Remington: the science andpractice of pharmacy (Easton, PA: Mack Publishing). Rijkers, G., T. Vervenne, and P. van der Pol. 2020. Cell. Mol.Immunol. 17:771–772. doi:10.1038 / s41423-020-0473-0. Stech, M., Nikolaeva, O., Thoring, L., Stöcklein, W.F.M., Wüstenhagen, D.A., Hust, M., Dübel, S., and Kubick, S. (2017). Sci. Rep. 7,12030. Strohl, W.R. (2009). Curr. Opin. Biotechnol. 20, 685–691. Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K.,Yoshida, M., and Arai, N. (1988). Mol. Cell. Biol. 8, 466–472. Urlaub, G., and Chasin, L.A. (1980). Proc. Natl. Acad. Sci. U. S. A.77, 4216–4220. Bharadwaj, A., et al. , 2022, Multidrug-Resistant Bacteria: TheirMechanism of Action and Prophylaxis. Biomed Res Int 2022:5419874. Bloom, D. E., et al., 2018, Antimicrobial resistance and the role ofvaccines. Proc Natl Acad Sci U S A 115(51):12868-12871. Bryson, C. J., et al. (2010). Prediction of immunogenicity oftherapeutic proteins: validity of computational tools. BioDrugs, 24(1), 1-8. Capuano, S. V., 3rd, et al., 2003, Experimental Mycobacteriumtuberculosis infection of cynomolgus macaques closely resembles the variousmanifestations of human M. tuberculosis infection. Infect Immun 71(10):5831-44. Catalano, A., et al., 2022, Multidrug Resistance (MDR): A WidespreadPhenomenon in Pharmacological Therapies. Molecules 27(3). Chen, C. Y., et al., 2013, Phosphoantigen / IL2 expansion anddifferentiation of Vgamma2Vdelta2 T cells increase resistance to tuberculosisin nonhuman primates. PLoS Pathog 9(8):e1003501. Chen, Z. W., 2016, Protective immune responses of majorVgamma2Vdelta2 T-cell subset in M. tuberculosis infection. Curr Opin Immunol42:105-112. Colson, A. R., et al., 2021 , Antimicrobial Resistance: Is HealthTechnology Assessment Part of the Solution or Part of the Problem? ValueHealth 24(12):1828-1834. Dai, W. et al., 2024, BSI-093, a best-in-class anti-BTN3A monoclonalantibody for cancer immunotherapy by activation of Vg9Vd2 T cells, Cancer Res(2024) 84 (6_Supplement): 5298. Das, H., et al., 2001, MICA engagement by human Vgamma2Vdelta2 Tcells enhances their antigen-dependent effector function. Immunity 15(1):83-93. Dieli, F., et al., 2002, Selective depression of interferon-gamma andgranulysin production with increase of proliferative response by Vgamma9 / Vdelta2 T cells in children with tuberculosis. J Infect Dis 186(12):1835-9. Dieli, F., et al., 2000 , Vgamma9 / Vdelta2 T lymphocytes reduce theviability of intracellular Mycobacterium tuberculosis. Eur J Immunol 30(5):1512-9. Dieli, F., et al., 2001 , Granulysin-dependent killing ofintracellular and extracellular Mycobacterium tuberculosis by Vgamma9 / Vdelta2T lymphocytes. J Infect Dis 184(8):1082-5. El Daker, S., et al., 2013 , An abnormal phenotype of lungVgamma9Vdelta2 T cells impairs their responsiveness in tuberculosis patients.Cell Immunol 282(2):106-12. Gay, L., et al., 2022 , Role of Vgamma9vdelta2 T lymphocytes ininfectious diseases. Front Immunol 13:928441. Gioia, C., et al., 2002, Lack of CD27-CD45RA-V gamma 9V delta 2+ Tcell effectors in immunocompromised hosts and during active pulmonarytuberculosis. J Immunol 168(3):1484-9. Gong, G., et al., 2009, Phosphoantigen-activated V gamma 2V delta 2 Tcells antagonize IL-2-induced CD4+CD25+Foxp3+ T regulatory cells inmycobacterial infection. Blood 113(4):837-45. Harly, C., et al., 2012, Key implication of CD277 / butyrophilin-3(BTN3A) in cellular stress sensing by a major human gammadelta T-cell subset.Blood 120(11):2269-79. Huang, D., et al., 2008 , Immune distribution and localization ofphosphoantigen-specific Vgamma2Vdelta2 T cells in lymphoid and nonlymphoidtissues in Mycobacterium tuberculosis infection. Infect Immun 76(1):426-36. Infectious Diseases Society of, America, et al., 2011, Combatingantimicrobial resistance: policy recommendations to save lives. Clin InfectDis 52 Suppl 5(Suppl 5):S397-428. Kauffman, K. D., et al., 2021, PD-1 blockade exacerbatesMycobacterium tuberculosis infection in rhesus macaques. Sci Immunol 6(55). Kulicke, C. A., D. A. Lewinsohn, and D. M. Lewinsohn, 2020 , Clonalenrichments of Vdelta2- gammadelta T cells in Mycobacterium tuberculosis-infected human lungs. J Clin Invest 130(1):68-70. Liang, J., et al., 2021, Allogeneic Vgamma9Vdelta2 T-Cell TherapyPromotes Pulmonary Lesion Repair: An Open-Label, Single-Arm Pilot Study inPatients With Multidrug-Resistant Tuberculosis. Front Immunol 12:756495. Lin, P. L., et al., 2013 , Radiologic Responses in CynomolgusMacaques for Assessing Tuberculosis Chemotherapy Regimens. Antimicrob AgentsChemother 57(9):4237-4244. Lin, P. L., et al., 2009, Quantitative comparison of active andlatent tuberculosis in the cynomolgus macaque model. Infect Immun 77(10):4631-42. Magiorakos, A. P., et al., 2012. Multidrug-resistant, extensivelydrug-resistant and pandrug-resistant bacteria: an international expertproposal for interim standard definitions for acquired resistance. Clinicalmicrobiology and infection, 18(3), 268-281. Meraviglia, S., et al., 2010 , Partial and ineffective activation ofV gamma 9V delta 2 T cells by Mycobacterium tuberculosis-infected dendriticcells. J Immunol 185(3):1770-6. Peloquin, C. A., and G. R. Davies, 2021 , The Treatment ofTuberculosis. Clin Pharmacol Ther 110(6):1455-1466. Picchi, H., et al., 2018 , Infectious complications associated withthe use of immune checkpoint inhibitors in oncology: reactivation oftuberculosis after anti PD-1 treatment. Clin Microbiol Infect 24(3):216-218. Qaqish, A., et al., 2017 , Adoptive Transfer of Phosphoantigen-Specific gammadelta T Cell Subset Attenuates Mycobacterium tuberculosisInfection in Nonhuman Primates. J Immunol 198(12):4753-4763. Sharpe, S. A., et al., 2017 , An aerosol challenge model oftuberculosis in Mauritian cynomolgus macaques. PLoS One 12(3):e0171906. Shen, Y., et al., 2002, Adaptive immune response of Vgamma2Vdelta2+ Tcells during mycobacterial infections. Science 295(5563):2255-8. Sibley, L., et al., 2022, Determination of the Pharmacokinetics andPharmacodynamics of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol in aCross-Over Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection.Pharmaceutics 14(12). Shukla, A. A., Hubbard, B., Tressel, T., Guhan, S.,&Low, D. (2007).Downstream processing of monoclonal antibodies—application of platformapproaches. Journal of Chromatography B, 848(1), 28-39. Spencer, C. T., et al., 2013, Granzyme A produced by gamma(9)delta(2)T cells induces human macrophages to inhibit growth of an intracellularpathogen. PLoS Pathog 9(1):e1003119. Szereday, L., Z. Baliko, and J. Szekeres-Bartho, 2003, Gamma / delta Tcell subsets in patients with active Mycobacterium tuberculosis infection andtuberculin anergy. Clin Exp Immunol 131(2):287-91. Tiberi, S., et al., 2018 , Tuberculosis: progress and advances indevelopment of new drugs, treatment regimens, and host-directed therapies.Lancet Infect Dis 18(7):e183-e198. White, A. D., et al., 2015, Evaluation of the Immunogenicity ofMycobacterium bovis BCG Delivered by Aerosol to the Lungs of Macaques. ClinVaccine Immunol 22(9):992-1003. Yang, R., et al., 2019, IL-12 Expands and Differentiates HumanVgamma2Vdelta2 T Effector Cells Producing Antimicrobial Cytokines andInhibiting Intracellular Mycobacterial Growth. Front Immunol 10:913.

Claims

1. BTN3A activated antibody for the treatment of multidrug-resistant bacterial infections in human subjects, preferably selected from multidrug-resistant Mycobacterium tuberculosis infections.

2. The BTN3A activating antibody for use according to claim 1, wherein the BTN3A activating antibody has one or more of the following properties: (i) As measured in a flow cytometry assay, it is at 50 μg / ml or less, preferably 10 μg / ml or less EC. 50 Combined with human PBMC; (ii) As measured by a granulation assay, its concentration in co-culture with cells expressing BTN3A is below 5 μg / ml, preferably 1 μg / ml or lower at EC50. 50 In vitro induction of γδ T cells, typically activation of Vγ9Vδ2 T cells; (iii) As measured by CFU (colony-forming unit) assay, it induces in vitro activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with Mtb-infected macrophages or Mtb bacterial suspension; or (iv) It induces the activation of circulating γδ T cells in vivo, typically Vγ9Vδ2 T cells.

3. The BTN3A activating antibody for use according to claim 1 or 2, wherein the BTN3A activating antibody comprises HCDR1 of SEQ ID NO: 5, HCDR2 of SEQ ID NO: 6 or 35-38 and HCDR3 of SEQ ID NO: 7, and LCDR1 of SEQ ID NO: 8, 39 or 40, LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10, preferably HCDR1-3 of SEQ ID NO: 5-7 and LCDR1-3 of SEQ ID NO: 8-10.

4. The BTN3A activating antibody for use according to any one of claims 1-3, wherein the BTN3A activating antibody is (i) Containing HCDR1-3 of SEQ ID NO: 11-13 and LCDR1-3 of SEQ ID NO: 14-16; (ii) comprising a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide, wherein the variable heavy chain (VH) polypeptide comprises at least about 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence identical to that of SEQ ID NO: 1, and the variable light chain (VL) polypeptide comprises at least about 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence identical to that of SEQ ID NO: 2; (iii) Containing a variable heavy chain (VH) polypeptide and a variable light chain (VL) polypeptide, wherein the variable heavy chain (VH) polypeptide contains at least about 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence as SEQ ID NO: 3, and the variable light chain (VL) polypeptide contains at least about 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence as SEQ ID NO:

4.

5. The BTN3A activating antibody for use according to any one of claims 1-4, wherein the BTN3A activating antibody comprises the variable heavy chain VH of SEQ ID NO: 1 and the light chain VL of SEQ ID NO:

2.

6. The BTN3A activating antibody for use according to any one of claims 1-5, wherein the BTN3A activating antibody comprises a mutated or chemically modified IgG1 constant region, wherein the mutated or chemically modified IgG1 constant region does not confer or confers reduced binding to the Fcγ receptor when compared with a corresponding antibody having a wild-type IgG1 isotype constant region, preferably the BTN3A activating antibody comprises a mutated IgG1 constant region, which is an IgG1 triple mutant Fc having mutations L247F, L248E and P350S.

7. The BTN3A activating antibody for use according to any one of claims 1-6, wherein the activated BTN3A antibody comprises the heavy chain of SEQ ID NO: 23 and the light chain of SEQ ID NO:

24.

8. The BTN3A activating antibody for use according to any one of claims 1-7, wherein the BTN3A activating antibody is combined with, concurrently with or sequentially administered with at least one second therapeutic substance selected from antiviral agents, antibacterial agents, anti-inflammatory agents, cytokines, therapeutic cells and / or immune checkpoint inhibitors.

9. The BTN3A activating antibody for use according to any one of claims 1-8, for treating symptoms caused by multidrug-resistant bacterial infection, preferably from Mycobacterium tuberculosis, wherein the BTN3A activating antibody is administered in combination with, simultaneously with, or separately from rifampin, isoniazid, ethambutol, and pyrazinamide.

10. The BTN3A activating antibody for use according to any one of claims 1-9, wherein the treatment reduces Vγ9Vδ2 T cells in the blood of the treated subject.

11. The BTN3A activating antibody for use according to any one of claims 1-10, for treating a condition caused by infection with multidrug-resistant Mycobacterium tuberculosis, wherein the treatment (i) reduces the risk of relapse after treatment interruption, (ii) increases the efficacy of standard care treatment, (iii) allows for a shorter duration of standard care treatment, and / or (iv) prevents progression from latent tuberculosis to active tuberculosis.

12. The BTN3A activating antibody for use according to any one of claims 1-11, for treating a condition caused by infection with multidrug-resistant Mycobacterium tuberculosis, wherein the subject is a human subject with active tuberculosis.

13. The BTN3A-activating antibody for use according to any one of claims 1-12, for the treatment of conditions caused by Mycobacterium tuberculosis infection resistant to at least isoniazid and rifampin.

14. The BTN3A activated antibody for use according to any one of claims 1-13, for the treatment of conditions caused by methicillin-resistant Staphylococcus aureus, MDR Escherichia coli, MDR Pseudomonas aeruginosa, carbapenem-resistant Klebsiella pneumoniae, MDR Streptococcus pneumoniae, and vancomycin-resistant enterococci (Enterococcus faecalis and Enterococcus faecium).

15. The BTN3A activating antibody for use according to any one of claims 1-14, wherein the BTN3A activating antibody is administered to a subject in need by intravenous infusion, preferably at a unit dose of 0.1 mg to 1 g, for example at a dose of 1 mg to 200 mg, preferably 2 to 6 times every 2 to 4 weeks.

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