Antituberculosis vaccine targeting selected Mycobacterium tuberculosis protective antigens to dendritic cells

Abstract

There is an urgent need for an effective therapeutic vaccine against tuberculosis (TB), which remains a major public health problem. Current "classical" strategies under development have failed or are suboptimal, and more effective vaccines are needed to achieve the World Health Organization's 2035 End TB Strategy. We have generated a post-exposure / therapeutic TB vaccine candidate (CD40.TB) whose heavy chain consists of an antibody directed against a surface antigen (i.e., CD40) of antigen-presenting cells (i.e., dendritic cells) conjugated to three relevant Mycobacterium tuberculosis (Mtb) antigens and which is liable to induce potent anti-TB humoral and cellular immunity.

Description

[Technical Field]

[0001] Field of the invention: The present invention is in the field of medicine, particularly infectious diseases. [Background technology]

[0002] Background of the invention: TB remains a major global health crisis, exacerbated by reduced preventive diagnostics during the COVID-19 pandemic, resulting in 1.5 million deaths in 2020, according to the 2021 WHO Global Tuberculosis Report. It is believed that 5–10% of individuals exposed to Mtb will develop active TB disease [1]. The majority of individuals infected with Mtb demonstrate immune reactivity to Mtb but remain asymptomatic and either clear the pathogen or maintain latent infection (LTBI). In patients with active TB disease, the low susceptibility of Mtb to anti-TB drugs is one reason for the prolonged duration of TB treatment. Indeed, current guidelines recommend a 6-month regimen for patients with TB susceptible to effective drugs, consisting of 2 months of treatment with a 3-drug regimen followed by an additional 4 months of treatment with a 2-drug regimen. Current TB treatments are associated with several problems, including significant toxicity due to long-term treatment, poor adherence to treatment leading to the emergence of antimicrobial resistance, and ongoing transmission risk, especially in patients with high bacterial burdens. Multidrug-resistant (MDR) TB cases (82 countries reported at least one case of extensively drug-resistant (XDR) TB in 2018 [2]) represent a continuing and significant threat to human health, despite encouraging progress in novel drug combinations [3], which may, however, exhibit significant toxic effects. In this context, post-exposure / therapeutic anti-TB vaccines could be a reasonable strategy to help reduce the risk of secondary transmission during initial treatment, to avoid drug toxicity through shortening treatment duration, and, overall, to prevent TB disease reactivation in LTBI.

[0003] Clinical evidence suggests that innate immunity can control TB infection in some individuals, but this phenomenon is observed in only a minority of patients [4]. After upper respiratory tract infection, Mtb infects macrophages in the lungs, using several components of their lung membranes as receptors and manipulating macrophages to promote their survival and reduce apoptosis [5]. Infected macrophages promote IL-10 and IFN-γ responses, but their membrane expression of MHC and costimulatory molecules is reduced [6]. These negative responses delay the priming and homing of CD4+ T cells to the lung. However, these early Th1 responses slow the infection and prevent its dissemination through granuloma formation [7]. CD8+ T cells also participate in the primary response but appear to play a minor role during the early stages of TB infection.

[0004] The importance of T cell immunity in controlling Mtb infection has been demonstrated in human and nonhuman primate (NHP) models. The impaired Th1 cytokine (i.e., IFN-γ) production and higher risk of progression from latent to active TB in immunocompromised HIV-infected patients highlight the role of T cell responses in controlling TB disease progression in humans. [8] Interestingly, latent and active TB have been shown to differ in terms of T cell responses; active TB is associated with an increased frequency of Mtb-specific CD4+ T cells with one or two functions (TNF-α positive, or TNF-α and IFN-γ positive), whereas triple-function Mtb-specific CD4+ T cells (IFN-γ positive, TNF-α positive, and IL-2 positive) are more frequently detected during latent infection. [9] Phenotypic and functional changes in CD8+ T cells during the transition from latent to active TB have also been described. Although Mtb-specific CD8+ T cells are more prevalent in active TB patients, these cells appear to be less functional and differentiated compared to LTBI [8, 9]. Finally, the involvement of IL-17-producing cells in Mtb immunology has been highlighted. Again, active TB is accompanied by a decrease in IL-17-producing cells in the blood. However, in this case, IL-17-producing cells appear to be trapped in the lungs during active disease

[10] . Several transcriptome analyses of whole blood from active TB patients or LTBI subjects have been performed in the past few years [11-13]. The signature of active TB is clearly dominated by the overexpression of type I and type II IFN-inducible genes and genes associated with myeloid or inflammatory cells, as well as the downregulation of genes encoding B and T cell functions.

[0005] Therefore, T cell responses appear to be the most plausible targets for an effective anti-Mtb therapeutic vaccine, and the observed differences in their function and / or phenotype in latent and active TB are highly significant, indicating which "appropriate" T cell responses must be elicited by an effective therapeutic vaccine to prevent TB disease reactivation in LTBI individuals.

[0006] Currently, the Bacillus Calmette-Guérin vaccine (BCG) is the only available vaccine against TB, with variable efficacy in reducing the incidence of pulmonary TB in adults and adolescents.

[14] BCG is a live, attenuated strain of Mycobacterium bovis that lacks the so-called Region of Difference 1 (RD1), which contains genes encoding the type VII secretion system "ESX-1" sequences that secrete key Mtb virulence factors, including the early secreted antigenic target (6 kDa) (ESAT-6) and culture filtration protein (10 kDa) (CFP-10). BCG induces both protective and anti-protective responses that may contribute to TB persistence. Although immunological correlates of protective efficacy and / or immune system control of TB have not been established to date, it is widely accepted that an effective TB vaccine must elicit antigen-specific and appropriate effector CD4+ (Th1) and CD8+ T cells.

[0007] To date, 14 vaccine candidates are in clinical trials, half of which are being evaluated in therapeutic regimens, and several of them are in Phase II / III trials [14-16]. Two different strategies based on vaccines derived from whole mycobacterial cells have been developed: live-attenuated or heat-killed bacteria, or subunit vaccines. Therapeutic vaccine strategies using inactivated mycobacterial species, such as Mycobacterium indicus pranii or Mycobacterium vaccae, have shown some efficacy in both mouse models and humans

[17] . The efficacy of these two strains correlates with enhanced T-cell immunity and modulation of the inflammatory response, preventing extracellular bacterial growth. A vaccine for recurrent urinary tract infections (RUTI) is another therapeutic vaccine that targets both replicating and non-replicating Mtb

[18] . Alternative strategies have been developed based on a narrowed repertoire of Ags (protein-based vaccines formulated with adjuvants) focusing on actively secreted mycobacterial proteins involved in TB pathogenesis, namely ESAT-6, CFP-10, TB10.4, Apa, Ag85 complex proteins, Mpt64, Mtb19, Mtb32, Mtb39, or PE / PPE superfamily proteins expressed at various stages of infection / disease [19-21]. Some of these Ags have yielded promising results in animal models and humans [14, 17, 22-28]. One group used Mtb proteins involved in iron sequestration and antigens produced by the bacteria under hypoxic conditions (Rv1738, Rv1909, Rv2032, Rv2359, Rv2711, Rv3130, and Rv3841). A single dose of this vaccine failed to reduce the bacterial burden in the lungs of mice, but it did extend the survival time of the animals after the boost

[25] .In contrast, the ID93 vaccine, which contains non-replicating Mtb Ags (Rv2608, Rv3619, and Rv3620) and replicating Mtb antigens (Rv1813), was able to induce both immunological and bacteriological responses in mouse and NHP TB models

[26] . More recently, eight protective antigens were selected to create an Mtb-specific subunit vaccine, designated H107, which, when coadministered with BCG, resulted in increased adaptive responses to both H107 and BCG, expanding the overall vaccine repertoire with Th17 responses and less differentiated Th1 cells

[27] . Advances in vaccine technology have enabled the development of DNA vaccines, the protective potential of which was initially controversial. However, when administered with anti-TB drugs, DNA vaccines encoding either Ag85B or Hsp (heat shock protein) 65 appear to have shown convincing evidence of therapeutic activity

[28] . Viral vector vaccines (poxvirus and adenovirus) developed for prophylactic purposes have shown only modest efficacy in NHP models, and the MVA (modified vaccinia virus, Ankara strain):Ag85A vaccine in particular has failed to demonstrate any clinical benefit in humans.

[29] Recently, L. Picker's team developed a TB vaccine based on RhCMV and immunized NHPs with the 68-1 RhCMV strain, which expresses nine distinct Mtb proteins (Ag85A, Ag85B, ESAT-6, Rv3407, Rv1733, Rv2626, RpfA, RpfC, and RpfD) and is known to elicit atypical CD8+ T cell responses restricted by MHC-II and MHC-E molecules.

[30] A 40% protection rate was observed, and in an additional 30% of animals, the vaccine helped control infection, demonstrating both prophylactic and therapeutic efficacy. Although encouraging, it remains to be determined whether this strategy can be used to design a vaccine that can be deployed in humans.Indeed, although RhCMV shares considerable homology with human CMV, it is a replication-competent virus that can potentially cause pathogenicity in vulnerable individuals, and it is unclear whether attenuated or replication-defective vectors can elicit the type of T cell responses that can mediate sterilizing immunity against Mtb in humans. GamTBvac, one of four vaccines in Phase III clinical trials, is a recombinant subunit TB vaccine containing the Ag85A and ESAT6-CFP10 antigens and a CpG ODN (oligodeoxynucleotide) adjuvant formulated with dextran.

[31] Another promising vaccine candidate in humans is M72 / AS01, which was first developed by GlaxoSmithKline for prophylactic purposes and contains a recombinant fusion protein derived from Mtb32A and Mtb39A Ags and a liposome-based adjuvant. E Its therapeutic properties were also explored in a Phase IIb trial in infected individuals without any signs of active TB, and data showed that two doses of the vaccine elicited an immune response that protected 49.7% against progression to pulmonary TB disease over a three-year follow-up period.

[32] This prophylactic vaccine will soon enter Phase III clinical trials.

[0008] Overall, these data highlight that i) even though the composition of the Ags likely to be most protective remains an open question in TB vaccinology, previous studies have identified several plausible T cell Ags for inclusion in therapeutic vaccines. These include antigens actively secreted through Mtb's diverse secretion systems, particularly those related to the ESX-1 type VII secretion system, which is involved in Mtb survival in the host, and expressed at high levels during both the acute and chronic phases of infection, namely ESAT-6 and CFP-10, as well as other secreted Ags, such as Mpt64 and Ag85 complex members; and ii) the effect of adjuvants appears to be important, highlighting the need to activate the immune system to elicit an appropriate immune response.

[0009] One way to enhance the immunogenicity of proteins is to improve their delivery to antigen-presenting cells, particularly dendritic cells (DCs), which capture, process, and present Ags to T cells as peptides bound to both major histocompatibility complex (MHC) class I and II [33-35]. In particular, WO 2012 / 129227 discloses fusion proteins with antigens for the production of DC-targeted vaccines by conjugating several Mycobacterium tuberculosis protein antigens with high-affinity monoclonal antibodies against several DC receptors, with the aim of developing a novel human vaccine based on in vivo targeting to DCs. Summary of the Invention

[0010] Summary of the Invention: The present invention is defined by the claims. In particular, the present invention relates to an anti-tuberculosis vaccine that targets selected protective antigens of Mycobacterium tuberculosis to dendritic cells.

[0011] Detailed description of the invention: Key definitions: As used herein, the terms "polypeptide," "peptide," and "protein" are used synonymously herein and refer to polymers of amino acids of any length. The terms also encompass amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When considered in the context of gene therapy, a polypeptide refers to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

[0012] As used herein, the phrase "derive from" refers to a process in which a first component (e.g., a first polypeptide) or information derived from that first component is used to isolate, derive, or create a different second component (e.g., a second polypeptide that is different from the first polypeptide).

[0013] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, comprising deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to make up the double-stranded form.

[0014] As used herein, the term "encoding" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a predetermined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a predetermined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand (the nucleotide sequence of which is identical to the mRNA and is usually provided in a sequence listing) and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0015] As used herein, the term "fusion protein" refers to a protein created through the attachment of two or more polypeptides originating from separate proteins. In particular, fusion proteins can be created by recombinant DNA technology and are typically used in biological research or therapeutics. Fusion proteins can also be created through chemical covalent conjugation, with or without a linker between the polypeptide portions of the fusion protein. The two or more polypeptides in the fusion protein are fused directly or via a linker.

[0016] As used herein, the term "directly" means that a first amino acid at the N-terminus of a first polypeptide is fused to the last amino acid at the C-terminus of a second polypeptide.

[0017] As used herein, the term "linker" has its general meaning in the art and refers to an amino acid sequence of sufficient length to ensure proper secondary and tertiary structure formation of a protein. In some embodiments, the linker is a peptide linker containing at least one but fewer than 30 amino acids, e.g., 2-30 amino acids, preferably 10-30 amino acids, more preferably 15-30 amino acids, even more preferably 19-27 amino acids, and most preferably 20-26 amino acids. In some embodiments, the linker has 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; or 30 amino acid residues. Typically, the linker allows the compound to adopt the proper conformation. The most suitable linker sequences (1) adopt a flexible, extended conformation, (2) do not tend to develop ordered secondary structures that may interact with the functional domains of the fusion protein, and (3) have minimal hydrophobic or charged features that may facilitate interactions with the functional protein domains.

[0018] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: one variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light (VL) and heavy (VH) chains determine binding recognition and specificity to antigens. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain assembly, secretion, placental transport, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or influence the overall domain structure and thereby the binding site. Complementarity-determining regions, or CDRs, refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a 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. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs derived from the heavy and light chain variable regions, respectively. Framework regions (FRs) refer to the amino acid sequences interposed between the CDRs.Thus, light and heavy chain variable regions typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al., as set forth in Kabat et al., 1987, "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, National Institutes of Health, USA (Kabat et al., 1992, hereafter "Kabat et al."). The Kabat residue designations do not always correspond directly to the linear numbering of the amino acid residues in the SEQ ID NO: sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening of, or insertion into, structural elements of the basic variable domain structure, whether framework regions or complementarity-determining regions (CDRs) The correct Kabat numbering of residues for a given antibody can be determined by aligning the homologous residues in the antibody's sequence with the "standard" Kabat numbering 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. For the agonist antibodies described hereinafter, the CDRs were determined using a CDR discovery algorithm from www.bioinf.org.uk. See the section entitled "Methods for identifying CDRs by sequence mining" in the antibody section.

[0019] As used herein, the term "chimeric antibody" refers to an antibody comprising the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In one embodiment, a "chimeric antibody" is an antibody molecule in which (a) the constant regions (i.e., heavy and / or light chains) or portions thereof have been modified, substituted, or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, agonist molecule (e.g., CD40 ligand), hormone, growth factor, or drug; or (b) the variable region or portions thereof have been modified, substituted, or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized, particularly humanized, antibodies. Furthermore, chimeric antibodies may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0020] As used herein, the term "humanized antibody" includes antibodies that have the six CDRs of a murine antibody but also have humanized framework and constant regions. More specifically, as used herein, the term "humanized antibody" can include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0021] As used herein, the term "antigen" or "Ag" has its common meaning in the art and refers to a molecule that, when processed and presented by MHC molecules, can specifically bind to an antibody or to a T cell receptor (TCR). An antigen is further capable of being recognized by the immune system and / or of eliciting a humoral and / or cellular immune response, leading to the activation of B and / or T lymphocytes. An antigen may have one or more epitopes or antigenic sites (B epitopes and T epitopes).

[0022] As used herein, the term "epitope" has its general meaning in the art and refers to a fragment of at least 8 amino acids that is recognized by an immune response component. As used herein, the term "immune response component" includes, but is not limited to, at least a portion of a macrophage, lymphocyte, T lymphocyte, killer T lymphocyte, immune response modulator, helper T lymphocyte, antigen receptor, antigen-presenting cell, cytotoxic T lymphocyte, T8 lymphocyte, CD1 molecule, B lymphocyte, antibody, recombinant antibody, genetically engineered antibody, chimeric antibody, monospecific antibody, bispecific antibody, multispecific antibody, diabody, chimeric antibody, humanized antibody, human antibody, heteroantibody, monoclonal antibody, polyclonal antibody, antibody fragment, and / or synthetic antibody. The term "epitope" may be used synonymously with antigen, paratope-binding site, antigenic determinant, and / or determinant.

[0023] As used herein, the term "polyepitopic polypeptide" refers to a polypeptide that includes at least two epitopes.

[0024] As used herein, the term "Mycobacterium tuberculosis" or "Mtb" has its general meaning in the art and is a pathogenic bacterial species of the family Mycobacteriaceae and the causative agent of tuberculosis.

[0025] As used herein, the term "Ag85B" refers to one protein in the Ag85 complex. The Ag85 complex is actually a family of three 30-32 kDa proteins (Ag85A, Ag85B, and Ag85C), all of which possess mycolyltransferase enzyme activity, involved in the attachment of mycolic acids to cell wall arabinogalactan and the biogenesis of the codon. Ag85B contains several immunodominant T cell epitopes (Huygen K, Front Immunol. 2014; PMID: 25071781), and Ags in the Ag85 complex are part of several vaccine candidates currently being tested for TB. In particular, BCG overexpressing Ag85B conferred greater protection against Mtb than BCG in guinea pigs (Horwitz MA, Proc Natl Acad Sci USA. 2000; PMID: 11095745), and DNA vaccination with a plasmid encoding Ag85B conferred robust Th1 immunity and protection in mice (Lozes E, Vaccine. 1997; PMID: 9234526). Furthermore, some Ag85B epitopes are recognized by human CD4+ and CD8+ T cells from PPD (tuberculosis skin reaction)-positive individuals (Silver RF, J Immunol. 1995; PMID: 7722319; Valle MT, Clin Exp Immunol. 2001; PMID: 11207652; Mustafa AS, Infect Immun. 2000; PMID: 10858206; Lindestam Arlehamn CS, PLoS Pathog. 2013; PMID: 23358848; Weichold FF, Genes Immun. 2007; PMID: 17429413).

[0026] As used herein, the term "ESAT-6" refers to one of the most immunogenic Ags of Mtb. This antigen is part of many live attenuated and subunit vaccines currently being tested for TB, including MTBVAC (Marinova D, Exp Rev Vaccines. 2017; PMID: 28447476), H1 (Mearns H, Vaccine. 2017; PMID: 27866772), and GamTBvac (Tkachuk AP, Vaccines 2020; PMID: 33153191), but is absent from BCG.

[0027] As used herein, the term "Mpt-64" refers to a secreted protein encoded in the divergence region (RD2) (Stamm CE, mSphere. 2019; PMID: 31167949), which is absent in "late" BCG strains, such as the most widely used Pasteur and Danish BCG subtypes (Brosch R, Proc Natl Acad Sci USA. 2007; PMID: 17372194).

[0028] As used herein, the terms "tuberculosis" or "TB" have their common meaning in the art and refer to a bacterial infection caused by Mtb. TB is a potentially serious infectious disease that primarily affects the lungs. The bacteria are spread from person to person through tiny droplets expelled into the air via coughing and sneezing.

[0029] As used herein, the term "APC" or "antigen-presenting cell" refers to cells that can activate T cells and B cells, including, but not limited to, some macrophages, B cells, and dendritic cells.

[0030] As used herein, the term "dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinct morphology and high levels of surface MHC class II expression (Steinman, et al., Ann. Rev. Immunol. 9:271 (1991)).

[0031] As used herein, the term "CD40" has its general meaning in the art and refers to the human CD40 polypeptide receptor. In some embodiments, the CD40 is an isoform of the human reference sequence reported by UniProtKB-P25942 (also referred to as human TNR5).

[0032] As used herein, the term "subject" or "subject in need thereof" refers to a human or non-human mammal. Typically, a patient is suffering from or is at risk of being infected with Mycobacterium tuberculosis.

[0033] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment, as well as curative or disease-modifying treatment (including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition), including the suppression of clinical recurrence. Treatment can be administered to patients with a medical disorder or who are likely to eventually develop a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurring disorder, or to extend the patient's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of disease treatment, e.g., a dosing pattern used during therapy. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used in the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. An induction regimen may use (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than a physician would use during a maintenance regimen, administering a drug more frequently than a physician would administer a drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain predetermined criteria (e.g., pain, symptoms of disease, etc.)).

[0034] As used herein, the term "pharmaceutical composition" refers to a composition described herein, or a pharmaceutically acceptable salt thereof, that includes other substances, such as carriers and / or excipients. Pharmaceutical compositions provided herein typically include a pharmaceutically acceptable carrier.

[0035] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, that are appropriate for the specific dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation.

[0036] As used herein, the term "vaccination" or "vaccinating" refers to, but is not limited to, the process of eliciting an immune response in a subject against a particular antigen.

[0037] As used herein, the term "vaccine composition" is intended to mean a composition that can be administered to a human or animal to induce an immune system response; this immune system response can activate certain cells, in particular antigen-presenting cells, T lymphocytes and B lymphocytes.

[0038] As used herein, the term "adjuvant" refers to a compound that, when administered to a subject or animal, can induce and / or enhance an immune response to an antigen. It is also intended to mean a substance that generally acts to accelerate, prolong, or enhance the quality of a specific immune response to a particular antigen. In the context of the present invention, the term "adjuvant" refers to a compound that enhances both the innate immune response by affecting the transient response of the innate immune response, and the longer-lasting effects of the adaptive immune response by activating and maturing antigen-presenting cells (APCs), particularly dendritic cells (DCs).

[0039] As used herein, the phrase "therapeutically effective amount" means a sufficient amount of the active ingredients of the invention to elicit an immune response at a reasonable benefit / risk ratio applicable to any medical treatment.

[0040] antibody: The present invention provides - a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 1), CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 2) and CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 3), a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L (CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 4), CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 5) and CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 6)); wherein the heavy chain is fused to a polyepitope polypeptide comprising the Ag85B epitope set forth in SEQ ID NO:7, the ESAT-6 epitope set forth in SEQ ID NO:8, and the Mpt64 epitope set forth in SEQ ID NO:9; and The present invention relates to antibodies directed against CD40, including:

[0041] [ka]

[0042] Anti-CD40 antibody: In some embodiments, the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody, or even more preferably an IgG4 antibody.

[0043] In some embodiments, the antibody is a chimeric antibody, particularly a chimeric mouse / human antibody.

[0044] In some embodiments, the antibody is a humanized antibody.

[0045] Chimeric or humanized antibodies can be prepared based on the sequences of mouse monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to create a chimeric antibody, mouse variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, mouse CDR regions can be inserted into a human framework using methods known in the art. See, e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.

[0046] In some embodiments, the heavy chain of the antibody comprises a VH domain set forth in SEQ ID NO:10 and the light chain comprises a VL domain set forth in SEQ ID NO:11.

[0047] [ka]

[0048] In some embodiments, the heavy chain of the antibody consists of the amino acid sequence set forth in SEQ ID NO:12 and the light chain consists of the amino acid sequence set forth in SEQ ID NO:13.

[0049] [ka]

[0050] Polyepitope polypeptide: In some embodiments, the polyepitopic polypeptide has the formula "Ag85B-L1-ESAT-6-L2-Mpt64," where L1 and L2 represent linkers.

[0051] In some embodiments, the linker is selected from the group consisting of FlexV1, f1, f2, f3, or f4, as described below. [ka]

[0052] In some embodiments, the polyepitope polypeptide has the formula "Ag85B-f1-ESAT-6-f4-Mpt64" and typically consists of the amino acid sequence set forth in SEQ ID NO:19. [ka]

[0053] fusion: In some embodiments, the heavy chain of the antibody is fused to a polyepitopic polypeptide to form a fusion protein.

[0054] In some embodiments, the polyepitopic polypeptide is fused to the heavy and / or light chain either directly or via a linker.

[0055] In some embodiments, the linker is selected from the group consisting of FlexV1, f1, f2, f3, or f4 described above. In particular, the linker is FlexV1.

[0056] Best form: In some embodiments, an antibody of the invention comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 20 and a light chain having the amino acid sequence set forth in SEQ ID NO: 13 ("CD40.TB" vaccine). [ka]

[0057] Preparation: The antibodies of the present invention can be produced by any technique known per se in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can easily produce the polypeptide by standard polypeptide production techniques. For example, the antibodies of the present invention can be synthesized by recombinant DNA techniques as currently known in the art. For example, these fragments can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into an appropriate eukaryotic or prokaryotic host that will express the desired polypeptide, from which they can subsequently be isolated using well-known techniques.

[0058] In some embodiments, the amino acid sequences described herein include one or more sequences originating from restriction enzyme cloning site(s) present in the polynucleotide encoding the amino acid sequence. Typically, the sequences may consist of two amino acid residues, and typically include AP, AS, AR, PR, SA, TR, and TS sequences.

[0059] In some embodiments, the amino acid sequences described herein may include a signal peptide. As used herein, the term "signal peptide" has its general meaning in the art and refers to a prepeptide present as an N-terminal peptide on a precursor protein. The function of a signal peptide is to facilitate the translocation of an expressed polypeptide attached to the signal peptide into the endoplasmic reticulum. The signal peptide is usually cleaved off during this process. The signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.

[0060] Polynucleotides, vectors and host cells of the invention: A further object of the present invention relates to polynucleotides encoding the heavy and / or light chains of the antibodies of the invention.

[0061] Thus, in certain embodiments, the present invention provides a method for producing a compound comprising: a heavy chain fused to a polyepitope polypeptide comprising the Ag85B epitope set forth in SEQ ID NO: 7, the ESAT-6 epitope set forth in SEQ ID NO: 8, and the Mpt64 epitope set forth in SEQ ID NO: 9, wherein the heavy chain comprises complementarity determining regions CDR1H, CDR2H, and CDR3H, wherein CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 1), CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 2), and CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 3), and / or a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L (CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 4), CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 5) and CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 6)); The present invention relates to a polynucleotide encoding

[0062] Typically, the polynucleotide is a DNA or RNA molecule, which may be comprised in any suitable vector, for example a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0063] Thus, a further object of the present invention relates to a vector comprising a polynucleotide of the invention.

[0064] As used herein, the terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequences.

[0065] Such vectors can contain regulatory elements, such as promoters, enhancers, transcription terminators, etc., that cause or direct the expression of the antibody upon administration to a subject.

[0066] As used herein, the term "promoter sequence / regulatory sequence" refers to a polynucleotide sequence (e.g., a DNA sequence) recognized by the synthetic machinery of a cell, or introduced synthetic machinery, required to initiate specific transcription of a polynucleotide sequence, thereby permitting expression of a gene product operably linked to the promoter sequence / regulatory sequence. In some cases, this sequence may be the core promoter sequence; in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter sequence / regulatory sequence may, for example, be one that expresses a gene product in a tissue-specific manner.

[0067] As used herein, the term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous polynucleotide sequence, resulting in expression of the latter. For example, a first polynucleotide sequence is operably linked to a second polynucleotide sequence when the first polynucleotide sequence is placed in a functional relationship with the second polynucleotide sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other, e.g., in the same reading frame, as necessary to connect two protein-coding regions.

[0068] Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer, the Moloney murine leukemia virus long terminal repeat (LTR) promoter and enhancer, and the immunoglobulin heavy chain promoter and enhancer. Any expression vector for animal cells may be used as long as a gene encoding a human antibody constant region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pKCR, and pSG1βd2-4. Other examples of plasmids include replicative plasmids containing an origin of replication or integrative plasmids, such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenoviral vectors, retroviral vectors, herpesvirus vectors, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfecting packaging cells or transiently transfecting them with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GP envelope-positive cells, and 293 cells. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO 94 / 19478.

[0069] A further object of the present invention relates to host cells transfected, infected or transformed with the polynucleotides and / or vectors according to the invention.

[0070] As used herein, the term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA sequence, or RNA sequence into a host cell, so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed."

[0071] Polynucleotides of the present invention can be used to produce antibodies of the present invention in a suitable expression system.

[0072] As used herein, the term "expression system" refers to a host cell and a suitable vector under appropriate conditions for the expression of a protein encoding, for example, foreign DNA carried by the vector and introduced into the host cell.

[0073] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast. Mammalian host cells include Chinese hamster ovary (CHO) cells, such as dhfr-negative CHO cells used with a dihydrofolate reductase (DFHR) selectable marker (described in Urlaub and Chasin, 1980), the CHOK1 dhfr-positive cell line, NS0 myeloma cells, COS cells, and SP2 cells, such as the GS (glutamine synthetase) CHO cell line with the Xceed™ Gene Expression System (Lonza), or HEK cells.

[0074] The present invention also relates to a method for producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of (i) introducing, in vitro or ex vivo, a recombinant polynucleotide or vector as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, the resulting recombinant host cell, and (iii) optionally selecting cells that express and / or secrete the antibody.

[0075] Such recombinant host cells can be used for the production of the antibodies of the present invention.

[0076] Thus, the host cells disclosed herein are particularly suitable for producing the antibodies of the present invention. Indeed, when recombinant expression is introduced into mammalian host cells, the polypeptide is produced by culturing the host cells for a time sufficient to express the antibody within the host cells and, optionally, secrete the antibody into the culture medium in which the host cells are growing. The antibodies can be recovered and purified, for example, from the culture medium following their secretion, using standard protein purification methods.

[0077] Pharmaceutical and vaccine compositions: The antibodies described herein may be administered as part of one or more pharmaceutical compositions. The use of any conventional carrier medium is considered within the scope of the present invention, provided that it is not incompatible with the antibodies of the present invention, such as by producing any undesired biological effects or by otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol. esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solution, as well as other non-toxic compatible lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0078] The antibodies described herein are particularly suitable for the preparation of vaccine compositions. A further object of the present invention therefore relates to a vaccine composition comprising an antibody of the present invention.

[0079] In some embodiments, the vaccine compositions of the invention comprise an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is incomplete Freund's adjuvant (IFA) or other oil-based adjuvant, present at a weight / weight ratio (w / w) of 30-70%, preferably 40-60%, and more preferably 45-55%. In some embodiments, the adjuvant is polyinosinic-polycytidylic acid (poly(I:C)) or polyinosinic-polycytidylic acid and poly-L-lysine (poly-ICLC). In some embodiments, the vaccine compositions of the invention comprise at least one Toll-like receptor (TLR) agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists.

[0080] Treatment and prevention: The antibodies as well as pharmaceutical or vaccine compositions described herein are particularly suitable for eliciting an immune response against Mtb and can therefore be used for vaccine purposes to prevent or treat infection with Mtb.

[0081] Therefore, a further object of the present invention relates to a method for vaccinating a subject in need thereof against Mtb, comprising administering a therapeutically effective amount of an antibody of the present invention.

[0082] In some embodiments, the antibodies and pharmaceutical or vaccine compositions described herein are particularly suited for the treatment of tuberculosis.

[0083] In some embodiments, the subject may be a human or any other animal (e.g., bird and mammal) susceptible to Mtb infection (e.g., domestic animals such as cats and dogs; livestock and agricultural animals such as horses, cows, pigs, chickens, etc.). Typically, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is an agricultural animal or a pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is an adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant.

[0084] In some embodiments, the subject may be symptomatic or asymptomatic.

[0085] In some embodiments, the vaccines of the invention may be administered to healthy subjects or to subjects (immunosuppressed through drugs, chemotherapy, immunotherapy, primary immunodeficiency, or secondary immunodeficiency) at risk of becoming infected with Mtb or developing TB.

[0086] Typically, the active ingredients of the present invention (i.e., antibodies and pharmaceutical or vaccine compositions described herein) are administered to a subject in a therapeutically effective amount. It is understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concomitantly with the specific polypeptide used; and similar factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range, from 0.01 to 1,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient, with dosage adjustment depending on the symptoms of the subject to be treated. Medicaments typically contain from about 0.01 mg to about 500 mg of the active ingredient, particularly from 1 mg to about 100 mg. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0087] The antibodies and pharmaceutical or vaccine compositions described herein may be administered to a subject by any route of administration, particularly oral, intranasal, rectal, topical, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and the nature of the particular active agent used.

[0088] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0089] [Figure 1] Schematic of a DC-targeted vaccine for CD40.TB. Clone 12E12 monoclonal antibody is a fully humanized anti-human CD40 IgG4 monoclonal antibody. Selected Ags from Mtb, chosen from our in vitro experiments and the literature, are assembled with flexible spacers to improve their synthesis and / or secretion. [Figure 2A] Targeting antigen-presenting cells in individuals with active TB (ATBi) via CD40 improves antigen presentation and induces specific MTB immune responses. PBMCs from six active TB patients were stimulated with various concentrations (0.03 pM to 3 nM) of CD40.TB (top) or IgG4.TB control (bottom). After 8 days of culture and stimulation with a 15-mer peptide pool derived from ESAT-6 or CFP-10 (not included in the vaccine), specific CD4+ T cells were analyzed for IFNγ production (mean ± standard deviation). A. Antigen-specific CD4+ T cells were analyzed for Th1 and Th2 cytokine production (data not shown) (mean ± standard deviation). [Figure 2B] B. Flow cytometry dot plots are representative of IFNγ, TNFα, IL-2, and MIP1-β1 gated on CD4 positive T cells. [Figure 2C] C. Quantification of cytokines in the supernatants of PBMCs 2 days after stimulation with αCD40.TB (blue) or IgG4.TB (black). Results are shown as mean ± standard deviation. [Figure 3A]CD40.TB vaccine elicits polyfunctional, antigen-specific CD4+ T cell responses in ATBi, LTBi, and HD in vitro. Frequency of total cytokines (IFN-γ ± IL-2 ± TNF) produced by specific CD4+ T cells from ATBi, LTBi, or HD (n=11) after in vitro stimulation with αCD40.TB vaccine (3 pM) on day 0 and restimulation with ESAT-6 (1 μg / mL) and a 15-mer peptide pool derived from Ag85b and MPT64 cell wall proteins (5 μg / mL) (A) or with each Mtb peptide separately (C). [Figure 3B] (B) Polymorphic composition of Mtb-specific CD4+ T cell responses elicited by the αCD40.TB vaccine. Responses are color-coded according to the combination of cytokines produced. Arcs identify cytokine-producing subsets (IFN-γ, IL-2, and TNF-α) within the CD4+ T cell population. Median ± interquartile ranges are shown. The Mann-Whitney test was used for comparison (*P<0.03, ***P<0.001, ****P<0.0001). [Figure 3C] CD40.TB vaccine elicits polyfunctional, antigen-specific CD4+ T cell responses in ATBi, LTBi, and HD in vitro. Frequency of total cytokines (IFN-γ ± IL-2 ± TNF) produced by specific CD4+ T cells from ATBi, LTBi, or HD (n=11) after in vitro stimulation with αCD40.TB vaccine (3 pM) on day 0 and restimulation with ESAT-6 (1 μg / mL) and a 15-mer peptide pool derived from Ag85b and MPT64 cell wall proteins (5 μg / mL) (A) or with each Mtb peptide separately (C). [Figure 4-1]Leukotriene proliferative and cytotoxic potential in vitro. Proliferation of CD4 and CD8 T cells in PBMCs from patients with ATBi (n=9) (A) or HD (n=9) (B) stimulated with αCD40.TB or IgG4.TB control. Results are presented as the proliferation index (ratio of stimulated to unstimulated cells). An index greater than 1 indicates induction of proliferation. Results are presented as median values, and the Mann-Whitney test was used for comparison (**P<0.008, ****P<0.0001). Cytotoxic potential of Mtb-specific CD4+ T cell responses in patients with ATBi (n=6). [Figure 4-2] (C) Flow cytometry profile showing CD107a expression on Mtb-specific IFN-γ-producing CD4+ T cells in a representative ATBi subject (unstimulated cells (negative control) and cells stimulated with CD40.TB or IgG4.TB are shown). [Figure 4-3] (D) The percentages (range and median) of IFN-γ-positive CD107a-positive expression among total CD4 cells or among CD4 T cells with low CFSE (carboxyfluorescein succinimidyl ester) staining are shown (Mann-Whitney test was performed for comparisons, **P<0.02). [Figure 4-4] (E) Representative flow cytometry examples of perforin, granzyme B, granzyme A, and CD107a expression on Mtb-specific IFN-γ-producing CD4 T cells after antigen-specific in vitro T cell expansion over 6 days, and [Figure 4-5] (F) Cumulative analysis. [Figure 4-6] (G) All possible combinations of various markers are shown in a pie chart, with each slice corresponding to the average proportion of Mtb-specific IFN-γ-producing CD4 T cells for a particular marker combination identified by the respective arc. [Figure 5A] In vivo immunogenicity of CD40.TB. A. Human CD40 transgenic mice (n=3-5 / group) were immunized twice with 1 μg of CD40.TB±polyICLC or phosphate-buffered saline (sham). [Figure 5B] The in vivo immunogenicity of the B.CD40.TB vaccine was tested for its induction of both humoral (top panel) and T cell (bottom panel) responses in serum and spleen, respectively, one week after the second immunization. Specific Mtb Ag antibodies secreted in serum were measured by Luminex (top panel). Results are shown as median + interquartile range. [Example]

[0090] Working Example: After decades of research, according to recent World Health Organization recommendations, there remains an urgent need to accelerate the development of a potent TB vaccine. Only a few protein / adjuvant vaccine candidates are currently in clinical development. To date, "classical" strategies have failed or are suboptimal, and one of the World Health Organization's top priorities involves innovative approaches to develop novel TB vaccine strategies, drugs, and other health-related technologies to reduce the risk of TB disease, which already infects approximately 2 billion people. We strongly believe that the key to success lies in diversifying tools and technologies, which is why we wish to exploit a DC-targeting strategy by using a humanized anti-human CD40 monoclonal antibody fused to a relevant Mtb Ag.

[0091] We generated a novel TB vaccine candidate by directly fusing three Mtb Ags, namely ESAT-6, Ag85B, and Mpt64, to the C-terminus of the heavy chain of the 12E12 monoclonal antibody specific for human CD40 (the "CD40.TB construct") (Figure 1).

[0092] ESAT-6 is one of the most immunogenic Ags of Mtb. This antigen is part of many live attenuated and subunit vaccines currently being tested for TB, including MTBVAC

[48] , H1

[49] and GamTBvac

[31] , but is absent from BCG.

[0093] Approximately 10 current TB vaccine candidates contain Ags of the Ag85 complex, namely Ag85A or Ag85B. In particular, Ag85B contains several immunodominant T cell epitopes

[50] , and Ags of the Ag85 complex are part of several vaccine candidates currently being tested for TB. In particular, BCG overexpressing Ag85B conferred greater protection against Mtb than BCG in guinea pigs

[51] , and DNA vaccination with a plasmid encoding Ag85B conferred strong Th1 immunity and protection in mice

[52] . Furthermore, several Ag85B epitopes are recognized by human CD4+ and CD8+ T cells from tuberculin-positive individuals [53-57].

[0094] Mpt64 is a secreted protein encoded by the divergence region RD2

[58] , which is absent in "late" BCG strains, such as the most widely used Pasteur and Danish BCG subtypes

[59] . DNA vaccines containing Ag85B and Mpt64 Ags, along with conventional TB chemotherapy, have been shown to be effective in preventing TB reactivation and may be a promising strategy for controlling Mtb infection in mice

[60] . Mpt64 and ESAT-6 were also included in the H107 vaccine, which was associated with a significant increase in long-term protection

[27] .

[0095] The CD40.TB construct was tested in transiently transfected 293F cells and selected for stable transfection of the CHO-S cell line. Antibodies produced in the supernatant were purified, quality-controlled, and tested by flow cytometry for binding to primary mononuclear cells derived from healthy donors. We also generated a negative targeting control, an IgG4 antibody fused to the same Mtb Ag, designated IgG4.TB.

[0096] We investigated the ability of CD40.TB to elicit Mtb-specific T cell responses by characterizing in vitro recall and de novo CD4+ and CD8+ T cell responses in PBMCs from six patients with active TB (ATBi), as previously performed with the CD40.SARS-CoV-2 vaccine containing PBMCs from convalescent COVID-19 individuals

[46] (Figure 2). Human PBMCs were cultured with various concentrations of CD40.TB or IgG4.TB and restimulated with recombinant proteins or 15-mer peptide pools covering the entire sequence of the ESAT-6 or CFP-10 antigens (BEI Resources, National Institutes of Health, USA). As shown in Figures 2A-2B, Mtb-specific CD4+ T cell responses are dramatically enhanced upon stimulation of cells with the CD40.TB vaccine compared to the IgG4.TB control. The difference is even more pronounced when using very low concentrations of the CD40.TB construct. As expected, very low responses were observed when cells were restimulated with CFP-10, an antigen not included in the CD40.TB vaccine.The CD40.TB vaccine induced the secretion of Th1 and pro-inflammatory cytokines, such as MIP1-β, IL-1β, and IL-6, as well as IL-17 cytokines (Figure 2C).

[0097] We investigated the immunogenicity of the three Mtb antigens contained in the vaccine (Figure 3). After 8 days of culture with 3 pmol / L of vaccine, PBMCs from ATBi, LTBi (latent tuberculosis individuals), and HD (healthy BCG-vaccinated donors from the Etablissement Français du Sang) were restimulated with a pool of Mtb 15-mer peptides, ESAT6, Ag85b, and MPT64 cell wall proteins, or with each peptide separately. On day 9, CD4+ T cells producing Th1 cytokines (IFN-γ, TNF-α, IL-2), Th2 cytokines (IL-10, IL-4, IL-13), and MIP1-β cytokines were analyzed by flow cytometry. In ATBi, LTBi, and HD, the frequency of Th1 cytokine (IFN-γ, TNF-α, and IL-2)-producing CD4+ T cells increased after restimulation with the three antigens compared with unstimulated cells (Figure 3A). Therefore, the vaccine can induce recall responses by Mtb-specific CD4+ T cells in both populations. Furthermore, multifunctional analysis of CD4+ T cells using Boolean gating to examine multiple cytokine combinations (i.e., multifunctional responses) at the single-cell level showed that the predominant cytokine combinations in response to stimulation with the three Mtb antigens were bifunctional, which simultaneously produced two cytokines, followed by trifunctional Mtb-specific CD4+ T cells, which simultaneously produced up to three cytokines (IFN-γ ± IL-2 ± TNF) (Figure 3B). Cells producing only one cytokine were not significantly more prevalent in any of the three groups. Regarding the ability of each Mtb antigen to individually expand memory cells, we observed that ESAT-6 responses were predominantly induced in both ATBi (22.44%) and LTBi (13.74%), followed by Ag85b and MPT64 (3.31% and 1.4%) in ATBi and LTBi (3.489% and 6.451%), respectively. In HD, the response to Ag85b was the largest and single most prevalent, accounting for 20.93% of all cytokine-producing CD4+ T cells (Figure 3C).In summary, these results demonstrated that the CD40.TB vaccine was able to stimulate memory cells specific for the three Mtb antigens in both ATBi and LTBi, whereas only Ag85b-specific memory cells were amplified in HD.

[0098] Another important feature is that CD8+ T cells, although to a lesser extent than CD4+ T cells, possess the proliferative capacity induced by the CD40.TB vaccine, as shown in Figure 4. The frequency of proliferating T cells is higher upon cell stimulation with the CD40.TB vaccine compared with IgG4.TB (Figure 4A). PBMCs from HDs stimulated with the CD40.TB vaccine also induce proliferation of CD4+ and CD8+ T cells (Figure 4B). We also evaluated whether Mtb antigen-specific T cells could exhibit cytotoxic properties and degranulation after stimulation with the CD40.TB vaccine for 6 days and overnight restimulation with the three Mtb antigens present in the CD40.TB vaccine. We first evaluated the coexpression of the lysosome-associated membrane glycoproteins CD107a and IFN-γ on the surface of Mtb antigen-induced CD4+ total cells or CD4+ T cells with low CFSE staining (Figure 4C). Mtb-specific CD4+ T cells expressing both the cytotoxic markers CD107a and IFN-γ after peptide stimulation were also frequently detected in both total CD4 cells and CD4 cells with low CFSE staining, indicating that these cell subsets indeed possessed both Th1 and cytotoxic characteristics (Figure 4D). We did not detect a significant specific response to the non-targeting control (IgG4.TB) compared with unstimulated cells. We characterized the degranulation marker CD107a and cytotoxic markers, such as granzyme A (GRZA), granzyme B (GRZB), and perforin (Perf), among specific IFN-γ-producing CD4+ T cells. Cytometry dot plots from one representative individual showing the cytotoxic profile of Mtb-specific IFN-γ-positive CD4+ T cells stimulated with Mtb peptides are shown in Figure 4E. We observed that IFN-γ-positive CD4+ T cells elicited by the CD40.TB vaccine were predominantly cytotoxic, importantly expressing CD107a, granzyme A, granzyme B, or perforin, compared with unstimulated cells or IgG4.TB controls (Figures 4F and 4G).

[0099] Finally, two immunizations of human CD40 transgenic mice with 1 μg of CD40.TB vaccine per mouse on days 0 and 21, in the presence or absence of poly-ICLC, elicited Mtb-specific humoral and T cell responses against the three Ags contained in the CD40.TB vaccine compared with unimmunized mice (Figure 5).

[0100] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated herein by reference into the present disclosure. [Table 1] TIFF2026506361000008.tif248165 TIFF2026506361000009.tif168165

Claims

1. a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 1), CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 2) and CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 3), a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 4), CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 5) and CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 6); An antibody directed against CD40, comprising: the heavy chain is fused to a polyepitope polypeptide comprising the Ag85B epitope set forth in SEQ ID NO:7, the ESAT-6 epitope set forth in SEQ ID NO:8, and the Mpt64 epitope set forth in SEQ ID NO:9; antibody.

2. The antibody of claim 1, wherein the heavy chain of the antibody comprises a VH domain set forth in SEQ ID NO: 10 and / or the light chain comprises a VL domain set forth in SEQ ID NO:

11.

3. The antibody of claim 2, wherein the heavy chain of the antibody consists of the amino acid sequence set forth in SEQ ID NO: 12 and / or the light chain of the antibody consists of the amino acid sequence set forth in SEQ ID NO:

13.

4. The antibody of any one of claims 1 to 3, wherein the polyepitope polypeptide has the formula "Ag85B-L1-ESAT-6-L2-Mpt64", where L1 and L2 represent linkers.

5. The antibody of claim 4, wherein the linker L1 or L2 is selected from the group consisting of SEQ ID NO: 14 (FlexV1), SEQ ID NO: 15 (f1), SEQ ID NO: 16 (f2), SEQ ID NO: 17 (f3) and SEQ ID NO: 18 (f4).

6. The antibody of claim 5, wherein the polyepitope polypeptide has the formula "Ag85B-f1-ESAT-6-f4-Mpt64".

7. The antibody of claim 6, wherein the polyepitope polypeptide consists of the amino acid sequence set forth in SEQ ID NO:

19.

8. The antibody of any one of claims 1 to 7, wherein the heavy chain of the antibody is fused to the polyepitopic polypeptide to form a fusion protein.

9. The antibody of claim 8 , wherein the polyepitopic polypeptide is fused to the heavy chain either directly or via a linker.

10. The antibody of claim 9, wherein the linker is FlexV1 (SEQ ID NO: 14).

11. The antibody according to any one of claims 1 to 10, comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 20 and a light chain having the amino acid sequence set forth in SEQ ID NO:

13.

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

13. A vector comprising the polynucleotide of claim 12.

14. A host cell transfected, infected or transformed with a polynucleotide according to claim 12 and / or a vector according to claim 13.

15. A vaccine composition comprising the antibody of any one of claims 1 to 11.

16. A method for vaccinating a subject in need thereof against Mycobacterium tuberculosis, comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 21.

Images