EP4701733A1

EP4701733A1 - Methods for treating multidrug resistant bacterial infections

Info

Publication number: EP4701733A1
Application number: EP24723116.0A
Authority: EP
Inventors: Sophie AGAUGUE; Paul Frohna; Julien Lemaitre; Candie JOLY; Roger LE GRAND
Original assignee: Imcheck Therapeutics; Commissariat a lEnergie Atomique CEA; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Imcheck Therapeutics; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2023-04-27
Filing date: 2024-04-29
Publication date: 2026-03-04
Also published as: KR20260006622A; TW202506731A; AU2024260225A1; WO2024223943A1; CN121311243A

Abstract

The present disclosure relates to methods for treating multidrug resistant bacterial infectious disorders. In particular, the disclosure provides BTN3A activating antibodies, and their use in treating multidrug resistant bacterial infectious disorders in a human subject in need thereof, such as disorders caused by multidrug resistant Mycobacterium tuberculosis infections.

Description

METHODS FOR TREATING MULTIDRUG RESISTANT BACTERIAL INFECTIONS

BACKGROUND

Antimicrobial resistance is the capability of a microorganism to resist the action of the different antimicrobials. In this type of resistance, microbes can resist the medication that could once be successful against them (Colson, et al. 2021). When this resistance occurs to multiple drugs, it is known as multidrug resistance (MDR). There are different types of resistance mechanisms observed in microbes, like natural resistance in certain microbes against a particular antimicrobial, genetic mutation, 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 leading cause of community-acquired infections. The spread of MDR bacteria in society has resulted in an increase in morbidity, mortality, healthcare expenditure, and antibiotic use. Such overexploitation is responsible for the higher incidence of MDR and leads to the development of a vicious cycle. Resistance microbes are hard to treat, requiring alternative or higher doses of antimicrobials or lack/shortage of effective antimicrobials, adversely affecting countries at all levels of development. As per the statement of World Health Organization (WHO) and the Infectious Diseases Society of America (IDSA), MDR pathogens called ‘superbugs’ are one of the major public threats that yearly cause several million deaths global (Bloom, et al. 2018; Infectious Diseases Society of, et al. 2011). In 2021, WHO published the list of antibiotic-resistant pathogens (priority pathogens), especially highlighting the resistant gram-negative bacteria that pose maximum threat to human health (https://www.who.int/medicines/publications/WHO-PPL-

Short_Summary_25Feb-ET_NM_WHO.pdf). It has been estimated that if new novel drugs are not discovered or formulated, there would be no effective antibiotic available to treat these deadly resistant pathogens by 2050 (Bharadwaj, et al. 2022). Among those MDR bacterial strains, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Enterococci (Entorococcus faecium and Enterococcus Faecalis) and Mycobacterium tuberculosis are among the most frequently cited.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the leading causes of death worldwide with an increase incidence and death in the past two years. Globally, treatment success in patients with active tuberculosis range from 86% to 56%, mainly depending on the level of Mtb resistance to treatment (https://www.who.int/teams/global- tuberculosis-programme/tb-reports/global-tuberculosis-report-2022). In 2021 , 166 991 people were diagnosed with pulmonary tuberculosis with resistance to at least one antimicrobial agent resulting in less than 50% survival in these patients receiving WHO recommended treatment. Even new regimens such as BPaL (Bedaquiline, Pretonamid and Linezolid) effective in MDR- tuberculosis so far, are now associated with new bacterial resistances (Peloquin and Davies 2021). To overcome antimicrobial resistance, reduce treatment duration and achieve complete remission, novel therapeutic strategies and agents are urgently needed. Host- directed therapies represent innovative approaches to amplify host immune response and increase Mtb killing (Tiberi, et al. 2018). Immune checkpoint inhibitors, evaluated in different preclinical models of TB, could have the potential to restore efficient T cell responses against Mtb. Nevertheless, treatment with PD-1 blockade exacerbated tuberculosis in infected rhesus macaques and was associated with reactivation of tuberculosis in cancer patients (Kauffman, et al. 2021 ; Picchi, et al. 2018). Strategies activating and targeting more relevant immune cell populations might offer an alternative for tuberculosis immunotherapy. Especially, y952 T-cells are unconventional T cells recognizing phosphoantigens (pAg), metabolites of the mevalonate pathway produced by pathogens like Mtb (Chen, et al. 2013) and in eukaryotic cells in response to stress like infections (Harly, et al. 2012) and appearing as appealing targets for immunotherapy in infectious diseases (Gay, et al. 2022).

Arising as an anti-tuberculous component, y6 T cells and particularly y952 T-cells have been studied in vitro and in vivo using Non Human Primate (NHP) models. In vitro antimycobacterial effector properties of V<52 T cells (cytotoxicity through perforin and granulysin, granzyme and pro-inflammatory molecule production ...) and recognition of Mtb-infected cells have been shown 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, y952 T-cell loss in blood and BAL (BronchoAlveolar Lavage) of active TB patients with severe disease has been observed with a reduced frequency of T_EM and TEMRA and reduced functions (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. Decreased frequency of Vy9V62 T Cell Receptor (TCR) sequences within human TB-infected lung resections has also been described (Kulicke, et al. 2020). In an NHP model of tuberculosis, pAg-induced expansion, transendothelial migration and anti-bacterial function of Vy9V52 T cells in Mtb-infected macaques was associated with reduced TB burden (Huang, et al. 2008; Shen, et al. 2002). Furthermore, such studies allowed evaluation of the impact of y62 T cell expansion over TB pathology, which was regularly proved to be favorable (Chen, et al. 2013; Chen 2016). In particular, using IL-2 and the pAg HMBPP, it was shown that Vy9V52 T cells were able to inhibit IL2-mediated expansion of Tregs and to revert suppression of Mtb-specific T cell responses upon pAg stimulation in BCG-infected macaques (Gong, et al. 2009). Adoptive transfer of V52 T cells proved to be therapeutically efficient in a cynomolgus tuberculosis model (Qaqish, et al. 2017) which led to a clinical trial assessing allogeneic Vy9V62 T cell therapy in a clinical pilot study in MDR- tuberculosis patients (Liang, et al. 2021).

W02012/080351 reports BTN3A activating antibodies, such as murine mAb 7.2 or mAb 20.1 having the capacity to induce the proliferation and cytokine secretion of Vy9V52 T cells.

W02020/025703 further reports specific humanized BTN3A activating antibodies, in particular for their use in treating cancer disorders.

WO2020/136218 also discloses fragments derived from Fab fragment of an anti-BTN3A antibody mAb103.2 and their use as BTN3A activating antibody for inducing the proliferation and cytokine secretion of Vy9\/52 T cells.

However, to the knowledge of the inventors, there is no evidence of a plausible use of an activating compound of Vy9V52 T cells, and in particular, BTN3A activating antibodies, for treating MDR bacterial infectious disorders, in particular caused by Mtb infections.

SUMMARY

A first aspect of the present disclosure relates to a BTN3A activating antibody for use in treating multidrug resistant bacterial infectious disorders in a human subject in need thereof, preferably selected from multidrug resistant Mycobacterium tuberculosis infections.

Another aspect relates to methods for treating multidrug resistant bacterial infectious disorders in a human subject in need thereof, said method comprising administering a therapeutically efficient amount of BTN3A activating antibody, e.g. mAb3 as disclosed herein, optionally in combination with standard of care therapy, such as rifampicin, isoniazid, ethambutol and pyrazinamide (HRZE).

In specific embodiments, said BTN3A activating antibody has one or more of the following properties:

(i) it binds to human PBMCs with an EC50 of 50 pg/ml or below, preferably of 10 pg/ml or below, as measured in a flow cytometry assay; (ii) it induces in vitro the activation of y<5 T cells, typically Vy9V52 T cells, in co-culture with BTN3A expressing cells, with an EC50 below 5 pg/ml, preferably of 1 pg/ml or below, as measured with a degranulation assay;

(iii) it induces in vitro the activation of y6 T cells, typically Vy9V52 T cells, in coculture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by a CFU (Colony Forming Unit) assay; or

(iv) it induces in vivo the activation of y6 T cells, typically Vy9V<52 T cells.

Activation of y<5 T cells, typically Vy9V52 T cells, in vivo may be reflected by one or more of :

(i) a decrease of the count of circulating y6 T cells, typically Vy9V<52 T cells after injection of the anti-BTN3A antibody;

(ii) an increased expression of activation markers on circulating y6 T cells, in particular CD69, after injection of the anti-BTN3A antibody; or

(iii) an increased amount of pro-inflammatory cytokines in the circulation, in particular IL-6, IFNg, IL-15, MIP-1b, MCP-1 , G-CSF, after injection of the anti-BTN3A antibody.

Induction of the y<5 T cell activation in vivo is typically measured after injection of the anti- BTN3A antibody in a subject, for instance at a unit dose of 0.1 mg to 1g, in particular 1 mg to 200mg.

In specific embodiments, said BTN3A activating antibody comprises HCDRs1-3 of SEQ ID NO:5-7 and LCDRs1-3 of SEQ ID NO:8-10 In specific embodiments, said BTN3A activating antibody comprises HCDR1 of SEQ ID NO:5, HCDR2 of SEQ ID NO:6 or 35 to 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 variants of said HCDRs and LCDRs with 1 , 2 or 3 amino acid substitutions, deletions or insertions in the CDR sequence.

In specific embodiments, said BTN3A activating antibody either :

(i) comprises HCDRs1-3 of SEQ ID NO:11-13 and LCDRs1-3 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 of 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 of SEQ ID NO: 4;

(iv) competes for binding with mAb3 antibody having a heavy chain of SEQ ID NO:23 and a light chain of SEQ ID NO:24.

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

In specific embodiments, said infectious disorder is caused by multidrug resistant bacterial strains, preferentially Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Enterococci (Entorococcus faecium and Enterococcus Faecal is).

In preferred embodiments, said infectious disorder is tuberculosis caused by multidrug resistant Mycobacterium tuberculosis. In specific embodiments, said method of treatment increases the efficacy of standard of care treatment and shortens the duration of standard of care treatment. In specific embodiments, said method of treatment reduces the risk of relapse after treatment interruption. In specific embodiments, said method of treatment prevents progression from latent to active tuberculosis.

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

Other specific aspects and specific embodiments are disclosed hereafter.

LEGENDS OF THE FIGURES

Figure 1 : PK-PD study design. Pharmacokinetic and pharmacodynamic parameters are evaluated in 4 cynomolgus macaques. Especially, lung distribution and proper activation of y952 T cells are analyzed.

Figure 2 : PK-PD study. Decreased Frequency and Absolute Counts of circulating Vy9V62 T cells after mAb3 treatment in the blood of treated animals. Dosings of mAb3 are represented by dotted lines. (A) Frequency of total T cells, CD4+ and CD8+ a T cells, V61 and V62 T cells and NK cells within the CD45+ leukocyte population. (B) Absolute counts of each population were calculated with WBC count measured in the hematology parameters and percentage of each subset within the CD45+ leukocyte population.

Figure 3 : PK-PD study. In vivo activation of circulating Vy9V52 T cells by mAb3 as exemplified by an increased frequency of CD69+ cells within the Vy9V52T cell population. mAb3 dosings are performed at dO and d14 (dotted lines).

Figure 4 : PK-PD study. In vivo activation of circulating Vy9V52 T cells by mAb3 as exemplified by in vivo production of inflammatory cytokines and chemokines. mAb3 dosings are indicated by dotted lines.

Figure 5 : PK-PD study. Serie concentrations of mAb3 in treated healthy animals. mAb3 dosings have been performed at dO and d14.

Figure 6 : Study design and readouts to assess the therapeutic efficacy of mAb3 as a single agent.

Figure 7 : mAb3 administrations decrease lung lesions in Mtb-infected Cynomolgus macaques. Study was performed as described in Figure 6. (A) PET-CT images of lung granulomas (white dots) measured at baseline (before infection), at 4 WPI (before mAb3 treatment), at 8 WPI (after 1st mAb3 dosing and pre-2nd dosing) and at 12 WPI (after 3 mAb3 dosings and before the 4^th one). (B) quantification of the volume of lung granulomas measured in (A). mAb3 dosings are represented by dotted lines.

Figure 8 : Decreased Frequency and Absolute Counts of circulating Vy9V52 T cells after mAb3 treatment in the blood of Mtb-infected animals. Infection is represented by a broken vertical line, dosings of mAb3 are represented by vertical dotted lines, (top panels) Frequency of V<52 T cells within the total T cell population, (bottom panels) Frequency of CD69+ cells within the Vy9V52 T cell population.

Figure 9 : Study design to characterize the therapeutic efficacy of the combination of mAb3 with current standard of care in an NHP model of tuberculosis. Evaluation of the therapeutic efficacy of mAb3 and current standard of care (HZRE) will be initiated in MTB- infected NHP. If success in this first round, a second group of animals will be included to strengthen the results. Lastly, a relapse study could be considered in this particular clinical context. DETAILED DESCRIPTION

Definitions

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

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

As used herein, the term “BTN3A” has its general meaning in the art. In specific embodiments, it refers to human BTN3A polypeptides including either BTN3A1 of SEQ ID NO:32, BTN3A2 of SEQ ID NO:33 or BTN3A3 of SEQ ID NO:34.

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 term "antibody" or "immunoglobulin" have the same meaning and will be used equally in the present disclosure. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as 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, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the 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, antibody fragment, peptide or binding mimetic, such that a bispecific molecule result. Additionally, for the embodiment in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity, in addition to the first and second target epitope.

In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (A) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. In typical IgG antibodies, 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 both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate in the antibody binding site, or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding 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. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.

The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth 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, hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H- CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

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

As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules in order to reduce immunogenicity in human subject. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761 , and WO 90/07861 also propose four possible criteria which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid residue at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three-dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization. In some humanized forms of antibodies, some, most or all of the amino acids outside the CDR regions can be replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include lgG1 , lgG2, lgG3, lgG4, IgA and IgM molecules. A "humanized" antibody normally retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., Mol. Biol. 294:151 , 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591 ,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

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

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to full length or to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a BTN3A protein as above defined) as well as their BTN3A activating properties. In specific embodiments, a BTN3A activating antibody for use in treating infectious disorders as disclosed herein is an antibody fragment, and more particularly any protein including an antigen-binding domain of a BTN3A activating antibody as disclosed herein. Well known-antibody fragments comprise: a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain, or any fusion proteins comprising such antigen-binding fragments; a diabody, which refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., 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" of an antibody (also shortly named herein antibody fragment). More generally antibody fragments as herein intended also encompass single-domain antibodies that are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human singledomain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Well-suited antibody fragments include, but are not limited to, Fv, Fab, F(ab’)2, Fab’, dsFv, scFv, sc(Fv)2 and diabodies. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells as described herein.

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single specificity. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant for the binding specificity.

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

An "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of antibodies that specifically bind to other antigens than BTN3A). An isolated antibody that specifically binds to BTN3A may, however, have crossreactivity to other antigens, such as related BTN3A molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The phrases "an antibody recognizing an antigen" and "an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen”. The terms “an anti-BTN3A antibody” or “a BTN3A antibody” are also shortly used herein with the meaning of “an antibody recognizing BTN3A”.

As used herein, the term “activating antibody” refers to an antibody able to directly or indirectly induce immune functions of effector cells. In particular, as used herein, a BTN3A activating antibody has at least the capacity to induce the activation of yd T cells, typically Vy9V52 T cells, in co-culture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay (such degranulation assay is described in the Examples below). Typically, the degranulation assay measures activating effect of the BTN3A antibody on yd T cell degranulation against Daudi Burkitt's lymphoma cell line, e.g. by co-culturing such cells with a selected concentration of the antibody for 4 hours at 37°C.

As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope, notably BTN3A, means typically a binding with an affinity corresponding to a K_D of about 10’⁷ M or less, such as about 10'⁸ M or less, such as about 10’ ⁹ M or less, about 10'¹⁰ M or less, or about 10’¹¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore instrument using typically a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of SPR assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a Kothat is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1 ,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its K_D for binding to a non-specific antigen {e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen. An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, SPR technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

The term “affinity”, as used herein in the context of an antibody, means the strength of the binding of an antibody to an epitope.

The term "Kon" or "Kass" (Ka), as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdis" (Kd) or "Koff," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction.

The term "K_D", as used herein, is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k_Off to k_on (i.e. koff/kon) and is expressed as a molar concentration (M). The K_D value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the K_D value (lower concentration) and thus the higher the affinity of the antibody. K_D values for antibodies can be determined using methods well established in the art. Preferred methods for determining the K_D values of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. A method for determining the K_D of an antibody is by using SPR, or by using a biosensor system such as a Biacore® (see also for detailed information regarding affinity assessment Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE®. Anal Biochem. 2001 Sep 15;296(2): 197-207) or Octet® systems. The Octet® platform is based on bio-layer interferometry (BLI) technology. The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces - a layer of immobilized protein and an internal reference layer. The binding between a ligand immobilized on the biosensor tip surface and an 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 (AA) is a direct measure of the change in optical thickness of the biological layer, when this shift is measured over a period of time and its magnitude plotted as a function of time, a classic association/dissociation curve is obtained. This interaction is measured in real-time, allowing to monitor binding specificity, association rate and dissociation rate, and concentration, (see Abdiche et al. 2008). Affinity measurements are typically performed at 25 °C.

As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as BTN3A. In some embodiments, it is intended to refer to an antibody that binds to human BTN3A as expressed on peripheral blood mononuclear cells (PBMCs), preferably with an ECso below 50 pg/ml and more preferably below 10 pg/ml as determined by flow cytometry as described in the Examples. In other embodiments, it binds to an antigen recombinant polypeptide with a KD of 100nM or less, 10nM or less, 1nM or less, 100pM or less, or 10pM or less, as measured by SPR measurements as described in the Examples.

An antibody that "cross-reacts with an antigen other than BTN3A" is intended to refer to an antibody that binds that antigen other than BTN3A with a K_D of 10nM or less, 1 nM or less, or 100 pM or less. An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen, with a K_D of 1 M or greater, or a K_D of 10 pM or greater. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding against these proteins in standard binding assays.

Specificity can further be exhibited by, e.g., an about 10:1, about 20:1 , about 50:1 , about 100:1 , 10.000:1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is a BTN3A polypeptide).

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

As used herein, the term, "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence. The amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

Ther term “identity” as used herein in reference to polypeptide sequences, refers to the amino acid sequence identity between two molecules. When an amino acid position in both molecules is occupied by the same amino acid, then the molecules are identical at that position. The identity between two polypeptides is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained (including gaps if necessary). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using published techniques and widely available computer programs, such as BLASTP, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990), or the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm 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 either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

Generally, but not necessarily, it is preferable for amino acid substitutions relative to the reference polypeptide such as CDR regions to be conservative amino acid substitutions.

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

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

Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the disclosure in standard antigen binding assays such as an ELISA binding assay. The ability of a test antibody to inhibit the binding of antibodies of the present disclosure to the target demonstrates that the test antibody can compete with that antibody for binding to the target; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the target as the antibody with which it competes. Thus, another aspect of the disclosure provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein. As used herein, an antibody “competes” for binding when the competing antibody inhibits the target binding of an antibody or antigen binding fragment of the disclosure 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% in the presence of an equimolar concentration of competing antibody.

“combination therapy”, “co-administration”, “combined administration” or “concomitant administration” refers to a combined administration of at least two therapeutic agents, where a first agent, typically a BTN3A activating compound is administered at the same time or separately within time intervals, with a second agent, e.g. one or more anti-bacterial agents, in the same subject in need thereof, where these time intervals allow that the combined partners show a cooperative or synergistic effect for treating a disorder, e.g. multidrug resistant tuberculosis or other disorders caused by multidrug resistant bacterial infectious agents. It is not intended to imply that the therapeutic agents must be administered at the same time and/or formulated for delivery together although these methods of delivery are within the scope described herein. The BTN3A activating antibody, e.g. mAb3 as herein disclosed, can be administered concurrently with or prior to, or subsequent to one or more other additional therapies or therapeutic agents. The terms are also meant to encompass treatment regimens in which the agents are not necessarily administered by the same route of administration.

Activating BTN3A antibody for use of the present disclosure

The present disclosure relates to the use of a BTN3A activating antibody in methods for treating infectious disorders in a subject in need thereof, more specifically a disorder caused by multidrug resistant bacterial infection, e.g. Mycobacterium tuberculosis.

In specific embodiments, a BTN3A activating antibody for use according to the present disclosure exhibits one or more of the following properties:

(i) it binds to BTN3A with a KD of 10 nM or less, preferably with a KD of 1 nM or less, as measured by SPR, for example as described in the Examples;

(ii) it binds to human PBMCs with an ECso of 50 pg/ml or below, preferably of 10 pg/ml or below, as measured in a flow cytometry assay, for example as described in the Examples; and,

(iii) it induces in vitro the activation of yb T cells, typically Vy9V62 T cells, in coculture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured with a degranulation assay, for example as described in the Examples.

In some embodiments, antibodies for use of the present disclosure are chimeric, humanized, or human antibodies.

In preferred embodiments of the present disclosure, the BTN3A activating antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while having at least the same affinity (or superior affinity) as the parental non- human antibody. More particularly the BTN3A activating antibody is a humanized form of the murine antibodies 20.1, or 7.2, which murine antibodies are disclosed in W02012080351. In specific embodiments, a humanized antibody comprises all 6 CDRs, of non-human antibody, e.g., the CDRs of the murine mAb 20.1 or 7.2, and FRs (or portions thereof) derived from the murine antibody sequences with one or more mutations to reduce immunogenicity.

A humanized antibody optionally will also comprise at least a portion of a human constant region. Preferably, the recombinant antibody according to the disclosure is a humanized silent antibody, typically a humanized silent IgG 1 or lgG4 antibody. As used herein, the term “silent” antibody refers to an antibody that exhibits no or low FcyR binding and/or C1q binding as measured in binding assays such as those described in W02020/025703. In one embodiment, the term “no or low FcyR and/or C1q binding” means that the silent antibody exhibits an FcyR and/or C1q binding that is at least below 50%, for example below 80% of the FcyR and/or C1q binding that is observed with the corresponding antibody with wild type human I gG 1 or lgG4 isotype.

Examples of BTN3A activating antibodies are described in the paragraphs below. In some embodiments, the BTN3A activating antibody is selected from the group consisting of BTN3A activating antibodies such as those described in the International Patent Applications WO2012/080769; WO2012/080351, and WG2020/025703, WO2020/136218, WO 2020/033923, WO 2020/033926, WO2023/161457 and Dai et al, 2024, the content of which is herein entirely incorporated by reference.

In some particular embodiments, the BTN3A activating antibody is selected from the humanized antibodies described in W02020/025703 or is a humanized version of the BTN3A activating antibodies described in WO2012/080769, WO2012/080351 , WO2020/136218, WO 2020/033923, WO 2020/033926, WO2023/161457 and Dai et al, 2024.

In some embodiments, the BTN3A antibody can be selected from murine mAb 20.1, and mAb 7.2, which are described in W02012/080769 and WO2012/080351, or humanized versions thereof.

In some embodiments, the BTN3A activating antibody comprises the six CDRs (VH CDR1 (also called HCDR1), VH CDR2 (also called HCDR2), VH CDR3 (also called HCDR3), VL CDR1 (also called LCDR1), VL CDR2s (also called LCDR2), VL CDR3s (also called LCDR3)) of the antibody 20.1 , or 7.2 described in W02012/080769 and W02012/080351 or any of mAbs 1-5 as described in W02020/025703, or any of the antibodies described in WO 2020/033923, WO 2020/033926, WO2023/161457 and Dai et al, 2024.

In particular embodiments, the BTN3A activating antibody comprises HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as shown in Table 1 below: Table 1 : CDR regions of mAb 20.1, mAb 7.2, or mAb1 - mAb 6 or variants as defined in W02020/025703 or WO2023/161457, according to Kabat numbering.

In some embodiments, the antibodies for use as disclosed herein comprise 6 CDR regions which are respectively 100% identical to the 6 CDR regions of mAb 20.1, or mAb 7.2 as described in Table 1 , notably of mAb 20.1.

Other antibodies as disclosed herein include those having amino acids that have been mutated by amino acid deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity in the CDR regions as compared to the 6 CDR regions of the antibodies described herein, in particular antibodies 20.1 , or 7.2 described in W02012/080769 and W02012/080351 , notably as compared to the 6 CDR regions defined in Table 1.

BTN3A activating antibodies for use of the present disclosure include also those having at least 90%, notably at least, 95, 96, 97, 98, 99 or 100 % identity with the VH and VL regions as defined in Table 2. More particularly, BTN3A activating antibodies for use of the disclosure include the 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 the Table 2 below: Table 2: Variable heavy and light chain amino acid sequences of mAb1-mAb6

The corresponding amino acid and nucleotide coding sequence of the constant isotype regions of lgG1 , lgG4 and their mutant versions lgG1 L247F/L248E/P350S and lgG4 S241 P/L248E used for generating mAb1 to mAb6 are well-known in the art (Oganesyan et al., 2008; Reddy et al., 2000).

The C-terminal lysine found in IgG may be naturally cleaved off and this modification does not affect the properties of the antibody; so, this residue may additionally be deleted in the constructs of mAb1 to mAb6.

Full length light and heavy chains and coding sequences for making preferred humanized antibodies for use of the present disclosure, mAb1 and mAb3, are shown in the Table 3 below.

Table 3: DNA coding sequences for mAb1 and mAb3

Functional variants of mAb 20.1 and mAb 7.2, for use according to the present disclosure

Analysis of epitope mapping indicates that the reference mAb 20.1 binds residues on positions: 79, 83 and 88 of the human BTN3A1 of SEQ ID NO:32. Thus the present disclosure encompasses use of BTN3A antibodies that bind an epitope comprising amino acid residues located in positions 79 to 88 of SEQ ID N°32, and that have one or more of the functional properties as previously defined and as further reminded below, in particular that have one or more of the functional properties of the reference mAb 20.1 or its humanized form mAb3.

As also indicated by epitope mapping analysis, the reference mAb 7.2 binds residues positions: 73, 79, 83, 88, 90, 93 of the human BTN3A1 of SEQ ID NO:32. Thus the present disclosure encompasses BTN3A antibodies that bind an epitope comprising amino acid residues located in positions 73 to 93 of SEQ ID N°32, and most particularly, an epitope comprising amino acid residues on positions: 73, 79, 83, 88, 90 and 93 of SEQ ID N°32 and that have one or more of the functional properties as previously defined and as further reminded below, in particular that have one or more of the functional properties of the reference mAb 7.2 or its humanized form mAb1 .

In yet other embodiments, a functional variant antibody of the disclosure has full length heavy and light chain amino acid sequences; or variable region heavy and light chain amino acid sequences, or all 6 CDR regions amino acid sequences that are homologous or more specifically identical to the corresponding amino acid sequences of any one of the reference antibody mAb 20.1 or mAb 7.2 or their humanized forms (mAb3 or mAb1 respectively), described above, and wherein such functional variant antibodies retain the desired functional properties of said reference antibody.

A functional variant of the reference mAb 20.1 antibody, or its humanized form mAb3, or of the reference antibody mAb 7.2 or its humanized form mAb1 , notably a functional variant having VH and VL, or all 6 CDRs used in the context of a monoclonal antibody of the present disclosure still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity (typically assessed by KD as measured by SPR of the parent antibody (e.g.: mAb3 or mAb1)) and in some cases such a functional variant may be associated with greater affinity, selectivity and/or specificity than the reference antibody (e.g.: mAb3 or mAb1).

Desired functional properties of the reference antibody, typically when the reference antibody is mAb3 or mAb1 , or of any example reference antibody as herein disclosed, may be selected from the group consisting of:

(i) specificity for BTN3A1 , in particular the property of binding to human BTN3A1 as measured by SPR assay; for example as described in the Examples;

(ii) in vitro induction of the activation of y<5 T cells, typically Vy9V52 T cells, in coculture with BTN3A expressing cells, as measured in a degranulation assay, for example as described in the Examples; (iii) in vitro induction of the activation of yd T cells, typically Vy9Vd2 T cells, in coculture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by a CFU (Colony Forming Unit) assay.

For example, functional properties according to points (i) to (iii) above of a functional variant of the reference mAb3 or 1 are substantially equal or superior to the corresponding functional properties of the corresponding reference antibody mAb3 or mAb1 as described above. By substantially equal it is herein intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95%, 96 %, 97 %, 98 %, 99 % or 100% of the corresponding functional property of the reference mAb3 or mAb1.

In specific embodiments, the BTN3A activating antibodies for use according to the present disclosure are functional variants of mAb 20.1 or its humanized form of mAb3, or mAb 7.2 or its humanized form of mAb1 , having not more than 1 , 2, 3 or 4 amino acid variations (including deletion, insertion, or substitution) in one or more CDRs, as compared to the CDR sequences of the antibodies 20.1, or 7.2 respectively or, more particularly as compared to the CDR sequences of mAb 20.1.

For example, the present disclosure relates to functional variant antibodies of the reference mAb 20.1 or its humanized form mAb3, comprising a variable heavy chain ( H) and a variable light chain (V ) sequences where the CDR sequences, i.e., the 6 CDR regions; HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, LCDR3 share at least 60, 70, 90, 95 or 100 percent sequence identity to the corresponding CDR sequences of mAb 20.1 or its humanized form mAb3 reference antibody, as defined in SEQ ID NO:5-10, wherein said functional variant antibody specifically binds to BTN3A, and the antibody exhibits at least one of the following functional properties i) to iii):

(i) specificity for BTN3A1 , in particular the property of binding to human BTN3A1 with a KD of 10 nM or less, preferably with a KD of 5 nM or less, or with a KD of 5 nM or less, as measured by SPR for example as described in the Examples;

(ii) binding to human PBMCs with an ECso of 50 pg/ml or below, preferably of 10 pg/ml or below, as measured in a flow cytometry assay, for example as described in the Examples;

(iii) in vitro induction of the activation of yd T cells, typically Vy9Vd2 T cells, in coculture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay, for example as described in the Examples. Most preferably it exhibits properties i) to iii).

The present disclosure also relates to functional variant antibodies of the reference mAb 7.2 or its humanized form mAb1 , comprising a variable heavy chain (VH) and a variable light chain (V ) sequences where the CDR sequences, i.e., the 6 CDR regions; HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, LCDR3 share at least 60, 70, 90, 95 or 100 percent sequence identity to the corresponding CDR sequences of mAb 7.2 or the mAb1 reference antibody, as defined in SEQ ID NO: 11-16, wherein said functional variant antibody specifically binds to BTN3A1, and the antibody exhibits at least one of the following functional properties:

(iii) in vitro induction of the activation of yb T cells, typically Vy9V<52 T cells, in coculture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay, for example as described in the Examples.

Preferably said functional variants exhibit all functional activities i) to iii).

It further relates to functional variant antibodies of the mAb3 reference antibody comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99% or 100% identical to the corresponding heavy and light chain variable regions of said mAb3 reference antibody, as defined respectively in SEQ ID NO: 1 and 2; the functional variant antibody specifically binds to BTN3A, and exhibits at least one of the following functional properties:

(i) specificity for BTN3A1 , in particular the property of binding to human BTN3A1 with a K_D of 10 nM or less, preferably with a K_D of 5 nM or less, or with a K_D of 5 nM or less, as measured by SPR for example as described in the Examples;

(ii) binding to human PBMCs with an ECso of 50 pg/ml or below, preferably of 10 pg/ml or below, as measured in a flow cytometry assay, for example as described in the Examples; (iii) in vitro induction of the activation of yb T cells, typically Vy9V52 T cells, in coculture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay, for example as described in the Examples.

Preferably it exhibits properties i) to iii).

It further relates to functional variant antibodies of the mAb1 reference antibody comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95, 96%, 97%, 98%, 99% or 100% identical to the corresponding heavy and light chain variable regions of said mAb1 reference antibody, as defined respectively in SEQ ID NO: 3 and 4; the functional variant antibody specifically binds to BTN3A, and exhibits at least one of the following functional properties:

(iii) in vitro induction of the activation of yb T cells, typically Vy9Vb2 T cells, in coculture with BTN3A expressing cells, with an EC50 below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay, for example as described in the Examples.

In some embodiments, said functional variants exhibit all functional activities i) to iii).

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

The sequences of CDR variants may differ from the sequence of the CDRs of the parent antibody sequences through mostly conservative substitutions; for instance, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. Functional variant antibodies with mutant amino acid sequences can be obtained by mutagenesis (e g., site-directed or PCR-mediated mutagenesis) of the coding nucleic acid molecules, followed by testing of the encoded altered antibody for retained function (/. e., the functions set forth above) using the functional assays described herein.

Antibodies that cross-compete with the reference mAb3 or mAb1

Additional antibodies with similar advantageous properties of the reference mAb3 or the reference mAb1 as disclosed herein can be identified based on their ability to cross-compete with (e.g., to competitively inhibit the binding of), in a statistically significant manner, said reference mAb3 or mAb1 as described above, in standard BTN3A1 binding assays.

Test antibodies may first be screened for their binding affinity to BTN3A1, for example from human recombinant antibody libraries using for example phage display technologies or from transgenic mouse expressing human variable region antibodies immunized with BTN3A1 antigens as assessed typically in the Examples (see Material and Methods section).

In another embodiment, antibodies for use according to the present disclosure include antibodies that bind to the same epitope as do at least the reference mAb3 or the reference mAb1.

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

For example, the following test can be used to screen a BTN3A1 antibody for its ability to cross-compete with mAb3 reference antibody and/or to screen an anti-BTN3A1 antibody for its ability to bind to the same epitope as said reference antibody: BTN3A-KO cells transfected with human BTN3A1 (typically HEK293T) can be stained with saturating concentration (e.g., 10 pg/mL) of the reference antibody mAb3. Different doses of test BTN3A1 mAbs can then be tested for their competitive potential with the mAb3 reference antibody. The mAbs that do compete with the reference antibody will not be able to recognize BTN3A1 in the presence of such reference antibody. The data can be expressed as mean fluorescence intensity. Alternatively, competition assay can be performed in a binning assay as described in the Example section. Typically, binning experiment can be performed by immobilizing recombinant human BTN3A1 on a Biosensor and by presenting the reference antibody followed by the competing antibody. The selected antibodies can be further tested and selected for the advantageous BTN3A activating properties as compared to mAb3 or mAb1 in particular as previously detailed.

In some embodiments, the antibodies for use of the present disclosure compete for binding to BTN3A antibodies described above, in particular an antibody for use of the present disclosure competes for binding with an antibody selected from mAb 20.1 , and mAb 7.2, which are obtainable from one of the hybridomas accessible under CNCM deposit number 1-4401, and I- 4402 such as described in W02012/080769 and WO2012/080351, as well as from mAbs 1-6 described in W02020/025703. In more particular embodiments, the antibodies for use of the present disclosure compete for binding with an antibody selected from mAb 20.1 as produced by the hybridomas deposited at the CNCM under deposit number 1-4401 , and an antibody having a heavy chain of SEQ ID NO:23 and a light chain of SEQ ID NO:24.

Accordingly, in one embodiment, the present disclosure provides an isolated antibody for use in treating infectious disorders as disclosed herein, wherein said isolated antibody competes with the reference mAb3 or the reference mAb1 , from binding to BTN3A1 , and wherein said antibody has one or more of the following properties:

(ii) in vitro induction of the activation of yb T cells, typically Vy9Vb2 T cells, in coculture with BTN3A expressing cells, as measured in a degranulation assay, for example as described in the Examples;

(iii) in vitro induction of the activation of yb T cells, typically Vy9Vb2 T cells, in coculture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by a CFU (Colony Forming Unit) assay.

In specific embodiments, functional properties according to points (i) to (iii) above of an antibody that competes for binding to BTN3A1 with the reference mAb3 or mAb1 are substantially equal or superior to the corresponding functional properties of the reference antibody mAb1 or mAb3 respectively, as described above. By substantially equal it is herein intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95% or 100% of the corresponding functional property of the reference mAb1 or mAb3.

In a certain embodiment, the cross-blocking antibodies or antibody that competes for binding to BTN3A1 with the reference mAb1 or mAb3, is a chimeric, humanized or human recombinant antibody. Framework or Fc engineering

The BTN3A activating antibodies for use of the disclosure can include modifications made to framework residues within VH and VL, to decrease its immunogenicity.

In some specific embodiments, the antibody for use of the disclosure is a humanized monoclonal antibody of the parent murine antibody mAb 20.1 , including at least the following amino acid mutations in the VH framework regions (as compared to VH parental framework regions): V5Q; V11L; K12V; V20L; R66K; M69L; T75S; M80I; E81Q; R83T; T87S; L108A and at least the following amino acid mutations in the VK framework regions (as compared to VK framework regions): T5N; V15L ; R18T; V19I ; K39R; K42N ; A43I ; D70G F73L ; V104L.

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

Furthermore, an antibody for use of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

As used herein, the term “isotype constant region’’ or “Fc region” is used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native seguence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl-terminus of the IgG antibody wherein the numbering is according to the EU numbering system. The C- terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody or its corresponding codon deleted in the recombinant constructs. Accordingly, a composition of antibodies of the disclosure may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

In other embodiments, the Fc region is modified to decrease the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody for an Fey receptor by modifying one or more amino acids. Such antibodies with decreased effector functions, and in particular decreased ADCC, include silent antibodies.

In certain embodiments, the Fc domain of the lgG1 isotype is used. In some specific embodiments, a mutant variant of the IgG 1 Fc fragment is used, e.g. a silent lgG1 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate ADCC and/or to bind to an Fey receptor.

In certain embodiments, the Fc domain of the lgG4 isotype is used. In some specific embodiments, a mutant variant of the lgG4 Fc fragment is used, e.g. a silent lgG4 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate ADCC and/or to bind to an Fey receptor.

Silenced effector functions can be obtained by mutation in the Fc constant part of the antibodies and have been described in the Art (Baudino et al., 2008; Strohl, 2009). Examples of silent lgG1 antibodies comprise the triple mutant variant lgG1 L247F L248E P350S. Examples of silent lgG4 antibodies comprise the double mutant variant lgG4 S241 P L248E.

In certain embodiments, the Fc domain is a silent Fc mutant preventing 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 the replacement of N314 by a glycine or an alanine.

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

Another modification of the antibodies herein that is contemplated for use according to the present disclosure is pegylation or hesylation or related technologies. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacting with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

In certain embodiments, the C-terminal lysine commonly present on human IgG heavy chain constant domains, is engineered out to reduce heterogeneity due to the cleavage of this residue commonly observed during manufacturing or storage. Such modifications do not perceptibly change the desirable functions of these antibodies, while conferring the benefit of stability to these molecules.

Nucleic acid molecules encoding antibodies of the disclosure

Also disclosed herein are the nucleic acid molecules that encode the BTN3A activating antibodies for use according to the present disclosure. Examples of variable light chain and heavy chain nucleotide sequences are those encoding the variable light chain and heavy chain amino acid sequences of any one of the above disclosed exemplary BTN3A activating antibodies, in particular mAb 7.2, mAb 20.1 and their humanized forms, such as mAb1 , mAb2, mAb3, mAb4, mAb5, and mAb6, some of them being easily derived from the Table 1 and Table 2, and using the genetic code and, optionally taking into account the codon bias depending on the host cell species.

The present disclosure also pertains to nucleic acid molecules that derive from the latter sequences having been optimized for protein expression in mammalian cells, for example, CHO cell lines.

Further disclosed herein are the nucleic acid molecules encoding respectively a heavy chain of humanized form of mAb 20.1 of SEQ ID NO:23 and a light chain of humanized form of mAb 20.1 of SEQ ID NO:24.

Further disclosed herein are the nucleic acid molecules of SEQ ID NO:28 and SEQ ID NO:29 encoding respectively a VH and a VL of humanized form of mAb 20.1.

The nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis and others well known in the art (Ausubel et al., 1988). A nucleic acid of the disclosure can be, for example, DNA or RNA and may or may not contain intronic sequences. In an embodiment, the nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector. Nucleic acids of the disclosure 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 by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to an scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment (for example VL and VH as defined in Table 2) is 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, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed under control of a desired promoter.

The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (Kabat et al., 1992) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an lgG1 , lgG2, lgG3, lgG4, IgA, IgE, IgM or IgD constant region. In some embodiments, the heavy chain constant region is selected among lgG1 isotypes, for example human lgG1 isotype. In other embodiments, the heavy chain constant region is selected among lgG4 isotypes, for example human lgG4 isotype. For a Fab fragment heavy chain gene, the VH- encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

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

To create an scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 - Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (Bird et al., 1988; Huston et al., 1988; McCafferty et al., 1990). Methods for producing recombinant antibodies for use according to the present disclosure

Antibodies of the present disclosure can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, 1985).

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

In addition to the antibody chain genes, the recombinant expression vectors disclosed herein carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) ethat control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel’s publication (Goeddel, 1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., 1988).

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

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

In one specific embodiment, a cloning or expression vector according to the disclosure comprises one of the coding sequences of the heavy and light chains of any one of mAb1 , or mAb3 operatively linked to suitable promoter sequences.

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

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

For example, the present disclosure relates to a host cell comprising at least the nucleic acids of SEQ ID NO:28 and 29 encoding respectively the VH and VL of mAb3.

The latter host cells may then be further cultured under suitable conditions for the expression and production of an antibody of the disclosure selected from the group consisting of mAb1 , or mAb3, respectively.

Alternatively, cell free expression systems may be used for the production of any of mAb3, or mAb1. Typically, methods of cell-free expression of proteins or antibodies are already described (Stech et al., 2017) .

Pharmaceutical compositions

In another aspect, the present disclosure provides a composition for use in treating multidrug resistant bacterial infectious disorders as disclosed hereafter, e.g., a pharmaceutical composition, containing BTN3A activating antibodies, in particular, a BTN3A activating antibody selected from the group consisting of mAb 20.1 , a BTN3A activating antibody having the 6 CDRs of mAb 20.1 of 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 which is humanized form of mAb 20.1 , and mAb3 having heavy chain of SEQ ID NO:23 and light chain of SEQ ID NO:24, and their antigen-binding portions, formulated together with a pharmaceutically acceptable carrier.

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

The pharmaceutical composition comprising the BTN3A activating antibody can be formulated at various concentrations. For example, the formulation may comprise the activating BTN3A activating antibody at a concentration of between 0.1 pM and 1 mM, more preferably between 1 pM and 500 pM, between 500 pM and 1 mM, between 300 pM and 700 pM, between 1 pM and 200 pM, between 100 pM and 200 pM, between 200 pM and 300 pM, between 300 pM and 400 pM, between 400 pM and 500 pM, between 500 pM and 600 pM, between 600 pM and 700 pM, between 800 pM and 900 pM, or between 900 pM and 1 mM. Typically, the formulation comprises the BTN3A activating antibody at a concentration of between 300 pM and 700 pM.

The pharmaceutical composition can be formulated at a single unit dose of 0.1 mg to 1 g. For example, the formulation may comprise the activating BTN3A activating antibody at a dose of between 0.5 mg and 1 g, more preferably between 0.1 mg and 1 mg, between 1 mg and 1 g, between 1 mg and 5 mg, between 5 mg and 50 mg, between 50 mg and 100 mg, between 100 mg and 200 mg, between 200 mg and 500 mg or between 500 mg and 1 g. The activating BTN3A activating antibody may also be formulated at a dose of between 1 mg and 1 g, between 5 mg and 1 g, between 5 mg and 500 mg, between 5 mg and 100 mg.

Pharmaceutical compositions disclosed herein can also include additional active therapeutic agents. For example, the pharmaceutical compositions can include an anti-BTN3A antibody of the present disclosure, for example one antibody selected from the group consisting of mAb 20.1 , a BTN3A activating antibody having the 6 CDRs of mAb 20.1 of 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 which is humanized form of mAb 20.1 , and mAb3 having heavy chain of SEQ ID NO:23 and light chain of SEQ ID NO:24, or their antigen-binding portions, combined with at least one anti-viral, anti-inflammatory or anti-bacterial agent. Examples of such other active therapeutic agents that can be used are described in greater detail below in the section on uses of the antibodies of the disclosure.

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

Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.

Other suitable carriers are well-known to those in the art (Remington and Gennaro, 1995). Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, the inflammatory status of the patient, etc.

The pharmaceutical compositions for use according to the disclosure can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like, e.g. by injection, infusion or aerosolization.

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

To prepare pharmaceutical compositions, an effective amount of the BTN3A activating antibody may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

Use of the BTN3A activating antibodies in treating infectious disorders

The present disclosure relates to BTN3A activating antibodies, in particular the specific BTN3A activating antibodies as described above, for use in treating multidrug resistant bacterial infectious disorders, more specifically disorders caused by multidrug resistant Mycobacterium tuberculosis.

As used herein, the terms “treat”, "treating" or "treatment" refers to one or more of (1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i. e. , arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i . e. , reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease. In particular, with reference to the treatment of an infectious disorder, the term “treatment” may refer to the prevention of infection by the infectious agent, inhibition of the replication of the infectious agent, reduction of the severity of one or more of the symptoms associated to the infection, or eradication of the infectious agent. In specific embodiments, in particular for subject suffering from tuberculosis and further treated in combination with rifampicin, isoniazid, ethambutol and pyrazinamide treatments (HZRE treatments), the term “treatment” refers to either shortening combined chemotherapy duration, reduce the risk of relapse after interruption of the treatment, and preventing progression from latent to active tuberculosis.

Latent tuberculosis infection relates to a state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens with no evidence of clinically manifest active tuberculosis.

Active tuberculosis relates to disease that occurs in someone infected with Mycobacterium tuberculosis and is characterized by signs or symptoms of active disease, or both, and is distinct from latent tuberculosis infection, which occurs without signs or symptoms of active disease. Active state of tuberculosis may be determined, e.g. by clinical, imaging or laboratory evidence.

In preferred embodiments, the subject is a human subject.

The BTN3A activating antibodies for use as disclosed above may be administered as the sole active ingredient or in conjunction with, e.g. as an adjuvant or in combination to other drugs e.g. cytokines, anti-viral, anti-bacterial, anti-inflammatory agents, for the treatment or prevention of diseases mentioned above.

For example, the antibodies for use as disclosed above may be used in combination with cytokines, anti-viral agents, anti-bacterial agents, anti-inflammatory agents, therapeutic cells and/or immune checkpoint inhibitors.

Examples of cytokines include cytokines for expanding and/or activating Vy9V52 T cells in vivo, including without limitation 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 their derivatives. The term derivative is used for any cytokine modifications that can rely on PEGylation (e.g. conjugation to polyethylene glycol (PEG) chains), mutation such as amino acid deletion, substitution or insertion, or association with potentiating agents (for example IL15/IL15Ra complexes fused to an lgG1 Fc, in which IL-15 is additionally mutated (asn72asp) that further increase biological activity making this complex an IL-2 and I L-15R|3Y 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 ).

The term “IL-2” has its general meaning and refers to the human interleukin-2. IL-2 is part of the body's natural immune response. IL-2 mainly regulates lymphocyte activity by binding IL- 2 receptors.

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

A method of use as defined herein may therefore comprises co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of a BTN3A activating antibody, and at least one second drug substance, said second drug substance being an antiviral or anti-bacterial, anti-inflammatory agents or cytokines, e.g. IL-2 or IL-15, or a cell therapy product (such as yd T cells), e.g. as indicated above.

In specific embodiments, anti-bacterial agents are standard of care treatments for multidrug resistant bacterial infectious disorders, for example, the combination of rifampicin, isoniazid, ethambutol and pyrazinamide (HZRE).

In specific embodiments, said infectious disorder is caused by MDR Staphylococcus aureus, MDR Escherichia coli, MDR Pseudomonas aeruginosa, MDR Klebsiella pneumoniae, MDR Streptococcus pneumoniae, or MDR Enterococci Entorococcus faecium and Enterococcus Faecalis).

Magiorakos et al (2012) describe antimicrobial categories including antimicrobial agents relevant for antimicrobial susceptibility for bacterial organisms including Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae and Pseudomonas aeruginosa. In some embodiments, the multidrug resistant bacterial infectious disorders are resistant to at least one agent in three or more antimicrobial categories.

In preferred embodiments, said infectious disorder is caused by multidrug resistant Mycobacterium tuberculosis, in particular with resistance to at least rifampicin or isoniazid.

In specific embodiments, said BTN3A antibody treatment decreases Vy9V62 T cells in the blood of treated subjects, in particular by at least 25, 50, 60, 70, 80, 90 or 95 %.

Use of the BTN3A activating antibodies in treating disorder caused by multidrug resistant tuberculosis infection

In specific embodiments, the present disclosure relates to BTN3A activating antibodies, in particular the specific BTN3A activating antibodies as described above, for use in treating a disorder caused by multidrug resistant Mycobacterium tuberculosis infection in a subject in need thereof.

In specific embodiments, the present disclosure relates to a method for treating a disorder caused by MDR Mycobacterium tuberculosis infection in a subject in need thereof, said method comprising administering a therapeutically efficient amount of a BTN3A activating antibody, in said subject.

In specific embodiments, the present disclosure relates to the use of a BTN3A activating antibody in the preparation of a medicament for treating a disorder caused by MDR Mycobacterium tuberculosis infection, in a subject in need thereof.

In specific embodiments, the subject eligible for such treatment has been diagnosed as being infected by MDR Mycobacterium tuberculosis infection.

In specific embodiments, the subject is selected among subjects having active tuberculosis disease. In particular, said BTN3A activating antibody delays or prevents progression of tuberculosis in a subject having active tuberculosis disease. Said BTN3A activating antibody may, in particular, reduce the Mtb infection burden in a subject having active tuberculosis disease. Said BTN3A activating antibody may also stop the progression of, or reduce clinical symptoms of active tuberculosis disease.

In particular, the Mycobacterium tuberculosis infection is resistant to at least isoniazid and rifampin.

In specific embodiments, the subject is selected among the subject at high risk of progression to severe tuberculosis.

For example, persons at high risk for developing tuberculosis disease fall into two categories:

(i) persons who have been recently infected with Mycobacterium tuberculosis bacteria and persons with medical conditions that weaken the immune system. Persons who have been recently infected with Mycobacterium tuberculosis bacteria include: close contacts of a person with infectious tuberculosis disease; persons who have immigrated from areas of the world with high rates of tuberculosis; children less than 5 years of age who have a positive tuberculosis test; groups with high rates of tuberculosis transmission, such as homeless persons, injection drug users, and persons with HIV infection;

(ii) persons who work or reside with people who are at high risk for tuberculosis in facilities or institutions such as hospitals, homeless shelters, correctional facilities, nursing homes, and residential homes for those with human immunodeficiency virus (HIV). Persons with medical conditions that weaken the immune system include babies and young children, people with HIV infection, substance abuse; silicosis; diabetes mellitus, severe kidney disease, low body weight, have received an organ transplant, have cancer, are under medical treatments such as corticosteroids or specialized treatment for autoimmune disorders like rheumatoid arthritis or Crohn’s disease (https://www.cdc.gov/tb/default.htm).

In specific embodiments, anti-bacterial agents may be used in combination with BTN3A activating antibody for treating said disorder caused by multidrug resistant Mycobacterium tuberculosis infection.

Examples of such anti-bacterial agents include without limitation rifampicin, isoniazid, ethambutol, pyrazinamide, levofloxacin, bedaquiline, pretomanid, linezolid and moxifloxacin (https://apps.who.int/iris/rest/bitstreams/1211676/retrieve).

In specific embodiments, said anti-bacterial drugs is HZRE treatment, a combination of rifampicin, isoniazid, ethambutol and pyrazinamide, for example as disclosed in https://www.who. int/publications/i/item/9789240065116. The disclosure having been fully described is now further illustrated by the following examples, which are illustrative only and are not meant to be further limiting.

EXAMPLES

Assays fortesting functional properties of anti-BTN3A activating antibodies

1. Assay for determining binding affinity by SPR of a BTN3A activating antibody

Multi-cycle kinetic analysis can be performed with any BTN3A antibody candidate using a Biacore T200 (serial no. 1909913) instrument running Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden).

Purified antibodies are diluted to a concentration of 2 pg/ml in 2 % BSA/PBS. At the start of each cycle, each antibody is captured on the Protein A at a density (RL) of ~ 146.5 RU (theoretical value to obtain an RMax of ~ 50 RU). Following capture, the surface is allowed to stabilize before injection of the BTN3A1 antigen (Sino Biological cat. no. 15973-H08H). BTN3A1 is titrated in 0.1% BSA/HBS-P+ (running buffer) in a two-fold dilution range from 25 to 0.78 nM. The association phase is monitored for 400 seconds and the dissociation phase for 35 minutes (2100 seconds). Kinetic data are obtained using a flow rate of 50 pl/min to minimize any potential mass transfer effects. Regeneration of the Protein A surface is conducted using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle. Two blanks (no BTN3A1) and a repeat of a single concentration of the analyte were performed for each tested antibody to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 is subtracted from that of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to a reference surface. Additionally, blank runs are subtracted for each Fc to correct any antigen-independent signal variation, such as drift. Sensorgrams were fitted using a one-to-one binding mathematical model with a global RMax parameter and no bulk signal (Constant Rl = 0 RU).

2. Assay for determining binding by flow cytometry on human PBMCs of a BTN3A activating antibody

The BTN3A activating antibodies for use according to the present disclosure can be characterized for their binding to BTN3A as expressed in human PBMCs, isolated from blood of healthy donors. PBMCs are isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs can be used fresh or are frozen and stored at - 80°C or in liquid nitrogen until required. 100 l cells at 1 x10⁶ cells/ml are transferred to each well of a fresh U-shaped bottom 96-well plate, then the plate is centrifuged and supernatant discarded.

A serial dilution of the antibodies, 0.001 pg/ml to 150 pg/ml is prepared in PBS 2 mM EDTA. Human PBMCs are resuspended in 50 pl of the diluted test antibody titration series prepared.

After incubation for 30 minutes at 4°C in the dark, the plate is centrifuged and washed twice with 150 pl/well of PBS 2 mM EDTA following which the wells are resuspended in 50 pl of a mix composed of goat anti-human antibody (PE labelled) diluted 1/100 and Live/dead neat IR diluted 1/500 in PBS 2 mM EDTA.

After incubation for 15 minutes at 4°C in the dark, the plate is centrifuged and washed once with 150 pl/well PBS 2 mM EDTA following which the wells are resuspended in 200 pl PBS 2 mM EDTA. Cells are analyzed on a BD LSR Fortessa Cytometer. Data are analyzed using a FlowJo software (Version 10, FlowJo, LLC, Ashland, USA) (Data not shown).

Same protocol can be performed to test binding on cynomolgus BTN3A as expressed on cynomolgus PBMCs and on Daudi Burkitt's lymphoma cell line.

3. In vitro functional efficacy: yS T cell degranulation assay

The assay consists of measuring activating effect of BTN3A antibodies on yb T cell degranulation against Daudi Burkitt's lymphoma cell line (Harly et al., 2012). yb T cells are expanded from PBMCs of healthy donors by culturing with zoledronic acid (1 pM) and IL2 (200 lU/ml) for 11-13 days. IL2 is added at day 5, day 8 and every 2 days thereafter. The percentage of yb T cells is determined at the initiation of culture and assessed for the time of culture by flow cytometry until it reached at least 80%. Frozen or fresh yb-T cells are then used in degranulation assays against Daudi cell line (E:T ratio of 1 :1), whereby the cells are co-cultured for 4 hours at 37°C in presence of 10 pg/ml of the mAb1 and mAb3 and their chimeric versions. Activation by PMA (20 ng/ml) plus lonomycin (1 pg/ml) served as positive control for yb T cell degranulation, and medium alone as negative control. At the end of a 4 hour co-incubation, cells are analyzed by flow cytometry to evaluate the percentage of yb T cells positive for CD107a (LAMP-1 , lysosomal-associated membrane protein-1) + CD107b (LAMP-2). CD107 is mobilized to the cell surface following activation-induced granule exocytosis, thus measurement of surface CD107 is a sensitive marker for identifying recently degranulating cytolytic T cells. y962 T cells expanded as described in the previous paragraph are cocultured with Mtb- infected macrophages at different ratios for 24 h at 37°C in the presence of different concentrations of mAb1 or mAb3. Mtb-infected macrophages can be obtained after infection of primary monocyte-derived macrophages or TH P-1 cells by Mtb H37Rv bacteria at a MOI of 10. Cells are then washed to eliminate bacilli released from macrophages, lysed with saponin and sonicated 20 seconds to release intracellular bacteria before mycobacteria viability is determined. Alternatively, expanded y9<52 T cells are cultured for 24 h at 37°C with 2x10⁴ bacteria (in 7H9 medium supplemented with Albumin Dextrose Catalase) in the presence or not of increasing concentrations of mAb1 or mAb3 for 24 h at 37°C. For mycobacteria viability assessment, serial 10-fold dilutions of cell lysate are made in 7H9 broth and plated on 7H10 agar plates. Plates are sealed in plastic, kept at 37°C, and the number of colonies (CFUs) is counted after 14-21 days.

EXAMPLE 1 : Humanization of mAb 20.1 and characterization

1. Description of humanization strategies a. Design of Composite Human Antibody™ Variable Region Sequences

Structural models of the murine 7.2 and 20.1 antibody V regions were produced using Swiss PDB and analyzed in order to identify important “constraining” amino acids in the V regions that were likely to be essential for the binding properties of the antibodies. Most residues contained within the CDRs (using both Kabat and Chothia definitions) together with a number of framework residues were considered to be important. From the above analysis, Composite Human sequences of 7.2 and 20.1 antibodies have been created. b. CD4+ T Cell Epitope Avoidance

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

2. Generation of humanized variants and preliminary characterization a. Construction of humanized variants plasmids

7.2 and 20.1 humanized variants were synthesized with flanking restriction enzyme sites for cloning into an expression vector system for human lgG4 (S241 P, L248E) heavy and kappa light chains. All constructs were confirmed by sequencing. b. Expression of Antibodies

Chimeric 7.2 and 20.1 (VHO/VKO), two control combinations (VH0/VK1 , VH 1/VK0) and combinations of humanized heavy and light chains were transiently transfected into Freestyle™ CHO-S cells (ThermoFisher, Loughborough, UK) using a MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA) from corresponding endotoxin-free DNA. Transfections were undertaken for each antibody using OC-400 processing assemblies. Following cell recovery, cells were diluted to 3 x10⁶ cells/mL into CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-Glutamine (ThermoFisher, Loughborough, UK) and 1 x Hypoxanthine-Thymidine (ThermoFisher, Loughborough, UK). 24 hours post-transfection, the culture temperature was reduced to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. Cultures were fed daily by the addition of 3.6 % (of the starting volume) feed (2.5% CHO CD Efficient Feed A (ThermoFisher, Loughborough, UK), 0.5% Yeastolate (BD Biosciences, Oxford, UK), 0.25 mM Glutamax (ThermoFisher, Loughborough, UK) and 2 g/L Glucose (Sigma, Dorset, UK)). IgG supernatant titers were monitored by IgG ELISA and transfections were cultured for up to 14 days prior to harvesting supernatants. c. Selection of mAb3, a humanized form of mAb 20.1

2 humanized variants of mAb 20.1 were selected out of 20 humanized candidates for further characterization.

The table 4 below summarizes the comparative data between the murine parent antibodies mAb 7.2, mAb 20.1 , and a humanized version of mAb 20.1 having the VH of SEQ ID NO:1 and VL of SEQ ID NO:2. Table 4: Functional properties of humanized mAb 20.1

EXAMPLE 2: Evidence for use of activating BTN3A antibody for treating active multidrug bacterial infection The inventors surmise that Vy9V52 T-cells are important effector against Mtb and potentially other MDR bacterial strains, which could be safely activated by mAb3 to control bacteria development.

For tuberculosis, the hypothesis was that local lung environment during active tuberculosis is associated with reduced responsiveness and infiltration of y962 T-cells. Restoring and boosting y962 T cell responses in lungs using mAb3 host-directed therapy may contribute to the control of tuberculosis pathology and infection. Indeed, one could expect that mAb3 could trigger Mtb clearance through direct killing of infected cells and local production of proinflammatory cytokines by y962 T cells, which in turn could revert immunosuppressive mechanisms within tuberculosis granulomas, enhance local recruitment of antimycobacterial effector cells and boost their antimycobacterial activity.

Used as a single agent or in combination with standard of care treatments (HZRE: rifampicin, isoniazid, ethambutol and pyrazinamide), mAb3 may shorten chemotherapy duration, reduce the risk of relapse after treatment interruption and prevent progression from latent to active tuberculosis. At the end, mAb3 treatment should increase the proportion of cured patients, in particular for rifampicin-resistant and extensively drug-resistant tuberculosis. This should also apply to other MDR bacterial infections.

In vitro and in vivo proof of concept of mAb3 therapeutic activity in a tuberculosis cynomolgus model as a single agent or in combination with current standard of care

• Evaluation of mAb3 monotherapy activity in a cynomolgus macaque model of active tuberculosis

A cynomolgus macaque model of tuberculosis infection allowing the exploration of disease heterogeneity observed in human has been developed using broncho-instillation or aerosolization (Capuano, et al. 2003; Lin, et al. 2009; White et al, 2015; Sharpe et al, PLoS One, 2017). Tuberculosis is a chronic lung infection characterized by granuloma formation, in which the dynamic of immune response is complex and heterogeneous.

Previous studies using pAg stimulation of Vy9V52 T cells prior to Mtb infection showed a significant reduction of clinical signs, disease burden and CFU count in lesions of macaques (Gong, et al. 2009; Huang, et al. 2008; Shen, et al. 2002). Nevertheless, in these studies, the activation of Vy9V62 T cells alone did not appear to be sufficient to achieve a cure. The experiments described in the next paragraphs are aiming to show the therapeutic benefit of activating Vy9V52 T cells in infected macaques.

PK-PD study of mAb3:

Since mAb3 has never been injected in an animal, a PK-PD study was necessary to validate the posology, the half-life and the in vivo pharmacodynamic activity of mAb3 (i.e. activation of Vy9V52 T cells) in cynomolgus macaques.

In this study, two doses, 0.3 and 10mg/kg, were evaluated in parallel. Two groups of two macaques received two intravenous (iv) infusions performed two weeks apart at week 0 and 2. mAb3 concentration was measured in the circulation and in bronchoalveolar lavages (BAL) using a generic ELISA, along with Vy9V52 T cell activation measured by mass cytometry at different time points after IV infusion (Figure 1).

Treatment items were thawed at 4°C. Once thawed, item was gently shaken before use to homogenize the solution. Stock solution of mAb3 (20mg/mL) was diluted with Vanguard buffer (PBS 1X, 125mM NaCI, 0.02% Tween 80, 25mM L-His and distilled water) to 0.2 mg/mL for the 0.3 mg/kg dose treatment and 5 mg/mL for the 10 mg/kg dose treatment. Syringes were filled with appropriate volume of treatment preparation according to the last data of animal weight. Animals were observed 7 days a week. At each time of bleeding, clinical examinations were performed, and body weight and rectal temperature were recorded. Whole blood was collected in 1 mL EDTA tubes for complete blood count (CBC) and Immuno phenotyping.

Serum was obtained by centrifugation of dry tubes at 1800 g during 10 minutes, at room temperature. The following cytokines were quantified in serum using NHP Milliplex (PCYTMG-40K-PX23, Millipore) and a Bioplex 200 analyzer (Bio-Rad) according to manufacturer’s instructions: Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), IFN (Interferon)-y, IL-10, IL-12/23 (p40), IL-13, IL-15, IL-17a, IL-18, IL-1 , IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8, MCP-1 , MIP1a, MIP1 , TGF (Transforming Growth Factor) a , TNF (Tumor Necrosis Factor) a, VEGF (Vascular Endothelial Growth Factor) and soluble CD40 Ligand. mAb3 quantification was performed on serum samples by ELISA.

Whole blood collected on an EDTA tube was analyzed by flow cytometry using the panel described below:

After staining and red blood cell lysis, cells were acquired on a ZE5 cytometer (Biorad).

The first mAb3 injection induced rapid and transient decrease of V52 T cells at D1 post injection in both experimental groups with a return to baseline D7 post injection in the 10mg/ kg group and at D14 for one animal. However, no impact on the Vy9V52 T cell count was observed after the second mAb3 treatment at 10 mg/kg and a less marked effect was observed at 0.3 mg/kg. The decrease after injection is indicative of an activation of these T cell subset (Figure 2).

A rapid decrease of V51 y5 T cells in the circulation was also observed after each mAb3 treatment only for the 0.3 mg/kg group, followed by an increase at day D7 and D21 post treatment in both groups (7 days after each treatment) with a return to baseline 14 days after the first injection and 28 days after the second injection.

An expansion of NK cells was observed after each injection with a peak at D7 and D21 in both groups. A decrease of CD4+ T cell count was observed 24 hours after the first injection in the 0.3 mg/kg group followed by an expansion that was stable until the second injection where a decrease was again observed. CD4+ T cell count decrease was observed in both groups after the second injection at D15 followed by a return to baseline count that stayed stable until the end of the study. An increase in CD8+ T cell count was observed 7 days after the first injection for all animals. The levels of CD8+ T cells decreased 24 hours after the second injection in all animals and came back to the levels of D15. These data suggest that the activation of Vy9\/52 T cells by mAb3 also induces bystander activation of all the other circulating immune populations.

This was confirmed by evaluating expression of activation markers, notably CD69 (Figure 3) which was highly increased 24h after the first injection of mAb3 at both doses. This was less marked after the second injection. The other immune subsets were not majorly impacted.

Increases of circulating pro nflammatory IL-6, IFNg, IL-15, MIP-1 b and MCP-1 were observed after the 1st injection of mAb3 at both doses in all animals (Figure 4), indicative of Vy9V52 T cell activation. G-CSF and IL1-RA were also induced.

The PK of the antibody was then measured in treated animals by ELISA. After the first treatment, in the 0.3 mg/kg group, mAb3 was no more detected from D7 whereas mAb3 was still detected at D14 in animals from the 3 mg/kg group (Figure 5). After the second treatment, clearance of mAb3 was faster than after the first injection in animals from the 0.3 mg/kg group since mAb3 was not detected 24 hours after the injection. Kinetics for the 3 mg/kg group were similar between the first and the second injections. Cmax values were comparable between the first and the second injections for both groups.

This study is important to define the proper therapeutic range to be used in the therapeutic efficacy studies and the timing of administration of mAb3.

Therapeutic activity of mAb3 as a single agent (study 1):

The objective was to evaluate the capacity of mAb3 to reduce infection burden in cynomolgus macaques infected by Mtb. Mauritian cynomolgus macaques were infected with 3x10⁵ CFU (Colony Forming Units)/mL of Erdman strain by aerosol route using CH technology nebulizer (Sharpe, et al. 2017; White, et al. 2015). mAb3 treatment was initiated at 6 weeks postinfection, when first pulmonary granulomas were visualized by PET-CT imaging of the lung. mAb3 was injected every two weeks. Two different doses were tested according to the PK/PD properties of the antibody, i.e 0.3 and 3 mg/kg. One group of infected untreated animals was also included (Figure 6).

Inoculum of Mtb was prepared by diluting the stock to obtain a bacterial suspension at 6.5x10⁵ CFU/mL in PBS-Tween 80 (0.05%), in a total volume of 7 mL for each animal. The cynomolgus macaques were exposed one by one to Mtb using the CH Technology jet nebulizer for 10min. All imaging acquisitions were performed on the Digital Photon Counting (DPC) PET-CT system (Vereos-lngenuity, Philips) implemented in BSL3 laboratory. For imaging sessions, animals were anesthetized with a mix of 5mg/kg ketamine and 0.05mg/kg medetomidine by IM route and placed in a supine position on a patient warming blanket (Bear Hugger, 3M). Anesthesia monitoring was performed using heart rate, oxygen saturation, capnography and temperature follow up. During the imaging session, the anesthesia was maintained with 1 to 5% of isoflurane delivered with 100% oxygen through the endotracheal tube. CT was performed 5 minutes prior to PET scan for attenuation correction and anatomical localization. The CT detector collimation used was 64 x 0.6 mm, the tube voltage was 120 kV and intensity of about 150mAs. A whole-body PET scan (4-5 bed positions, 3 min/bed position) was performed 40 min post injection of 3MBq/kg of [18F]-FDG via the saphenous vein.

Animals were randomized at 4 WPI in 3 different groups taking into account the number of granulomas measured by PET-CT:

Stock solution of mAb3 (20mg/mL) was diluted with Vanguard buffer (PBS 1X, 125mM NaCI, 0.02% Tween 80, 25mM L-His and distilled water) to 0.2 mg/mL for the 0.3 mg/kg dose treatment and 2 mg/mL for the 3 mg/kg dose treatment. Anti-BTN3A mAb3 antibody was injected by IV route starting week 6 PI every two weeks at an infusion rate of 2ml_/min. 2 animals were injected with Vanguard Buffer.

Disease progression monitoring was based on clinical scoring, PET-CT imaging, immune monitoring and Vy9V52 T cell phenotype (Figure 6).

The infected cynomolgus macaques were monitored as follows:

Progression of the pulmonary lesions and inflammation was evaluated by analyzing lesion volume and the [¹⁸F]-FDG signal on PET CT images taken at baseline and at 4, 8 and 12 weeks post infection (WPI).

Immune phenotyping by flow cytometry was performed on whole blood collected on EDTA tubes at baseline and at 2, 4, 6, 8, 10 and 12 WPI.

Serum was collected after centrifugation of dry tubes (at 1800 rpm for 10 min at room temperature), aliquoted and stored at -80°C until anti-BTN3A (mAb3) dosage was performed. Serum was collected at baseline and at 4, 6, 8 and 12 WPI. For weeks 4, 6, 8, 10 and 12, sampling was performed prior mAb3 administration and at tmax (30 minutes), 24 hours, 96 hours and 168 hours after treatment.

For broncho-alveolar lavage (BAL) collection at baseline and at 4, 6, 8, 10 and 12 WPI, animals were anesthetized using ketamine hydrochloride (Imalgene 1000, 10 mg/kg, intramuscular route). The animal was placed in left side recumbancy. A laryngoscope was used to visualize the epiglottis and larynx and a spray of 5% lidocaine was applied to the larynx. Two minutes after spray application, the animal was intubated using an endotracheal tube (COVETO ref: 706188). Pressure was made inside the balloon using a 5 mL syringe. The intubation was validated by controlling the breath at the extremity of the tube. 30 mL of 0.9% NaCI was injected into the endotracheal tube with a syringe. A 50 mL conic tube (Falcon®) was placed quickly in front of the endotracheal tube. The macaque diaphragm was compressed to collect a first part of the lavage. Then, the rest of the saline was very quickly recovered directly from the endotracheal tube. BAL were kept at 4°C until centrifugation at 4°C at 500g for 10 min. BAL cells were used for immunophenotyping.

As shown in Figure 7, infected animals treated with vehicle developed lung granulomas indicative of an active Mtb infection. 2 treated animals died during or shortly after the experiment. It is suspected that the disease progression stage was already too advanced in these two animals when the first dose was administered. 4 out of the 6 animals treated with mAb3 (2 in the 0.3 mg/kg and 2 in the 3 mg/kg groups) did not develop an active TB infection as measured by the volume occupied by lung granulomas. Out of these 4 animals, 3 did develop very few small lesions and one started to develop some granulomas at the end of the study. This indicates that mAb3 treatment can prevent the progression of an active Mtb infection. The extent of the eradication of MTb infection in the majority of the treated animals is unexpected and unprecedented in this MTb animal model of MDR-Tb infection.

The phenotypic analysis of circulating cells revealed that right after the first mAb3 injection, Vy9V52 T cell frequency and counts decreased in the blood of treated animals in both groups as early as 24 hours after injection, indicative of activation of these cells (Figure 8). This was confirmed by an increased frequency of CD69+ cells for the Vy9V62 T cells that were remaining in the circulation. After this decrease, Vy9V52 T cells did not come back in the circulation, indicative of a constant trafficking of these cells out of the circulation.

• Evaluation of mAb3 combined with standard-of-care chemotherapy (study 2):

The evaluation of mAb3 in combination with HZRE chemotherapy is also relevant for translation to the clinic. Standard of care HZRE chemotherapy in NHP models of tuberculosis have been proven to recapitulate treatment efficacy and risk of relapse at treatment interruption in humans (Lin, et al. 2013; Sibley, et al. 2022). Cynomolgus macaques are infected with 3x10⁵ CFU/mL of Erdman strain by aerosol route and the same efficacy criteria than in study 1 assessing mAb3 as single agent (PET-CT imaging, clinical monitoring, immune monitoring and Vy9V62 T cell phenotype) are used. T reatment strategy and regimen are defined according to the results obtained in the study assessing therapeutic efficacy of mAb3 as a single agent. As an example (figure 9), mAb3 + HZRE treatment start after 8 weeks of infection and for 8 weeks, modelling the treatment of an active tuberculosis. NHP are euthanized after treatment completion to evaluate lesion sterilization and drug penetration, as well as immune response in tissues. In case of success of the regimen, few NHP are followed after treatment interruption, until human endpoints are reached, to assess mAb3 capacities to delay or prevent relapse (relapse study). Untreated infected animals are included in this study as controls. The dynamic and function of y952 T cell are followed longitudinally as mentioned for study 1 .

• Characterization of anti-tuberculosis activity of NHP Vy9V52 T cells

Due to the low frequencies of Vy9V62 T cells in blood of cynomolgus, both classical intracellular cytokine staining (IFN-y, TNF-a, IL17A) and surface staining for CD69 activation and degranulation evaluation are performed with NHP PBMC or cells from BALs using fresh isolated bulk cells and ex vivo amplification of y952 T cells. Basically, PBMC are incubated for 6 hours with whole Mtb lysate, IPP, HMB-PP or other pAg of interest in the presence or not of mAb3, before performing staining and acquisition.

Co-culture assays using amplified or purified Vy9V52 T cells and Mtb-infected macrophages are used to evaluate intracellular killing of Mtb bacteria. Briefly, monocyte-derived macrophages are infected with Mtb for 4 hours and then co-cultured with autologous 52+ T cell for 3 days. CPU counts are determined on 7H11 plates or on BD BACTEC MGIT (automated mycobacterial detection system). These tests are first established on Vy9V62 T cells amplified from healthy NHP using different pAg combined with rlL2. Then in vitro potency of mAb3 to increase Vy9V52 T cell activation, cytokine production, degranulation and intracellular mycobacterial killing are evaluated in cells from non-infected and infected macaques. These data will support the fact that Vy9V52 T cells from infected animals are still responsive to mAb3 treatment.

HZRE impact can also be measured on amplified or purified Vy9V52 T cells from human or cynomolgus blood. Viability, activation phenotype and function (cytokine production and degranulation) is measured at different timepoints after in vitro treatment. Once HZRE in vitro impact has been established, an in vitro treatment with HZRE and mAb3 can be envisaged and same readouts as the ones described for assessing the in vitro impact of mAb3 are performed.

Ex vivo Translational Support for mAb3 using tuberculosis Patient samples

Vy9V52 T cells from healthy donors can be activated by different strains of bacteria (E coli, Mtb, S Aureus, C difficile) with is further increased by BTN3A activating antibodies as exemplified by increased expression of CD154, CD137 and IFNy (unpublished data). Differences in ex vivo short-term activation of V52 T cells in response to Mtb lysate, HMBPP or Zoledronate, mAb3, BCG, bacterial antigens (PPD, ESAT6/CFP-10) are evaluated between healthy donors, tuberculosis infected patients (latent, no disease), tuberculosis diseased patients and tuberculosis diseased patients with antibiotic-resistant Mtb strain. Diagnosis of tuberculosis is based on IFNy-release assay, clinical symptoms, positive culture, and positive PCR. Blood from confirmed tuberculosis patients are available as treatment-naive and for re-analysis after initiation of the anti-mycobacterial therapy.

The stability of the reactivity patterns of Vy9V52 T cells, changes over time/upon treatment for TB-diseased patients is assessed in a longitudinal study. The ability of V52 T cells from the 4 groups to be differentially activated by mAb3 short term, and phenotypical (differentiation marker, co-stimulatory/inhibitory receptors) or functional (IFNy, TNFa, granulysin/perforin) differences within the antigen-reactive y6 T cells are tested. Finally, V62 T cells from the four groups are activated by mAb3 and sorted according to CD154/CD137 induction to see if they differ in their functional/specificity profile after in vitro cloning/expansion.

For the short term activation of V<52 T cells, PBMC are activated with Mtb lysate, HMBPP or zoledronate, mAb3, BCG, bacterial antigens (PPD, ESAT-6/CFP-10) during 16-24h. After this period, an extensive immunophenotyping is performed by mass cytometry assessing the markers described below:

For the functional analysis of mAb3-activated and expanded 6 T cells, short term activation (o/n) of PBMC from tuberculosis-infected (latent infection) and tuberculosis-diseased patients are performed in the presence of mAb3. Activated (i.e. CD154+ and/or CD137+) V52 T cells are FACS-sorted, cloned into 96-well plates (100 cells/well) and expanded with IL-2 for 2-3 weeks. Testing of the reactivity pattern of expanded clones is done after restimulation with Mtb lysate, HMBPP, mAb3, BCG, bacterial antigens (PPD, ESAT-6), and selected additional bacteria (S aureus, C. difficile, E. coif). The reinduction of CD137/CD154, proliferation and intracellular expression of I FNy, TNFa, granulysin/perforin are analyzed.

From selected patients, it might be possible to obtain BAL and comparison of the reactivity pattern of BAL versus blood yd T cells particularly in the context of what has been described in the literature is performed.

Table 5 (mAbs sequences and in particular CDR sequences are according to Kabat):

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Claims

1. A BTN3A activating antibody for use in treating multidrug resistant bacterial infectious disorders in a human subject in need thereof, preferably selected from multidrug resistant Mycobacterium tuberculosis infections.

2. The BTN3A activating antibody for use according to Claim 1, wherein said BTN3A activating antibody has one or more of the following properties:

(i) it binds to human PBMCs with an ECso of 50 pg/ml or below, preferably of 10 pg/ml or below, as measured in a flow cytometry assay;

(ii) it induces in vitro the activation of yd T cells, typically Vy9V52 T cells, in co-culture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured with a degranulation assay;

(iii) it induces in vitro the activation of yd T cells, typically Vy9Vd2 T cells, in coculture with Mtb-infected macrophages or Mtb bacterial suspension, as measured by a CFU (Colony Forming Unit) assay; or

(iv) It induces in vivo the activation of circulating ydT cells typically Vy9V52 T cells.

3. The BTN3A activating antibody for use according to Claim 1 or Claim 2, wherein said BTN3A activating antibody comprises HCDR1 of SEQ ID NO:5, HCDR2 of SEQ ID NO:6 or 35 to 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 NQ:10 , preferably HCDRs1-3 of SEQ ID NO:5-7 and LCDRs1-3 of SEQ ID NQ:8-10.

4. The BTN3A activating antibody for use according to any one of Claims 1-3, wherein said BTN3A activating antibody either

(i) comprises HCDRs1-3 of SEQ ID NO:11-13 and LCDRs1-3 of SEQ ID NO:14-16;

(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 of SEQ ID NO: 4.

5. The BTN3A activating antibody for use according to any of Claims 1-4, wherein said BTN3A activating antibody comprises a variable heavy chain VH of SEQ ID NO:1 and a light chain VL of SEQ ID NO:2.

6. The BTN3A activating antibody for use according to any one of Claims 1-5, wherein said BTN3A activating antibody comprises a mutant or chemically modified I gG1 constant region, wherein said mutant or chemically modified lgG1 constant region confers no or decreased binding to Fey receptors when compared to a corresponding antibody with wild type lgG1 isotype constant region, preferably said BTN3A activating antibody comprises mutant lgG1 constant region which is an lgG1 triple mutant Fc with mutations L247F L248E and P350S.

7. The BTN3A activating antibody for use according to any one of Claims 1-6, wherein the activating BTN3A antibody comprises a heavy chain of SEQ ID NO: 23 and a light chain of SEQ ID NO: 24.

8. The BTN3A activating antibody for use according to any one of Claims 1-7, wherein said BTN3A activating antibody is administered in combination with, concomitantly or in sequence, at least one second therapeutic substance selected from an anti-viral agent, an anti-bacterial agent, an anti-inflammatory agent, cytokines, therapeutic cells and/or an immune checkpoint inhibitor.

9. The BTN3A activating antibody for use according to any one of Claims 1-8, for treating a disorder caused by multidrug resistant bacterial infections, preferably from Mycobacterium tuberculosis, wherein said BTN3A activating antibody is administered in combination, simultaneously or separately with rifampicin, isoniazid, ethambutol and pyrazinamide.

10. The BTN3A activating antibody for use according to any one of claims 1-9, wherein said treatment decreases Vy9V<52 T cells in the blood of treated subjects.

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

12. The BTN3A activating antibody for use according to any one of Claims 1-11, for treating a disorder caused by multidrug resistant Mycobacterium tuberculosis infection, wherein said subject is a human subject which has active tuberculosis disease.

13. The BTN3A activating antibody for use according to any one of Claims 1-12, for treating a disorder caused by Mycobacterium tuberculosis infection resistant to at least isoniazid and rifampin.

14. The BTN3A activating antibody for use according to any one of Claims 1-13, for treating a disorder caused by methicillin-resistant Staphylococcus aureus, MDR Escherichia coli, MDR

Pseudomonas aeruginosa, Carbapanem-resistant Klebsiella pneumoniae, MDR Streptococcus pneumoniae, Vancomycin-resistant Enterococci (Entorococcus faecium and Enterococcus Faecal is).

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