Antibodies and compositions for treating candidiasis

Abstract

The present invention relates to antibodies and compositions that can be used to treat candidiasis.

Description

[0001] Antibodies and compositions for the treatment of candidiasis

[0002] TECHNICAL FIELD

[0003] The present invention relates to antibodies and compositions, useful for the treatment of candidiasis.

[0004] TECHNOLOGICAL BACKGROUND

[0005] Candida albicans is an opportunistic pathogenic yeast that lives in humans as a commensal saprophyte in the oropharyngeal sphere, the digestive tract, and the urogenital system. In situations of disruption of natural defense barriers, dysbiosis, or dysfunction of the immune system, C. albicans can be responsible for superficial infections of the skin and mucous membranes, or acute disseminated and deep infections (Clancy and Hong Nguyen 2011; Revankar and Sobel 2011). At the level of a vaginal epithelium, C. albicans colonizes the mucous membranes in two morphological states: in the form of yeast, it lives in the non-inflammatory commensal saprophyte state, while in the form of hyphae (filament) it passes into the inflammatory pathological state (Richardson, Willems, et al. 2018). C. albicans then expresses a set of genes encoding specific proteins involved in hyphal elongation and adhesion to host cells.More recently, it has been shown that the hyphal stage is accompanied by the secretion of toxins involved in the inflammatory process of vaginal candidiasis (Richardson, Willems, et al. 2018). An alteration of the plasma membrane of the host epithelial cells, the release of lactate dehydrogenase (LDH) and a massive entry of Ca. 2+ are observed during candidiasis. Activation of the p38 / MKP1 / c-Fos signaling pathway leads to the synthesis of proinflammatory cytokines (11-6, G-CSF, GM-CSF) involved in the recruitment of macrophages and polymorphonuclear neutrophils (PMN) to epithelial cells, and IL-1a involved in the multiplication of T lymphocytes. The Thl inflammatory immune response would then be relayed and amplified by Thl 7 lymphocytes.

[0006] Vulvovaginal candidiasis (CW), and its recurrent form CWR (more than 4 episodes of CW per year, or "CWC" for chronic vulvovaginal candidiasis) are thought to be the consequence of an immunopathological state of the vaginal mucosa, in other words, a dysfunction of the immune system that does not recognize the pathogen (anergy), or that overreacts to pro-inflammatory cytokine signaling (Y ano et al. 2018). A therapeutic alternative to this dysfunction would be to modulate the inflammatory response by stimulating antibody-mediated adaptive immunity. Several studies have indeed demonstrated that the production of protective antibodies could be effective in the treatment of CW, and vaccine trials have been conducted (Cassone 2015; Ibrahim et al. 2013; De Bemardis et al. 2007; De Bernardis et al. 2012).However, these clinical trials are difficult to evaluate because they are based on a classic vaccine approach, i.e. the injection of Candida antigens intradermally or subcutaneously, while the target of the antibodies is at the level of the epithelia.

[0007] Current treatments for CW and CVVR are primarily based on the use of fungistatic imidazole and triazole antifungals in topical or systemic form, or, more rarely, on the use of polyene antifungals in topical form. More recently, a new class of triterpenoid antifungals inhibiting glucan synthase was approved by the FDA in 2021 for the treatment of vaginal candidiasis in the United States. However, these treatments are not entirely satisfactory. On the one hand, the use of antifungals does not provide long-lasting protection, which can lead to the development of CWC. Furthermore, antimicrobials exert a selective pressure that can lead to the emergence of resistant pathogens.

[0008] The present invention provides an effective alternative to the use of antifungal agents in the treatment of candidiasis.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors have developed a new treatment strategy for candidiasis using antibodies directed against specific targets.

[0011] Thus, in a first aspect, the invention relates to an antibody directed against candidalysin, or a fragment of this antibody. According to a particular embodiment, the anti-candidalysin antibody is a neutralizing antibody. According to another particular embodiment, the anti-candidalysin antibody is a human or humanized antibody. In a second aspect, the invention relates to an antibody directed against a protein of the IFF family (IFF family file F), or a fragment of this antibody. According to a particular embodiment, the protein of the IFF family is the Hyrl protein. According to a particular embodiment, the antibody directed against a protein of the IFF family is an opsonizing antibody. According to another particular embodiment, the antibody directed against a protein of the IFF family is a human or humanized antibody.

[0012] In a third aspect, the present invention relates to a pharmaceutical combination comprising one of the antibodies according to the invention, or a combination of an antibody directed against candidalysin and an antibody directed against a protein of the IFF family. According to another particular embodiment, in the combination according to the invention, the anti-candidalysin antibody and / or the antibody directed against a protein of the IFF family is a human or humanized antibody. According to one embodiment, the combination of the invention is in the form of a composition comprising said antibodies, or of a kit comprising a first composition comprising the antibody directed against candidalysin, and a second composition comprising the antibody directed against a protein of the IFF family. According to a particular embodiment, the composition according to the invention, or the compositions of the kit according to the invention, are in injectable form or in the form of a tablet.According to another preferred embodiment, the composition according to the invention is a tablet, or the first and second compositions of the kit are tablets, optionally mucoadhesive.

[0013] Finally, according to another aspect, the invention relates to one of the antibodies described herein, or the combination of the invention, for their use in the treatment of candidiasis, in particular CW or CVVR.

[0014] DETAILED DESCRIPTION

[0015] 1. Antibody directed against candidalysin

[0016] The first aspect of the invention relates to an antibody directed against candidalysin, or a fragment thereof.

[0017] Candidalysin is a cytolytic peptide secreted by C. albicans when it is in its filamentous, pathogenic, form (Moyes et al. 2016, Nature volume 532, pages 64-68). Candidalysin is obtained by the Kexl-dependent proteolytic maturation of the EceI-III peptide, itself generated by the Kex2-dependent proteolytic cleavage of the EceI protein (Uniprot, Q07730). Candidalysin participates in damaging vaginal epithelial cells, which, in response, secrete pro-inflammatory cytokines. The secreted cytokines contribute to the recruitment of innate immune cells, macrophages and neutrophils, to the surface of the vaginal epithelium, and whose effects, combined with those of candidalysin, may contribute to the establishment of an inflammatory loop.

[0018] The amino acid sequence of mature candidalysin is: SIIGIIMGILGNIPQVIQIIMSIVKAEKGNK (SEQ ID NO:1).

[0019] According to a particular embodiment, the invention therefore relates to an antibody directed against candidalysin, or a fragment thereof, candidalysin having an amino acid sequence consisting of SEQ ID NO:1. The antibody directed against candidalysin according to the invention makes it possible to reduce its cytolytic capacities, and to reduce the production of proinflammatory cytokines.

[0020] The inventors have more particularly identified candidalysin antigen binding domains that are particularly advantageous in the treatment of candidiasis. Under these conditions, the invention relates to a candidalysin binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), in which the VH comprises SEQ ID NO:2 (CDR1-H1), SEQ ID NO:3 (CDR2-H2) and SEQ ID NO:4 (CDR3-H3), respectively; and the VL comprises SEQ ID NO:5 (CDR1-L1), SEQ ID NO:6 (CDR2-L2) and SEQ ID NO:7 (CDR3-L3), respectively.

[0021] According to a particular embodiment, the invention relates to an antibody directed against candidalysin, comprising such a candidalysin binding domain.

[0022] According to another embodiment, the antibody directed against candidalysin is a monoclonal or polyclonal antibody, preferably monoclonal.

[0023] According to a particular embodiment, the antibody directed against candidalysin is preferably a neutralizing antibody. The term "neutralizing antibody" refers to an antibody which, when bound to an epitope, interferes with the normal activity of candidalysin. For example, a neutralizing antibody interferes with the cytolytic activity of candidalysin and reduces its function. Such neutralizing antibodies can be identified using techniques known in the art.

[0024] The preferably neutralizing antibody directed against candidalysin may therefore comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO:2 (CDR1-H1), SEQ ID NO:3 (CDR2-H2) and SEQ ID NO:4 (CDR3-H3), respectively; and the VL comprises SEQ ID NO:5 (CDR1-L1), SEQ ID NO:6 (CDR2-L2) and SEQ ID NO:7 (CDR3-L3), respectively.

[0025] The amino acid sequences of the complementarity determining regions (CDRs) are listed below:

[0026] CDR1-H1: GFSLSTSGMG (SEQ ID NO:2)

[0027] CDR2-H2: IYWDDDK (SEQ ID NO:3)

[0028] CDR3-H3: ARRARYYGNWFYFAV (SEQ ID NO:4)

[0029] CDR1-L1: QSVSNV (SEQ ID NO:5)

[0030] CDR2-L2: YAS (SEQ ID NO:6)

[0031] CDR3-L3: QQDYSSPPT (SEQ ID NO:7).

[0032] According to a particular embodiment, the antibody directed against candidalysin is an IgG1, IgG2, IgG3 or IgG4 isotype, more particularly IgG1.

[0033] According to a particular embodiment, the neutralizing antibody directed against candidalysin comprises a VH whose amino acid sequence consists of the sequence presented in SEQ ID NO: 8 and a VL whose amino acid sequence consists of the sequence presented in SEQ ID NO: 9:

[0034] - SEQ ID NO:8:

[0035] QVTLKESGPGILRPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDD KRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARRARYYGNWFYFAVWG AGTTVTVSS;

[0036] - SEQ ID NO:9:

[0037] SIVMTQTPKFLLVSAGDRVTITCKASQSVSNVVTWYQQKPGQSPKLLIYYASNRYTG VPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPPTFGGGTKLEIK. Preferably, the antibody directed against candidalysin is a human or humanized antibody.

[0038] Advantageously, the antibody is humanized.

[0039] 2. Antibody directed against a protein of the IFF family

[0040] The second aspect of the invention relates to an antibody directed against a protein of the IFF family, or a fragment of such an antibody.

[0041] The IFF family of proteins represents the largest family of glycosylphosphatidylinositol (GPI)-anchored proteins in C. albicans. This protein family consists of 12 members that are characterized by a highly conserved N-terminal region for their first 350 amino acids.

[0042] According to a particular embodiment, the antibody directed against a protein of the IFF family is capable of binding to the Hyrl protein (HYphally Regulated; Uniprot, P46591) (Richard and Plaine 2007, Eukaryotic Cell 6(2): 119-133). The Hyrl protein is specifically expressed in the hyphal wall of C. albicans during its filamentation (Bailey et al. 1996, Journal of Bacteriology 178(18): 5353-5360). As indicated above, the failure of Candida recognition by the immune system could be the consequence of PMN anergy. Among the different molecular mechanisms that could be responsible for this dysfunction, the Hyrl protein could have the function of masking immunogenic antigens of C. albicans in order to escape phagocytosis (Luo et al. 2010).

[0043] According to a particular embodiment, the invention therefore relates to an antibody directed against a protein of the IFF family, in particular against the Hyrl protein, or a fragment of such an antibody. According to a particular embodiment, the antibody is directed against a fragment of a protein of the IFF family, more particularly a peptide fragment included in the amino-terminal region of 350 amino acids highly conserved in the IFF family (Boisramé et al., 2011), more particularly a fragment found in the Hyrl protein. According to a particular embodiment, the fragment found in the Hyrl protein corresponds to amino acids 274-322 of Hyrl according to the sequence presented in SEQ ID NO: 10: LQLRADALPQYFKIGKGYDSKLFRIVNSRGLKNAVTYDGPVPNNEIPAV. The inventors have more particularly identified domains for binding to a protein of the IFF family, in particular Hyrl, which are particularly advantageous in the treatment of candidiasis.Thus, the invention relates more particularly to a binding domain for a protein of the IFF family, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 11 (CDR1-H1), SEQ ID NO: 12 (CDR2-H2) and SEQ ID NO: 13 (CDR3-H3), respectively; and the VL comprises SEQ ID NO: 14 (CDR1-L1), SEQ ID NO: 15 (CDR2-L2) and SEQ ID NO: 16 (CDR3-L3), respectively.

[0044] According to a particular embodiment, the invention relates to an antibody directed against a protein of the IFF family, in particular against the Hyrl protein, comprising such a binding domain.

[0045] According to another embodiment, the antibody directed against a protein belonging to the IFF family is a monoclonal or polyclonal antibody, preferably monoclonal.

[0046] According to a particular embodiment, the antibody directed against a protein of the IFF family is preferably an opsonizing antibody. The term "opsonizing antibody" refers to an antibody that promotes the phagocytosis of Candida albicans by innate immune cells, in particular macrophages. Such opsonizing antibodies can be identified using techniques known in the state of the art.

[0047] The antibody, preferably opsonizing, directed against an IFF family protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 11 (CDR1-H1), SEQ ID NO: 12 (CDR2-H2) and SEQ ID NO: 13 (CDR3-H3), respectively; and the VL comprises SEQ ID NO: 14 (CDR1-L1), SEQ ID NO: 15 (CDR2-L2) and SEQ ID NO: 16 (CDR3-L3), respectively.

[0048] The amino acid sequences of the complementarity determining regions (CDRs) are listed below:

[0049] CDR1-H1: GFTFNTNA (SEQ ID NO: 11)

[0050] CDR2-H2: IRSKSNNYAT (SEQ ID NO: 12)

[0051] CDR3-H3: VREGYYAWFAY (SEQ ID NO:13)

[0052] CDR1-L1: QSLLYSSNQKNY (SEQ ID NO: 14)

[0053] CDR2-L2: WAS (SEQ ID NO: 15) CDR3-E3: QQYYSYPFT (SEQ ID NO: 16).

[0054] According to a particular embodiment, the antibody directed against a protein of the IFF family is an IgG1, IgG2, IgG3 or IgG4 isotype, more particularly IgG2.

[0055] According to a particular embodiment, the opsonizing antibody directed against a protein of the IFF family comprises a VH whose amino acid sequence consists of the sequence presented in SEQ ID NO: 17 and a VE whose amino acid sequence consists of the sequence presented in SEQ ID NO: 18:

[0056] - SEQ ID NO: 17:

[0057] EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSN NYATYYADSVKDRFTISRHDSQSMLYLQMNNLKTEDTAMYYCVREGYYAWFAYW GQGTLVTVSA;

[0058] - SEQ ID NO:18:

[0059] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFTGSGSGTDFTETISSVKAEDEAVYYCQQYYSYPFTFGSGTKEEIK.

[0060] Preferably, the antibody directed against a protein of the IFF family is a human or humanized antibody. Advantageously, the antibody is humanized.

[0061] 3. Antibody fragment

[0062] According to a particular embodiment, the invention also relates to a fragment of one of the antibodies described above.

[0063] "Antibody fragment" means any part of an immunoglobulin obtained by enzymatic digestion or obtained by bioproduction comprising at least two disulfide bridges, for example Fab', F(ab')2, scFv or scFv-Fc.

[0064] The terms "Fab", "F(ab')2" and "scFv-Fc" refer to antibody fragments that retain the ability of the antibody to bind to an antigen. The F(ab')2 fragment is obtained by enzymatic digestion of immunoglobulins with pepsin. F(ab')2 is formed from two Fab' fragments linked by interchain disulfide bridges. The Fab' fragment consists of the Fab region (consisting of the variable regions and the CH1 and CE domains) and a hinge region. The scFv-Fc fragment is a fragment resulting from protein engineering, and consists of the scFv fragment (single chain variable fragment) and linked to an Fc fragment. The scFv fragment consists of only the variable domains VH and VL with a structure stabilized by a short flexible peptide arm, called a linker, which is placed between the two domains.

[0065] Of course, the person skilled in the art is able to select the type of fragment adapted to the use that will be made of the antibody. In particular, to the extent that opsonization involves binding of the antibody to an Fc receptor present on the surface of certain immune cells, the person skilled in the art will take care to use antibodies or antibody fragments comprising an Fc region if the action sought for the antibody is an opsonization property, in particular in the case of an antibody directed against the Hyrl protein.

[0066] 4. Combination

[0067] In a third aspect, the present invention relates to a pharmaceutical composition comprising an antibody directed against candidalysin, or a fragment thereof, and / or an antibody directed against a protein of the IFF family, or a fragment thereof. According to one embodiment, the pharmaceutical composition is a pharmaceutical combination. In this embodiment, the pharmaceutical composition comprises an antibody directed against candidalysin, or a fragment thereof, and an antibody directed against a protein of the IFF family, or a fragment thereof. According to a particular embodiment, the antibody directed against candidalysin or the fragment thereof, and the antibody directed against the protein of the IFF family or the fragment thereof, are distinct. According to this embodiment, the combination does not cover a bispecific antibody directed against both candidalysin and the protein of the IFF family.According to one embodiment, the antibody directed against candidalysin is a neutralizing antibody, more particularly a monoclonal neutralizing antibody. According to another embodiment, the antibody directed against a protein of the IFF family is an opsonizing antibody, more particularly a monoclonal opsonizing antibody. According to another embodiment, the pharmaceutical combination is in the form of a kit, comprising a first composition comprising the antibody directed against candidalysin, or a fragment of this antibody, and a second composition comprising the antibody directed against a protein of the IFF family, or a fragment of this antibody. 4.1.

[0068] According to a particular embodiment, the pharmaceutical composition is an injectable composition. According to another particular embodiment, the pharmaceutical composition is a composition for parenteral injection, more particularly intramuscularly, intravascularly or subcutaneously.

[0069] Thus, according to another embodiment of the invention, the pharmaceutical composition is an injectable composition comprising an antibody directed against candidalysin or a fragment of this antibody, in particular a neutralizing antibody, more particularly a monoclonal neutralizing antibody.

[0070] According to another embodiment, the pharmaceutical composition is an injectable composition comprising an antibody directed against a protein of the IFF family, or a fragment of this antibody, in particular an opsonizing antibody, more particularly a monoclonal opsonizing antibody.

[0071] According to another embodiment, the pharmaceutical composition is an injectable composition comprising an antibody directed against candidalysin, or a fragment thereof, and an antibody directed against a protein of the IFF family, or a fragment thereof. According to a further embodiment, the injectable composition comprises a neutralizing antibody directed against candidalysin and an opsonizing antibody directed against a protein of the IFF family. According to a more particular embodiment, the injectable composition comprises a monoclonal neutralizing antibody directed against candidalysin and a monoclonal opsonizing antibody directed against a protein of the IFF family.

[0072] According to a particular embodiment relating to the kit according to the invention, the first and / or the second composition is an injectable composition.

[0073] 4.2.

[0074] According to another aspect, the invention relates to a pharmaceutical composition comprising one or more antibodies, said pharmaceutical composition being a formulation for mucosal administration. More particularly, according to one embodiment, the pharmaceutical composition is in the form of a tablet, in particular a vaginal or buccal tablet. According to a particular embodiment relating to the kit according to the invention, the first and / or the second composition is in the form of a tablet. More particularly, the two compositions are in the form of a tablet.

[0075] However, the inventors were able to show that two antibodies can be formulated in the same tablet without their activity being significantly impacted by the processes, in particular lyophilization and compression, necessary for their formulation in a tablet. Thus, preferably, the combination according to the invention is in the form of a single pharmaceutical composition comprising the antibody directed against candidalysin, or a fragment of this antibody, and the antibody directed against a protein of the IFF family, or a fragment of this antibody.

[0076] According to a particular embodiment, the invention relates in particular to a pharmaceutical composition which is a tablet comprising an antibody directed against candidalysin, or a fragment of this antibody, in particular a neutralising antibody, more particularly a monoclonal neutralising antibody, and / or an antibody directed against a protein of the IFF family, or a fragment of this antibody, in particular an opsonising antibody, more particularly a monoclonal opsonising antibody. According to another embodiment, the pharmaceutical composition is a tablet comprising an antibody directed against candidalysin, or a fragment of this antibody, and an antibody directed against a protein of the IFF family, or a fragment of this antibody.According to a further embodiment, the tablet comprises a neutralizing antibody directed against candidalysin, or a fragment thereof, and an opsonizing antibody directed against a protein of the IFF family, or a fragment thereof. According to a more particular embodiment, the tablet comprises a monoclonal neutralizing antibody directed against candidalysin, or a fragment thereof, and a monoclonal opsonizing antibody directed against a protein of the IFF family, or a fragment thereof. Preferably, the tablet comprises a monoclonal neutralizing antibody directed against candidalysin and a monoclonal opsonizing antibody directed against a protein of the IFF family.

[0077] According to a particular embodiment, the tablet is a mucoadhesive tablet, in particular a mucoadhesive tablet for vaginal, buccal or oral administration of an antibody, preferably a vaginal mucoadhesive tablet. In order to give the tablet adhesive properties, the tablet comprises an excipient suitable for giving the tablet its properties of mucoadhesion to the vaginal wall, and possibly prolonged release properties. Such excipients are known to those skilled in the art. Examples of excipients that may be suitable are described in particular in Advanced Drug Delivery System Reviews, 57 (2005), 1692-1712. The excipient may be chosen in particular from chitosan and its derivatives, pectins, polyethylene glycols, sodium alginate, polyacrylic acids, cellulose derivatives such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or microcrystalline cellulose, gums, Carbopol® and combinations thereof.Preferably, the mucoadhesive tablet according to the present invention comprises hydroxypropylmethylcellulose (HPMC). HPMC of different viscosity grades may be used, in particular grades K4 to K1 00. However, according to a preferred embodiment, the mucoadhesive tablet according to the invention comprises HPMC of grade K1 5. The amount of excipient suitable for giving the tablet its mucoadhesion properties may vary widely, the person skilled in the art being able to choose the appropriate amounts depending on the desired mucoadhesion properties. According to a particular embodiment, in which the excipient conferring mucoadhesion properties is HPMC, said HPMC is in particular between 5 and 40% (m / m), in particular between 7.5 and 20% (m / m) in the tablet.Advantageously, and quite surprisingly, the tests carried out by the inventors have shown that in a large quantity of HPMC of different viscosity grades, the application of a low compression pressure, in particular 25 MPa, is sufficient to obtain a cohesive tablet (breaking stress > 1 MPa).

[0078] The invention therefore also relates to a process for the preparation of a tablet comprising an antibody, characterized in that it comprises the following steps:

[0079] (a) preparation of an antibody solution;

[0080] (b) lyophilization of the antibody solution; and

[0081] (c) compression of the lyophilized powder.

[0082] According to a particular embodiment, step (a) may comprise the mixing in solution of one or more antibodies, in particular between 100 pg and 10 mg of each antibody, with one or more saccharide excipient(s). The saccharide excipient may in particular be chosen from monosaccharides, disaccharides, oligosaccharides, cyclic oligosaccharides, polysaccharides and alcoholic saccharides. According to one embodiment, the saccharide excipient is a disaccharide, in particular a non-reducing disaccharide, more particularly trehalose or sucrose. According to a particular embodiment, the saccharide excipient is chosen from trehalose and sucrose, said excipient being more particularly trehalose.According to one embodiment, the antibody solution comprises between 1 and 10% (m / v) of saccharide excipient, in particular trehalose or sucrose, in particular between 2 and 8% (m / v), more particularly between 4 and 6% (m / v), the antibody solution more particularly comprising 5% (m / v) of saccharide excipient. According to a particular embodiment, the antibody solution comprises 5% (m / v) of trehalose or sucrose, more particularly 5% (m / v) of trehalose.

[0083] The antibody solution may also comprise a surfactant, in particular a non-ionic surfactant, more particularly polysorbate 20. According to a particular embodiment, the surfactant, in particular non-ionic, more particularly polysorbate 20, is present in the antibody solution at a concentration of between 0.001 and 0.01% (v / v), more particularly between 0.002 and 0.008% (v / v), even more particularly between 0.004 and 0.006% (v / v), the concentration being more particularly 0.005% (v / v).

[0084] Step (b) of the process is a freeze-drying step. Freeze-drying is a low-temperature, vacuum drying process that allows the removal by sublimation of the water contained in a previously frozen product. According to a particular embodiment, the freeze-drying carried out may comprise the following steps:

[0085] - freezing of the antibody solution;

[0086] - primary desiccation (or sublimation); and

[0087] - secondary desiccation.

[0088] According to a particular embodiment, the freezing step implemented during the lyophilization corresponds to the freezing of the antibody solution described above. The freezing is carried out so as to preserve the biological activity of the antibody or antibodies. The freezing parameters are controlled to promote amorphization and / or limit the crystal growth of the constituents of the mixture. Preferably, the cooling rate is between -5°C / min and -0.1°C / min, more particularly between -2°C / min and -0.5°C / min, the cooling rate being more particularly -1°C / min. In a particular embodiment, the solution is cooled to a temperature between -40°C and -60°C, in particular between -45°C and -55°C, more particularly down to -50°C. During the freezing, intermediate temperature maintenance plateaus may be produced.For example, holding for 10 to 30 minutes, particularly for 20 minutes, at a temperature of 5°C and / or -5°C, more particularly a 20-minute plateau at 5°C and a 20-minute plateau at -5°C, may be considered.

[0089] After reaching the target freezing temperature (or product temperature), in particular -50°C, said target temperature can be maintained for a variable time of between 15 minutes and 3 hours, in particular between 45 minutes and 1h30, more particularly between 50 minutes and 70 minutes, in particular for one hour. Then, a primary drying, also called sublimation, can be implemented. The parameters of the freeze-drying process used make it possible to preserve the biological activity of the antibodies. In particular, the operating parameters which control the freeze-drying cycle (shelf temperature, product temperature, pressure in the drying chamber and cycle duration) are chosen appropriately to preserve the biological activity of the antibodies, and can be adapted according to the volume of the aqueous mixture comprising said complexes (more particularly according to the volume of water to be sublimated).In a particular embodiment, the sublimation is carried out at a shelf temperature of between -15°C and -25°C, more particularly at -20°C. According to another embodiment, the sublimation is carried out under vacuum, at a pressure lower than atmospheric pressure, in particular at a pressure of between 0.005 mbar and 0.5 mbar, more particularly at a pressure of 0.1 mbar. The sublimation duration can vary to a large extent, and can in particular be between 5h and 25h, more particularly between 10 and 20h, in particular 14h. The end of the sublimation can be determined more precisely by a person skilled in the art, in particular by means of probes adapted to monitor the total pressure of the enclosure and the partial pressure of water vapor.

[0090] Secondary drying can be carried out in particular under the following conditions:

[0091] - the temperature is gradually increased to a target temperature of between 15°C and 30°C, in particular up to 20°C, at a speed of between +0.01°C / min and +1°C / min, more particularly between +0.05°C / min and +0.3°C / min, the speed of increase in temperature being more particularly +0.1°C / min;

[0092] - the target temperature is maintained for 2 to 10 hours, more particularly between 3 and 5 hours, more particularly for 4 hours.

[0093] Compared to the pressure conditions during sublimation, a slightly higher vacuum can be used during secondary drying at a pressure below atmospheric pressure, namely at a pressure between 0.001 mbar and 0.3 mbar, more particularly at a pressure of 0.05 mbar.

[0094] The containers containing the lyophilisate are then sealed in situ in the lyophilizer, under vacuum.

[0095] The lyophilisate may then be mixed with one or more useful excipients to facilitate the manufacture of a tablet. Examples include excipients for compression, in particular excipients for direct compression such as a mixture of 97% sucrose and 3% maltodextrin (in particular the DI-PAC® product), in particular in the case where the saccharide excipient used in step a) described above is trehalose. The quantity of excipient for compression, in particular direct compression, may vary widely. However, according to a particular embodiment, the compression excipient is present in an amount of between 10 and 90% (m / m), in particular between 20 and 80% (m / m), more particularly between 30 and 70% (m / m), even more particularly between 40 and 60% (m / m), and even more particularly in an amount of 50% (m / m) relative to the weight of the final tablet.Other excipients may be used during the compression step, including a lubricating agent. Furthermore, a lubricating agent may be applied by external spraying to facilitate the compression step. In one embodiment, the lubricating agent is selected from magnesium stearate, talc, sodium stearyl fumarate, and stearic acid, optionally in a mixture. Preferably, the lubricating agent is magnesium stearate. It is also at this step that the excipient suitable for imparting mucoadhesion properties may be mixed with the lyophilizate, before compression.

[0096] Step (c) of the process is a compression step, carried out under conditions allowing the biological activity of the antibodies to be preserved.

[0097] The mechanical stresses applied during the compression step can be controlled and monitored. More particularly, the pressure applied to the mixture comprising the antibody(ies) is controlled and monitored during the compression step. Preferably, the compression of step (c) is carried out at a pressure of between 5 and 200 MPa, in particular between 10 and 150 MPa, more particularly between 25 and 100 MPa, even more particularly between 25 and 50 MPa. According to a preferred embodiment, the compression step (c) is a direct compression, the direct compression not requiring a prior granulation step. According to this embodiment, the lyophilizate obtained at the end of step (b), slightly redissociated by mechanical action and optionally mixed with compression excipients, has properties suitable for direct compression.

[0098] According to a particular embodiment, the method according to the invention aims at the preparation of a tablet comprising an antibody, the antibody being an antibody directed against candidalysin as described in the present invention.

[0099] According to a particular embodiment, the method according to the invention aims at the preparation of a tablet comprising an antibody, the antibody being an antibody directed against a protein of the IFF family, in particular an antibody directed against the Hyrl protein as described in the present invention.

[0100] According to a particular embodiment, the method according to the invention aims at the preparation of a tablet comprising two antibodies, the first antibody being an antibody directed against candidalysin as described in the present invention, the second antibody being an antibody directed against a protein of the IFF family, in particular an antibody directed against the Hyrl protein as described in the present invention.

[0101] Furthermore, the invention relates to a tablet obtainable by means of the method as described above.

[0102] The antibodies, compositions and combinations described above are advantageously used in therapy.

[0103] Thus, according to one aspect, the invention relates to an antibody directed against candidalysin and / or an antibody directed against a protein of the IFF family, or a fragment of such antibodies, according to any of the embodiments described above, for use in the treatment of candidiasis, in particular candidiasis associated with a mucosa of the digestive candidiasis type or CW, more particularly CW, even more particularly CVVR. In a particular embodiment, the treatment relates to mucosal candidiasis, i.e. candidiasis affecting the mucous membranes, such as the oral, intestinal, or vaginal mucosa, preferably candidiasis affecting the vaginal mucosa.The invention also relates to a pharmaceutical combination as described above, for its use in the treatment of candidiasis, in particular candidiasis associated with a mucosa of the digestive candidiasis type or CW, more particularly CW, even more particularly CWR. In a particular embodiment, the treatment is aimed at mucosal candidiasis, that is to say candidiasis affecting the mucous membranes, such as the oral, intestinal, or vaginal mucosa, preferably candidiasis affecting the vaginal mucosa.

[0104] According to one embodiment, the treatment is carried out by passive immunization. In the context of the invention, the treatment may be a preventive (or prophylactic) treatment or a curative treatment. According to a preferred embodiment, the treatment is a preventive (or prophylactic) treatment.

[0105] In a particular embodiment, the treatment comprises administering to the patient a composition described above, in particular a tablet, in particular a vaginal, buccal or oral tablet, depending on the candidiasis to be treated, said tablet comprising an antibody directed against candidalysin, or a fragment of such an antibody, and an antibody directed against a protein of the IFF family, or a fragment of such an antibody. According to a particular embodiment, the tablet is a vaginal tablet, for the treatment of CW or CWR.

[0106] The pharmaceutical composition, depending on its formulation, can be administered enterally, parenterally, or locally, particularly locally in the mouth or vagina, particularly in the vagina.

[0107] According to another embodiment, the antibody, composition or combination according to the invention can be administered in combination with an antifungal agent. The combined treatment can be carried out by simultaneous or sequential administration of the antibody according to the invention and an antifungal agent. FIGURE LEGEND

[0108] [Fig. 1]: Monoclonal antibodies against candidalysin inhibit its hemolytic activity. a) The free hemoglobin level was measured spectrophotometrically (X = 540 nm) in the supernatant of sheep red blood cell suspension (equivalent to 100 pL of sheep red blood cells containing 50% erythrocytes in Alsever buffer, Boehringer Ingelheim Therapeutics GmbH) resuspended in PBS and co-incubated with candidalysin at different concentrations for 2, 4 or 6 h at 37°C. A positive hemolysis control was performed by suspending red blood cells in water under the same incubation conditions.

[0109] The % hemolysis indicated represents the ratio: (Abs sample / Abs positive control (H2O)) x 100. b) The free hemoglobin level was measured by spectrophotometry (X = 540 nm) from 100 pL of hybridoma culture supernatant previously supplemented with candidalysin (20 pM) and then used to treat sheep red blood cells for 6 h at 37°C. A positive hemolysis control was performed by resuspension of candidalysin-treated red blood cells in a hybridoma supernatant not producing anti-Cdlys antibodies (Ctl IgG).

[0110] The % hemolysis indicated represents the ratio: (Sample Abs / Control hybridoma supernatant Abs) x 100.

[0111] Values ​​represent the mean (+ / - standard error) of biological replicates (n > 3). For statistical analysis, an ANOVA test was used; *** P < 0.001.

[0112] [Fig. 2]: Neutralizing antibodies against candidalysin inhibit its cytolytic activity. The neutralizing property of the pre-selected antibodies was evaluated by quantifying the lactate dehydrogenase activity released by A-431 epithelial cells co-incubated with candidalysin alone (5 pM) or a mixture of candidalysin (5 pM) in combination with the purified antibodies (1.25 pM) for 24 h. Values ​​represent the mean (+ / - standard error) of biological replicates (n > 3). For statistical analysis, a student t-test was used. Comparison of means was made versus the 5 pM Cdlys control; *P < 0.05, **P < 0.01.

[0113] [Fig. 3]: Determination of the affinity constant (KD) of selected anti-Cdlys antibodies. Surface plasmon resonance (SPR) kinetic analysis of antibodies 3B 11.1.1, 5E2.2.1, 7B3.1, and 7D4.4 binding to candidalysin. The binding results are represented by the red curves. The black lines represent the theoretical fit to a set of in vitro kinetic titration data of three concentrations of the antibodies obtained by the Biaevaluation software.

[0114] [Fig. 4]: Neutralizing antibody 5E2.2.1 inhibits the pro-inflammatory activity of candidalysin. A-431 vaginal epithelial cells were incubated with 10 μM of synthetic candidalysin peptide alone or in combination with 5E2.2.1 antibody at different concentrations (0.625, 1.25 and 2.5 μM) for 24 h. The culture medium was analyzed to quantify the secretion of pro-inflammatory cytokines IL-1a (a) and IL-8 (b) by sandwich ELISA. Values ​​represent the mean (+ / - standard error) of biological replicates (n > 3). For statistical analysis, a student t-test was used. Comparison of means was performed versus the control Cdlys 5 μM; *P < 0.05, **P < 0.01

[0115] [Fig. 5]: Immunofluorescence analysis of the antigenic specificity of the 5E2.2.1 antibody for candidalysin secreted by C. albicans. Yeasts were incubated on a cell lawn of adhered vulvar epidermoid cells for 3 h (MOI 1:2.5) and cultured for 3 h at 37°C. Once fixed, the cells were labeled with calcofluor-white to reveal fungal components external to the epithelial cells (cyan labeling) and to discriminate the invasion pocket (no labeling). The cells were then incubated with the 5E2.2.1 antibody and the antibody was revealed by a fluorescein-labeled secondary antibody

[0116] [Fig. 6]: Western blot analysis of the specificity of anti-Hyrl antibodies on denatured proteins. Culture supernatants of hybridoma clones (1 / 10 dilution) eme ) were used for the detection of Hyrl protein on 1x10 total protein extracts 6C. albicans WT cells in yeast or hyphal form and C. albicans hyrl A / A mutant in hyphal form. The Hyrl protein is 94 kDa in size.

[0117] [Fig. 7]: Analysis of antigenic targeting of the 4F3.2.1 antibody by immunofluorescence, a) Filamentation of C. albicans WT yeasts (strain SC5314) was induced by incubation in RPMI / 10% FCS medium for 180 min at 37°C. The filaments were incubated with the antibodies contained in the culture supernatant of clone 4F3.2.1 (1 / 10 dilution eme ) then the cells were labeled with a fluorescein-coupled anti-mouse IgG secondary antibody, b) Filamentation of C. albicans (strain SC5314) at different incubation times in RPMI / 10% FCS medium at 37°C and pseudo-filamentation in YNB medium (pseudo-hyphae are indicated by a white arrow). After incubation with purified antibody 4F3.2.1 diluted 1 / 500 eme, the cells were labeled with a secondary anti-mouse IgG antibody coupled to fluorescein. The 4F3.2.1 antibody labels the filament from the early phase of its differentiation (30 min), and does not label either the yeast form or the pseudofilament form

[0118] [Fig. 8]: Opsonization of C. albicans by 4F3.2.1 increases the phagocytic activity of macrophages. The diagram shows the percentage of internalized fungal cells within a macrophage population after 1 h of infection (1 macrophage:1 yeast infection ratio) in the presence of 4F3.2.1 antibody (IxlO 4antibody / fungal cell) or in the absence of antibodies. The internalized fungal mass was assessed by fluorimetry, measuring the fluorescence emitted by yeasts labeled with Calcofluor White, before and after quenching with trypan blue (excitation wavelength 350 nm, emission wavelength 460 nm). The % phagocytosis indicated represents the ratio: (fluorescence after quenching with trypan blue / fluorescence before quenching with trypan blue) x 100. The values ​​represent the mean (+ / - standard error) of 4 independent technical replicates (n > 12). For statistical analysis, a student t-test was used; *** P < 0.001

[0119] [Fig. 9a], [Fig. 9b], [Fig. 9c]: Efficacy of prophylactic treatment of 5E2.2.1 and 4F3.2.1 antibodies in combination in the treatment of vulvovaginal candidiasis in the murine model, a) Chronogram of the experiments b) Quantification of bioluminescence emitted by C. albicans cells (strain CAI4-gLuc59) after intravaginal injection of Coelenterazine. Measurements were performed 2, 4 and 7 days after infection and treatment discontinuation. For statistical analysis, a Mann-Whitney test was used; *P < 0.05, **P < 0.01 c) Quantification of immunomodulators IL-1a and MIP-2 in vaginal washes (diluted 1 / 10 eme in PB S) performed 8 days after infection and treatment cessation. Values ​​represent the mean (+ / - standard error). For statistical analysis, an ANOVA test was used; *P < 0.05, **P < 0.01.

[0120] [Fig. 10]: Structure of lyophilisates of sucrose 5% (m / v) + 0.005% Tween-20 (a) and trehalose 5% (m / v) + 0.005% Tween 20 (b) visualized by scanning electron microscopy at x500 magnification.

[0121] [Fig. 11]: Study of the impact of the lyophilization process on the ability of the TAT antibody to bind to its epitope from formulations containing sucrose or trehalose by indirect ELISA a) Formulation containing 100 pg of antibody b) Formulation containing 500 pg of antibody

[0122] [Fig. 12]: Study of the behavior of formulations under compression a) Compressibility profile of formulations b) Tabletability profile of formulations [Fig. 13]: Study of the influence of compression pressure on the ability of the TAT antibody to bind to its epitope during compression of antibody lyophilisates mixed with Di-Pac® a) antibody lyophilized with sucrose b) antibody lyophilized with trehalose

[0123] [Fig. 14]: Study of the influence of compression pressure on the ability of the TAT antibody to bind to its epitope during compression of crude antibody lyophilisates a) antibody lyophilized with sucrose b) antibody lyophilized with trehalose

[0124] [Fig. 15]: Study of the impact of the compression process (25 MPa) on the ability of the TAT antibody to bind to its epitope during the compression of lyophilisates of a) sucrose or b) trehalose containing 500 pg of antibody

[0125] [Fig. 16]: Study of the influence of lyophilization and compression processes on the neutralizing activity of the 5E2.2.1 antibody. The neutralizing property of the 5E2.2.1 antibody was evaluated by quantifying the activity of lactate dehydrogenase released by A-431 epithelial cells co-incubated with candidalysin (10 pM) and the antibody in solution or after reconstitution in liquid phase after the lyophilization and compression processes after 24 h of incubation at 37°C. The values ​​represent the mean (+ / - standard error) of biological replicates (n > 3). For statistical analysis, an ANOVA test was used; ***P < 0.001.

[0126] [Fig. 17]: Study of the influence of lyophilization and compression processes on the capacity of the opsonizing antibody 4F3.2.1 to bind to the Hyrl protein in its physiological form by immunofluorescence

[0127] [Fig. 18]: Study of the effect of the combination of anti-Cdlys 5E2.2.1 and anti-Hyrl 4F3.2.1 antibodies in the formulation on their ability to bind their antigenic target during lyophilization and compression processes

[0128] [Fig. 19]: Stability study followed for 6 months evaluating the ability of the TAT antibody in tablets to bind to its epitope during its storage at 5, 25, 30 and 40°C by indirect ELISA.

[0129] [Fig. 20]: Study of the influence of the physicochemical properties of the female vaginal environment on the integrity of the TAT antibody formulated in tablet form

[0130] [Fig. 21]: Tablability profile of different powder formulations incorporating HPMC [Fig. 22]: Study of the effect of integrating HPMC into the formulation on the ability of the TAT antibody to bind to its epitope.

[0131] [Fig. 23]: Study of the mucoadhesive property of lyophilized trehalose tablets containing 10, 20 or 30% HPMC K15 after 24 hours of hydration. [Fig. 24]: Release profile of the TAT-1 antibody from lyophilized trehalose tablets formulated with HPMC of different grades and integrated at different proportions. The values ​​represent the mean (+ / - standard error) measured on 3 tablets.

[0132] EXAMPLES

[0133] 1. Production of anti-Cdlvs and anti-Ayr antibodies /

[0134] Hybridoma technology was used for the production of antibodies against candidalysin (Cdlys; KEGG accession number, CAALFM C403470CA) and against the N-terminal part of the Hyrl protein (KEGG accession number, CAALEM Cl 13450WA). This technique is based on the artificial hybridization of antibody-producing B lymphocytes with an immortalized myeloma cell line. Secretory B lymphocytes were collected from mice (9-week-old Balb / c) immunized with the candidalysin peptide (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNK; SEQ ID NO: 1) or the peptide sequence from the N-terminal part of the Hyrl protein (amino acids 274-322, LQLRADALPQYFKIGKGYDSKLFRIVNSRGLKNAVTYDGPVPNNEIPAV; SEQ ID NO: 10).

[0135] Hybridoma cells resulting from the fusion of murine lymphoblasts with P3U 1 myeloma cells were selected by ELISA for their ability to secrete antibodies specifically directed against the antigen, then cloned by the limiting dilution method to ensure the monoclonality of the hybridoma. At the end of this selection step, 24 hybridomas secreting anti-Cdlys antibodies and 8 hybridomas secreting anti-Hyrl antibodies were retained.

[0136] 2. Selection of a candidalysin-neutralizing antibody

[0137] Candidalysin, discovered in 2016, is the first fungal peptide toxin described to date. This 31-amino acid peptide is secreted by the hyphae and plays a major role in the development of CW since, once accumulated in the lumen of the vaginal mucosa, it permeabilizes the membranes of epithelial cells, causing cellular damage and triggering an inflammatory response.

[0138] 2.1. Functional characterization of antibodies 2.1.1 Neutralization of candidalysin by specific antibodies inhibits its hemolytic activity on sheep red blood cells

[0139] In order to select biologically active antibodies, their potential to neutralize candidalysin was studied in vitro through a hemolysis test.

[0140] To do this, sheep red blood cells were treated with candidalysin at different concentrations (10, 20, 30 pM) for 2, 4, or 6 h at 37°C. The level of hemoglobin released into the supernatant following hemolysis of red blood cells by candidalysin was measured by spectrophotometry (X = 540 nm).

[0141] This first experiment made it possible to demonstrate that candidalysin, in addition to its cytolytic activity on epithelial cells, also exhibits haemolytic activity and that the latter is dependent on the concentration of candidalysin and the incubation time with red blood cells (Figure 1a).

[0142] The ability of the 24 previously selected monoclonal antibodies to neutralize candidalysin was subsequently evaluated by this hemolysis test. To do this, culture supernatant from the 24 hybridomas containing the specific antibodies directed against candidalysin was supplemented with candidalysin (20 μM) and then used to resuspend the red blood cells. The suspension was then incubated for 6 h at 37°C.

[0143] The hemoglobin level detected in the different supernatants was measured by spectrophotometry and compared to that measured from a control suspension of red blood cells treated with candidalysin in hybridoma supernatant not secreting antibodies directed against candidalysin.

[0144] Of the 24 supernatants tested, the percentage of hemolysis after candidalysin treatment was significantly reduced when red blood cells were incubated in 8 of them, suggesting the presence of secreted antibodies capable of neutralising candidalysin and significantly limiting its hemolytic activity (Figure 1b). Among the most effective, hybridomas 7D4.4, 7B3.1, 5E2.2.1, 3G8.3 and 3B11.1.1 were selected for further selection experiments. 2.1.2 Neutralisation of candidalysin by specific antibodies inhibits its cytolytic activity on A-431 epithelial cells

[0145] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme present in all tissues. This enzyme, which catalyzes the reversible pyruvate-lactate conversion, is released from the cell when the integrity of the cell membrane is impaired. LDH is thus classically used as a marker of cellular damage and its assay for the characterization of the cytolytic activity of candidalysin on human oral (TRI 46) or vaginal (A-431) cell lines has already been reported.

[0146] To confirm the biological activity of the five selected antibodies, their neutralizing property was evaluated by assaying the enzymatic activity of LDH released into the supernatant by vulvar squamous cell carcinoma cells (A-431) co-incubated with candidalysin alone (control condition) or in combination with the different antibodies for 24 h. A condition including a non-specific antibody to candidalysin was included in the study and used as an additional control condition.

[0147] The results presented in Figure 2 show that the presence of antibodies 7B3.1, 5E2.2.1, 3G8.3 or 3B 11.1.1 during treatment of A-431 cells with candidalysin significantly reduces, with comparable efficacy, the release of LDH by vaginal epithelial cells into the culture medium, which reflects a reduction in cellular damage caused by candidalysin and therefore the ability of these antibodies to neutralise candidalysin.

[0148] 2.2 Biochemical characterization of antibodies

[0149] 2.2.1 Isotyping

[0150] Monoclonal antibody typing was performed by immunodiffusion to determine the antibody class or subclass and to detect, where appropriate, non-monoclonality of the hybridoma (Table 1).

[0151] [Table 1]

[0152] Isotypic characterization of the five clones revealed that hybridoma 3G8.3 was polyclonal (mixture of IgG1 and IgG3) despite the cloning steps. This clone was therefore not retained for further selection. Furthermore, all other clones had the same IgG1 / K isotype. Isotype was therefore not used as a selection criterion.

[0153] 2.2.2 Determination of affinity constants (KD)

[0154] Surface Plasmon Resonance (SPR) is a very fine physical technique that was used to determine the affinity constants between antibodies and candidalysin. The measurements carried out (Figure 3) indicate that the 4 antibodies have very good affinities for candidalysin ranging from 1.4 nM for the most affinity antibody 5E2.2.1, to 3.15 nM for the 7D4.4 antibody. Although the affinity constant values ​​for the 4 clones are very close and not very discriminating, the 5E2.2.1 antibody was selected for the continuation of the project.

[0155] Sequencing of this 5E2.2.1 antibody was performed from the hybridomas described above. The following sequences could be determined:

[0156] >VH

[0157] DNA sequence (369 bp: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4); SEQ ID NO: 19:

[0158] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCGGCCCTCCCAGACCCTCA GTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGC TGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGG ATGATGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGA TACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACT GCCACATACTACTGTGCTCGAAGAGCTCGTTACTACGGGAACTGGTTTTACTTCG

[0159] CTGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA Séquence en acides aminés (123 aa, CDRs soulignées: FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4) ; SEQ ID NO:8: QVTLKESGPGILRPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDD KRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARRARYYGNWFYFAVWG AGTTVTVSS

[0160] >VL

[0161] Séquence ADN (321 pdb: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) ; SEQ ID NO:20:

[0162] <h2 style=";text-align:left;direction:ltr">AGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGG TTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGTTGTAACTTGGTACCA ACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTAC ACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCA CCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTA TAGCTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0163] <h2 style=";text-align:left;direction:ltr"> Sequence in amino acids (107aa, CDRs saturated: FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4); SEQ ID NO:9:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0164] <h2 style=";text-align:left;direction:ltr"> SIVMTQTPKFLLVSAGDRVTITCKASQSVSNVVTWYQQKPGQSPKLLIYYASNRYTG VPDRFTGSGYGTDFTFTISTVQAEDLAVYFCOQDYSSSPPTFGGGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0165] <h2 style=";text-align:left;direction:ltr"> 2.3 Use in vitro the anti-inflammatory activity of anti-Cdlys 5E2.2.1 on infected cells<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0166] The ability of the selected antibody 5E2.2.1 to neutralize candidalysin was finally studied on its capacity to inhibit the pro-inflammatory response. For this, vulvar squamous cell carcinoma cells (A-431) were co-incubated with 10 μM candidalysin alone (control condition), or in combination with the antibody 5E2.2.1, for 24 h at different concentrations, or a control IgG antibody non-specific to candidalysin.

[0167] The results presented in Figure 4 show that when cells are treated with candidalysin neutralised by the 5E2.2.1 antibody, the secretion of pro-inflammatory cytokines by A-431 cells such as IL-1a and IL-8 is significantly reduced for almost all antibody concentrations tested, which should translate, in vivo, into a reduction of the inflammatory process. 2.4 Study of the specificity of the anti-Cdlys antibody 5E2.2.1 for candidalysin

[0168] A recent study has shown that during mucosal C. albicans infection, candidalysin secreted by C. albicans hyphae accumulates within the invasion pocket created during hyphal invagination within epithelial cells and that this accumulation at the invasion pocket is essential for the full potential of cellular damage observed during infection (Mogavero et al. 2021).

[0169] The specificity of the antibody for its target, candidalysin, was studied by immunofluorescence on a co-culture of A431 vulvar epidermoid cells and C. albicans. The results presented in Figure 5 show that the binding of the antibody for its target is concentrated at the invasion pocket and that this binding is specific for candidalysin since no labeling is observed during the co-culture of the A431 line with the mutant strain deleted for the ECE1 gene encoding candidalysin. Furthermore, these results highlight the ability of the selected antibody to reach the invasion pocket created by the yeast during its invagination, an essential prerequisite to ensure its role as a protective antibody with neutralizing activity during CW.

[0170] 3. _ Selection of an antibody targeting the hyrl protein

[0171] 3.1 Study of the antigenic specificity of antibodies

[0172] Hyrl is a 94 kDa cell wall protein anchored by glycosylphosphatidylinositol lipids. It is specifically expressed during filamentation of C. albicans and belongs to the ILE protein family. The first step in characterizing the different anti-Elyrl antibodies selected in hybridoma supernatants by ELISA was to determine the specificity of the antibodies for their target by Western blotting. For this purpose, we extracted total proteins from C. albicans in yeast and hyphal form, as well as those from the hyphal stage of a C. albicans hyrlE / E mutant strain constructed in the laboratory. The denatured total proteins were separated according to their size in a polyacrylamide gel and then transferred onto a PVDE membrane. The membranes were visualized after being incubated in culture supernatant of each selected hybridoma, followed by an anti-mouse Ig secondary antibody.Culture supernatant of the commercial hybridoma TAT-1 secreting antibodies against tubulin-α was used as a protein loading control condition.

[0173] The results presented in Figure 6 show that all the selected antibodies recognized the sequence of a protein (35 kDa) expressed in both morphological forms of C. albicans but in different proportions. Furthermore, the antibodies secreted by clones 4F3.2.1, 4G3.1.1 and 12C 10.4.2 recognized a protein whose size, between 70 and 100 kDa, could correspond to that of the Hyrl protein (94 kDa). However, the expression of this protein in the yeast and filament forms of the WT strain as well as in the filament form of the hyr J mutant indicates that it does not correspond to the Hyrl protein.

[0174] These initial results indicate that each of the monoclonal antibodies selected by ELISA are probably conformational antibodies since none of them is capable of binding to the denatured Hyrl protein.

[0175] The ability of the antibodies to detect the Hyrl protein expressed in its physiological form in vivo was studied by immunofluorescence (Figure 7). Among the 8 monoclonal antibodies tested, only 4F3.2.1 was able to obtain labeling of C. albicans hyphae by immunofluorescence (Figure 7a). To verify the kinetics of fixation of the 4F3.2.1 antibody to the inflammatory hyphal form of C. albicans, the yeasts were grown under conditions favoring their filamentation (RPMI medium at 37°C), then they were immunolabeled at different stages of filamentation (15, 30, 45, 60 or 120 min). The yeasts were also grown in YNB medium which favors their growth in the form of pseudo-hypha, a non-inflammatory form of C. albicans. The immunofluorescence results (Figure 7b) indicate that the antibody is specific for the hyphal form of C. albicans and does not bind to yeast or pseudohyphal forms. Antibody 4F3.2.1 can therefore be considered specific for the pathological and inflammatory form of C. albicans.

[0176] Sequencing of this 4F3.2.1 antibody was performed from the hybridomas described above.

[0177] The following sequences could be determined: >VH

[0178] DNA sequence (360 bp: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4); SEQ ID NO:21:

[0179] GAGGTGCAGCTTGTTGAGACTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGA

[0180] AACTCTCATGTGCAGCCTCTGGATTCACCTTCAATACCAATGCCATGAACTGGGT

[0181] CCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAG

[0182] TAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCC

[0183] AGACATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAACTGAGG

[0184] ACACAGCCATGTATTACTGTGTGAGAGAGGGTTACTACGCCTGGTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0185] Amino acid sequence (120aa, underlined CDRs: FR1-CDR1-FR2-CDR2-FR3-CDR3-

[0186] FR4) ; SEQ ID NO:17:

[0187] EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSN

[0188] NYATYYADSVKDRFTISRHDSQSMLYLQMNNLCTEDTAMYYCVREGYYAWFAYW GQGTLVTVSA

[0189] >VL

[0190] DNA sequence (339 bp: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) ; SEQ ID NO:22:

[0191] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGG

[0192] TTACTATGAGCTGCAAGTCCAGTCAGCCTTTTATAGTAGCAATCAAAAGAA

[0193] CTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTAC

[0194] TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTG

[0195] GGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTA

[0196] TTACTGTCAGCAATATTATAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTG GAAATAAA

[0197] Amino acid sequence (113aa, CDRs underlined: FR1-CDR1-FR2-CDR2-FR3-CDR3-

[0198] FR4); SEQ ID NO: 18:

[0199] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWA

[0200] STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCOOYYSYPFTFGSGTKLEIK

[0201] 3.2 Phagocytosis test The opsonizing activity of the 4F3.2.1 antibody was studied by implementing a phagocytosis test. Murine macrophages (J77.4 line) were infected for 1 h at 37°C with C. albicans cells labeled with Calcofluor-white in a medium inducing their filamentation, in the presence or absence of antibodies. To distinguish fungal cells internalized by macrophages from non-internalized cells, the fluorescence of extra-macrophagic cells was masked by the addition of trypan blue. The fluorescence measurement is therefore proportional to the internalized fungal mass.

[0202] The results presented in Figure 8 show that opsonization of C. albicans hyphae by the 4F3.2.1 antibody significantly increases its susceptibility to be phagocytosed by macrophages in vitro.

[0203] 4. Evaluation of 1' 5E2.2.1 and 4F3.2.1 antibodies in combination in a mouse model of candidiasis

[0204] The prophylactic therapeutic efficacy of the anti-Cdlys and anti-Hyrl antibodies selected in this work was studied in an experimental mouse model according to the chronogram in Figure 9a. Animals (10 mice per group) in pseudoestrus condition were treated intravaginally for 6 consecutive days, either with the antibodies in combination (1, 10 or 50 pg of each antibody), or with PBS (negative control). The animals were then infected intravaginally with 1x10 7CFU of a laboratory-constructed bioluminescent strain of C. albicans (strain CAI4-gLuc59) and the development of infection was followed by imaging.

[0205] The results presented in Figure 9b were obtained by measuring the bioluminescence of the vaginal region in live animals and show that a significant reduction in fungal load was observed 4 days after infection in mice treated with 10 and 50 pg of each of the antibodies, but not in the group treated with 1 pg of antibody, compared to the untreated control group (PBS). This reduction is statistically significant until day 4 post-infection. This trend is maintained 7 days after infection, although the reduction is no longer statistically significant compared to the PBS control group. However, a significant reduction in fungal load between the 10 and 50 pg groups and the 1 pg-treated group is still observed 7 days after treatment cessation. Overall, the results obtained show the prophylactic efficacy of the treatment.Vaginal lavages were performed on the animals 8 days after infection to quantify the pro-inflammatory cytokine IL-1a and the chemokine MIP-2 (CXCL2, functional equivalent of IL-8 in humans). The results presented in Figure 9c show that the amount of cytokine IL-1a detected is significantly lower in the group treated with 10 µg of antibodies than in the untreated group (PB S). Overall, the decrease in IL-1a indicates a reduction in the inflammatory state of the vaginal mucosa, which could be the consequence of the neutralization of candidalysin by the antibodies. Regarding the chemokine MIP-2, it is significantly decreased in animals treated with 50 µg of antibodies, which indicates in this case that the reduction in the inflammatory state could not only come from the neutralization of candidalysin, but also from a decrease in the recruitment of phagocytes to the infectious site.

[0206] Monoclonal antibodies used in immunotherapy are conventionally administered, in the form of sterile preparations, by parenteral route (IV, SC, IM). The in vitro and in vivo activity in the form of solution of the 2 selected antibodies has been demonstrated in the preceding paragraphs and the production of sterile solutions administrable by parenteral route is well known to those skilled in the art. Such administration does not, however, appear to be the most relevant because the antibodies must, after injection, be able to diffuse to the level of the vaginal cavity to be able to exert their therapeutic activity. In addition, there is the problem, beyond sterility, of the stability of antibodies known to be relatively unstable in a liquid medium. This instability often requires storage under controlled temperature conditions at 5 + / - 3 °C and therefore complex logistics.To overcome this instability, many antibodies are presented in a sterile lyophilized form to be reconstituted extemporaneously at the time of administration. The problem of local bioavailability remains. The possibility of administering the selected antibodies in the form of a solution (or gel) vaginally can be considered.

[0207] The idea behind this research was to combine the novel therapeutic approach for the treatment of superficial infections using monoclonal antibodies described previously with in situ delivery in an innovative compressed galenic form. This approach has the triple advantage of delivering the antibodies directly to their site of action, avoiding the problem of stability in a liquid medium, and overcoming the problem of sterility.

[0208] Several scientific and technological challenges arise in this compression-forming of monoclonal antibodies. First, it is necessary to determine and control the compression properties of lyophilized systems (extremely high porosity, amorphous state of the solid fraction, etc.) in order to obtain a tablet that meets the expected criteria for critical quality attributes (mechanical strength, friability, release, stability, etc.). There is very little literature on the compression of lyophilizates.The second point which constitutes an important technological barrier is linked to the fact that matrix compression, commonly used for the production of pharmaceutical tablets, generates macroscopically and especially locally, stresses under strong mechanical constraints, up to several hundred megapascals (MPa) macroscopically with local effects of stress concentration and a certain heterogeneity of the distribution of the constraints within the granular system. However, the molecules of interest of this invention are antibodies, that is to say biological macromolecules with a fragile three-dimensional structure likely to be altered under the effect of mechanical constraints. This is certainly one of the major reasons why compression is not, a priori, envisaged by those skilled in the art. It is therefore necessary to link the compression effects to the maintenance of the biological activity of the antibodies of interest.Finally, it is necessary to demonstrate that this process allows the obtaining of a stable form over time and under different storage temperature conditions, with, again, conservation of the biological activity of the antibodies after disintegration and dissolution of the tablet. It is this set of considerations that led our scientific approach to optimizing the form and delivery conditions of the selected monoclonal antibodies.

[0209] 5.1 Study of the influence of freeze-drying

[0210] Lyophilization represents the technique of choice for drying liquid formulations containing therapeutic proteins including monoclonal antibodies. However, during lyophilization, molecules are exposed to numerous stresses that can alter their structures and consequently their biological activities, which requires a judicious choice of excipients to be added to the formulation and process parameters. For the lyophilization step, two formulations were tested: trehalose 5% (w / v) + 0.005% Tween 20 and sucrose 5% (w / v) + 0.005% Tween 20. The model antibody was added to these formulations at two different dosages: 100 and 500 µg in order to evaluate the influence of the amount of antibody.

[0211] Lyophilization was carried out using a pilot-size freeze dryer (Cryotec) equipped with product temperature monitoring probes and two pressure probes: a Pirani-type probe and a capacitive probe. Regarding the different stages of freeze-drying, freezing was carried out directly on the freeze-dryer shelves in vials containing 1 mL of formulation by applying a cooling rate of 1°C / min until a product temperature of -50°C was reached. Intermediate trays were made with a hold at 5 and -5°C for 20 min to ensure temperature homogeneity between the different vials during cooling. Sublimation was carried out by choosing a shelf temperature of -20°C (temperature increase from -50°C to -20°C at a rate of 1°C / min) and a pressure of 0.1 mbar and was carried out for 14 h.The duration of this step can however vary depending on the number of vials to be lyophilized and can be monitored by following the evolution of the two pressure probes. The process parameters for the secondary drying step were programmed as follows: increase of the shelf temperature from -20 to 25°C at a rate of 0.1°C / min then maintain at 25°C for 4 hours. Finally, at the end of the lyophilization cycle all the vials were vacuum sealed before unloading from the device.

[0212] Several characterizations were carried out on the lyophilisates obtained. For the placebo lyophilisates used for cycle optimization, the characterizations are as follows: determination of the glass transition temperature by differential scanning calorimetry (DSC), observation of the structure of the lyophilisate by scanning electron microscope (SEM) and determination of the residual moisture content by the Karl Fischer method. For the lyophilisates containing the antibodies, the affinity of the antibody for its target was determined by indirect ELISA and SPR.

[0213] The physicochemical characterizations of the placebo lyophilizates demonstrated the production of an amorphous product characterized by a higher glass transition temperature for the trehalose-based lyophilizates (~ 100°C) compared to those containing sucrose (~ 60°C). Macroscopically, an adapted texture of the cakes was obtained without visible signs of collapse. A porous structure characteristic of the lyophilizates was observed by SEM (Figure 10). The lyophilizates had a moisture content of 0.5% on average for the trehalose formulations and 1.3% for the sucrose formulations.

[0214] Regarding indirect ELISA experiments aimed at studying the binding capacity of the TAT antibody to its antigenic target after the lyophilization process, it is commonly accepted that a coefficient of variation (CV) of the order of 10%, or even 20% (supplier sites) is frequently obtained and is considered satisfactory for the indirect ELISA method due to the numerous steps performed during this assay. In our experimental protocol, a CV of up to 10% was calculated on triplicates. Thus, to analyze our results, a variation in absorbance values ​​of less than 10% was not considered as a significant variation in the ability of the antibody to bind to its epitope.

[0215] In this test, tubulin is fixed to the bottom of the wells of the ELISA plate, then the control TAT antibody (non-lyophilized) or reconstituted after lyophilization is added. After washing, a secondary anti-mouse IgG antibody labeled with HRP (horseradish peroxidase) allows the quantification of the TAT antibodies fixed on the epitope in each well thanks to the oxidation of diamino benzidine catalyzed by HRP. The results are therefore expressed as a percentage of the HRP enzymatic activity, taking as reference 100% of activity measured for the control (non-lyophilized TAT antibody, in solution). The results of the ELISA tests are presented in Figures 11 and show that the ability of the TAT antibody to bind to tubulin after its lyophilization in the trehalose and sucrose formulations containing both 100 µg of antibody (Figure 11a) and 500 µg of antibody (Figure 11b), is similar to that observed under control conditions.

[0216] 5.2 Study of the influence of compression

[0217] The obtained crude lyophilisates were compressed to characterize their mechanical properties using a StylOne evolution press simulator equipped with 6 mm diameter flat cylindrical punches. To facilitate tablet manufacturing, the lyophilisates were redispersed by light mechanical action and then also mixed with a direct compression excipient (Di-Pac®) at a level of 50% (w / w) initially to improve flow properties and facilitate handling. External lubrication with magnesium stearate was used. Preliminary compressibility (Figure 12a) and tabletability (Figure 12b) tests on placebo formulations without antibodies were performed to determine the process parameters to be used.These tests, focusing on breaking stress and porosity, demonstrated that when using lyophilized disaccharides (trehalose or sucrose), with and without the addition of the excipient for direct compression Di-Pac®, low compression pressures were sufficient to obtain cohesive tablets (breaking stress of 1 MPa) compared to those commonly used in pharmaceutical compression. Indeed, in our experiments, pressures of around 25 MPa are sufficient to obtain a cohesive tablet, despite high porosity within the tablet. This result is in itself surprising and very far from the characteristics of the same disaccharide in its native form. These results guided the choice of a compression pressure range from 25 to 100 MPa for the compression of lyophilizates containing TAT.

[0218] Antibody tablets were then manufactured by compressing the lyophilisates at 25, 50 and 100 MPa. An ELISA test was then used to evaluate the influence of compression pressure on the ability of the TAT antibody to bind to tubulin after reconstitution of the tablets in liquid medium. As previously described, the amount of TAT antibody bound to the tubulin epitope is revealed by a secondary antibody labeled with HRP, and by the oxidation of DAB, the HRP substrate. For this study, the absorbance values ​​measured from the TAT antibody lyophilisates after reconstitution in liquid medium were normalized to 100% of the enzymatic activity.

[0219] The results presented in Figure 13a show that when sucrose is used as a cryo- and lyoprotectant excipient in the formulation, compression of the lyophilizate-Di-pac® mixture under a compression pressure of 25 MPa does not impact the antibody binding affinity compared to its lyophilized form. However, it is observed that when compression pressures of 50 and 100 MPa are applied, the antibody binding activity is significantly impaired with, respectively, losses of -14 and -16% of enzymatic activity evaluated in comparison with the lyophilized form of the antibody.

[0220] Regarding the formulation containing trehalose as a cryoprotectant in admixture with Di-Pac®, the results presented in Figure 13b indicate that the compression process does not seem to affect the binding affinity of the antibody regardless of the compression pressure applied (between 25 and 100 MPa). In order to further investigate the results obtained and to determine the influence of the presence of Di-Pac® on the binding affinity of the TAT antibody to its target, tubulin, it was decided to compress the lyophilisates alone without the addition of a direct compression excipient. The results of the ELISA tests (Figure 14) show that following the compression of the lyophilisates containing the antibody under compression pressures ranging from 25 to 100 MPa, the binding capacity of the antibody to its epitope is fully preserved in both the sucrose and trehalose formulations.For the system formulated with trehalose as a cryoprotectant, the addition of Di-Pac® therefore has no influence on the maintenance of the biological activity of the antibody.

[0221] Finally, the influence of the amount of antibody in the formulation was studied by increasing the amount from 100 to 500 µg. Compression was performed without the addition of Di-Pac® only at the pressure of interest (25 MPa) allowing sufficient cohesion (breaking stress of 1.5 MPa). The ELISA test shows the same binding affinity of the antibody after compression at 25 MPa compared to the lyophilizate and the positive control for both formulations (Figure 15).

[0222] To further investigate these results, the binding affinity of the TAT, 5E2.2.1 and 4F3.2.1 antibodies after their lyophilization (trehalose formulation containing 500 µg of antibody) and then after their compression (25 MPa) was measured by SPR, a very sensitive and precise technique that allowed us to measure the binding affinity (KD) of the Fab fragments of each antibody to their respective epitope, and that of their crystallizable Fc fragment to streptococcal protein G. The results of these analyses are presented in Table 2 and show that the antibodies reconstituted after lyophilization (freeze-dried) but also after compression (tablet) have affinity constants for their Fab and Fc fragments that are similar to those of the antibodies before their formation by lyophilization (solution).This study demonstrates that neither the lyophilization process nor the compression process have a deleterious impact on the integrity of the antibody under these formulation and process conditions and highlights that the development approach undertaken with the TAT model antibody is transposable to antibodies in general.

[0223] [Table 2]

[0224] Measurement of affinity constants (KD) by SPR of TAT, 4F3.2.1 and 5E2.2.1 antibodies in solution, lyophilized, and compressed, against their respective antigenic target (Fab fragments) and against the G protein (Fc fragment).

[0225] In order to confirm that, beyond their binding affinity (see results Table 2), the biological activity of the anti-Hyrl 4F3.2.1 and anti-Cdlys 5E2.2.1 antibodies of interest is also preserved after their compression forming, experiments similar to those used previously for the selection / characterization of these antibodies were carried out.

[0226] For the anti-cdlys antibody 5E2.2.1: study of the neutralizing property of the antibody by LDH dosage (see point 2.1.2) after lyophilization (FD) and compression (tablet) processes

[0227] Here, lyophilizates and tablets (25 MPa) containing 150 pg of the 5E2.2.1 antibody were prepared and then reconstituted in DMEM / SVF10% cell culture medium supplemented with Candidalysin (10 pM) before being applied to a cell lawn of A-431 epithelial cells. The amount of LDH released by the cells after 24 h of incubation at 37°C was measured and compared to a condition in the absence of the antibody (o mAb, 100% of the cytolytic activity of the toxin) and in the presence of the antibody having not undergone any shaping process (solution). The results presented in Figure 16 indicate that the presence of the reconstituted 5E2.2.1 antibody after lyophilization and after its compression shaping at 25 MPa significantly reduces, and with an efficiency comparable to that of the antibody in solution, the release of LDH by epithelial cells into the culture medium. This result demonstrates the neutralizing property of the 5E2.2 antibody.1 with respect to the candidalysin toxin is preserved following the freeze-drying and compression processes.

[0228] For the anti-Hyrl antibody 4F3.2.1: study of the antibody's ability to detect the Hyrl protein expressed in its physiological form in vivo by immunofluorescence (see point 3.1)

[0229] Here, lyophilizates and tablets (25 MPa) containing 100 pg of the 4F3.2.1 antibody were prepared, reconstituted in liquid medium, and used for immunolabeling of C. albicans hyphae. The antibody binding was then revealed by a fluorescein (FITC)-labeled secondary antibody. As shown in Figure 17, the antibody retains its ability to bind to the Hyrl protein expressed on the hyphal surface both after lyophilization and after an additional compression step at 25 MPa.

[0230] All these results demonstrate that neither the lyophilization process nor the compression process have any impact on the biological activity of the antibodies of interest.

[0231] Furthermore, the impact of the compression process during the combined compression of the two anti-Cdlys 5E2.2.1 and anti-Hyrl 4F3.2.1 antibodies was studied. To this end, the ability of the 5E2.2.1 and 4F3.2.1 antibodies to bind to their respective antigenic targets, the Candidalysin protein and the Hyrl protein, was evaluated by ELISA using (1) a 5% (w / v) trehalose + 0.005% Tween 20 solution containing a mixture of the two antibodies (100 μg of each antibody / mL), (2) lyophilisates reconstituted in liquid medium obtained after lyophilization of this solution and (3) tablets reconstituted in liquid medium obtained by compression of the lyophilisates at the compression pressure of interest of 25 MPa. As before, the measured absorbance values ​​were expressed as a percentage of the HRP enzymatic activity, taking as reference 100% activity the absorbance values ​​measured from the formulation of the two antibodies in solution.

[0232] The results presented in Figure 18 show that for each of the antibodies, neither their simultaneous lyophilization, nor even their simultaneous compression at a compression pressure of 25 MPa, significantly impacts their respective capacity to bind to their antigenic target. This result demonstrates that under these operating and formulation conditions, the different tablet forming processes integrating the 5E2.2.1 and 4F3.2.1 antibodies do not affect the integrity of these antibodies when they are combined in the formulation.

[0233] 5.3 Study of the stability of compressed antibodies

[0234] A stability study of the TAT antibody (100 ug) compressed at 25 MPa in a trehalose base was initiated. The stability of the reconstituted antibody was measured as previously by ELISA test after storage of the tablets at 4°C, 25°C, 30°C or 40°C for 15 days, 1 month, 2 months, 3 months and 6 months. The enzymatic activity measured by ELISA using the antibodies reconstituted from the tablets was compared to that obtained from a trehalose-based lyophilisate containing 100 ug of TAT antibody stored at 4°C for these same periods.

[0235] The results presented in Figure 19 indicate that the ability of the TAT antibody to bind to its epitope is not impaired after storage in tablet form for 6 months at 5 and 25°C and is also not impaired after storage in tablet form for 4 months at 30°C. The binding capacity of the TAT antibody after compression is impaired only after 3 months of storage at 40°C. These results show that antibody tablets can be stored at room temperature (temperature controlled 25 + / - 2 °C according to ICH recommendations) for long periods (6 months) without loss of activity. Thus, the stability / activity of the antibodies would not be affected if during storage or transport periods a relatively short temperature excursion occurred.

[0236] We were thus able to define conditions which allow, under low pressure, to obtain antibody tablets based on lyophilisate whose mechanical resistance is very satisfactory. The tablets produced are characterized by a high residual porosity, of the order of 35%. Under these conditions, the 2 process steps, lyophilization, then compression, allow to respect the molecular structure of the monoclonal antibodies tested and to maintain, after redispersion of the tablet form, the biological activity of the antibodies without modification of their binding affinity to their epitope. All of these results are clearly not trivial for those skilled in the art and complete the new therapeutic strategy of passive vaccination for the treatment of a superficial fungal infectious pathology based on the two monoclonal antibodies developed in this invention, the anti-Cdlys and anti-Hyrl antibodies. 5.4 Study of the influence of the vaginal environment on the release and preservation of the integrity of antibodies formulated in tablet form.

[0237] Considering its unique physiological and physicochemical properties (acidic pH 4-5), the influence of the female vaginal environment on the preservation of the integrity of antibodies formulated in tablet form was studied. For this purpose, compression tests were performed on lyophilisates formulated with trehalose containing 100 µg of the TAT antibody and then the ability of the antibody to bind to tubulin after reconstitution of the tablets in vaginal fluid simulant (VFS, described by Owen and Katz in 1999, pH 4.2) was evaluated by EEISA. For this experiment, different tablet reconstitution times were considered (0, 24, 48 h) and the absorbance values ​​measured from antibody tablets reconstituted in PBS were normalized and expressed as 100% of the enzymatic activity.

[0238] The results of the EEISA are presented in Figure 20 and show that the ability of the TAT antibody formulated in tablet form to bind to its antigen is not significantly altered following its reconstitution in an environment approaching vaginal environmental conditions, up to 48 hours after its reconstitution.

[0239] 5.5 Development of a mucoadhesive tablet formulation

[0240] The mucoadhesive tablet dosage form is already known and used for different treatments, including for prolonged release forms (for example, see application EP3116517). Many polymers can be used as formulation excipient for their bioadhesive property. Among these excipients, hydroxypropylmethylcellulose (HPMC), a semi-synthetic polymer derived from cellulose, can be mentioned. Preliminary tabletability study tests were carried out on placebo lyophilizates formulated with trehalose 5% (w / v) and Tween 20 (0.005%); mixed with HPMC of different viscosity grades (K15 and K100) and at different percentages (w / w) (10, 20, 30%) in order to characterize the mechanical properties of these formulations in compression.These tests demonstrated that at a level of 10 to 30% HPMC in the formulation, the application of a low compression pressure of 25 MPa is sufficient to obtain a cohesive tablet (breaking stress > 1 MPa) for both HPMC Kl 5 and Kl 00 (figure 21).

[0241] To determine whether the presence of HPMC in the formulation could impact the integrity of the antibody, compression tests (25 MPa) were performed on a formulation incorporating a trehalose lyophilisate containing 100 µg of the TAT model antibody and HPMC K15 powder at a concentration of 20% (w / w). The ability of the antibody to bind to its antigenic target, tubulin, before and after the compression process was then studied by ELISA after reconstitution of the samples in liquid medium.

[0242] The results presented in Figure 22 show that the absorbance values ​​measured on the raw lyophilisates and in the presence of HPMC are similar, indicating that the propensity of the TAT antibody to bind to tubulin is not altered when HPMC is added to the formulation in powder form (i.e. before compression). Furthermore, the binding capacity of the antibody is fully maintained following the compression process in the presence of HPMC. These results highlight that the addition of HPMC to the powder formulation for compression has no influence on the integrity of the antibody and encourage the use of this excipient for the development of mucoadhesive tablets containing antibodies.

[0243] Tests aimed at evaluating the mucoadhesive property of placebo tablets formulated from trehalose lyophilisates in the presence of HPMC were undertaken. The system setup, inspired by the work of Baloglu et al., (2011) consists of the slow hydration of the tablet, generated by a syringe pump with a controlled flow rate of 0.3mL / h. This method was used to mimic the flow rate of secretions in the vaginal cavity which amounts to 6mL / 24h. In our setup, the tablet is previously placed in contact with the surface of a moistened pig mucin tablet and immobilized by manual pressure for 10 sec in order to mimic a phenomenon of adhesion to a mucosa.

[0244] Experiments implementing this system were carried out on tablets obtained from trehalose lyophilisates incorporating 10, 20, and 30% of HPMC Kl 5 and showed mucoadhesion times of at least 24 hours (Figure 23). Longer times are not accessible by this experiment due to the disintegration of the mucin support, but the demonstration of the mucoadhesive character of the formulated tablets is obtained.

[0245] An in vitro release study of the TAT-1 model antibody after compression shaping was undertaken. Here too, slow hydration of the tablets (0.5 mL / h) was performed using a device similar to that used previously, with a syringe pump, in order to best reproduce the physiological conditions of the vaginal cavity. The tablets were hydrated with PBS and the amount of antibody released was measured over time. This release study was performed on different formulations of trehalose lyophilizate tablets incorporating HPMC of different grades (HPMC K4, Kl 5 or Kl 00) and in different proportions in the final formulation (10, 15 or 20%).

[0246] The release profiles of the different formulations tested are shown in Figure 24. It is observed that the antibody release kinetics can be modulated over a wide time range, from relatively short periods (>90% released after 10 hours for the 10% HPMC K4 condition) to periods exceeding four days for systems containing HPMC K1 00.

[0247] Specifically, both formulations incorporating HPMC Kl 00 show zero-order release kinetics, indicating a constant release of the antibody as a function of time, independent of its initial concentration in the tablet.

[0248] In contrast, the release profiles of formulations containing 10% HPMC grade K4 and K1 5 are less linear. Modeling of the release mechanism using Weibull and Korsmeyer-Peppas models suggests that, for these two formulations, antibody release is primarily driven by a complex release mechanism incorporating a tablet swelling step followed by tablet erosion.

[0249] All of these results show that the mucoadhesive tablet form opens up the possibility of modulating the conditions of antibody release over desired times and by controlling the mechanisms of availability.

[0250] Bibliographic references

[0251] Cassone, A. 2015, Vulvovaginal Candida Albicans Infections: Pathogenesis, Immunity and Vaccine Prospects. BJOG: An International Journal of Obstetrics and Gynecology 122(6): 785-794.

[0252] Clancy, Cornelius J., and M. Hong Nguyen. 2011, Systemic Candidiasis: Candidemia and Deep-Organ Infections. In Candida and Candidiasis Pp. 429-441. John Wiley & Sons, Ltd.

[0253] Boisramé, Cornu, Da Costa, Richard. 2011 , Unexpected role for a serine / threonine-rich domain in the Candida albicans Iff protein family. Eukaryot Cell. 2011 Oct; 10(10): 1317-30.

[0254] De Bernardis, Elavia, Haiqun Liu, Rachel O’Mahony, et al. 2007, Human Domain Antibodies against Virulence Traits of Candida Albicans Inhibit Eungus Adherence to Vaginal Epithelium and Protect against Experimental Vaginal Candidiasis. The Journal of Infectious Diseases 195(1): 149-157.

[0255] De Bernardis, Flavia, Mario Amacker, Silvia Arancia, et al. 2012, A Virosomal Vaccine against Candidal Vaginitis: Immunogenicity, Efficacy and Safety Profile in Animal Models. Vaccine 30(30): 4490-4498.

[0256] Ibrahim, Ashraf S., Guanpingsheng Luo, Teclegiorgis Gebremariam, et al. 2013, NDV-3 Protects Mice from Vulvovaginal Candidiasis through T- and B-Cell Immune Response. Vaccine 31(47): 5549-5556.

[0257] Luo, Guanpingsheng, Ashraf S. Ibrahim, Brad Spellberg, et al. 2010. Candida Albicans Hyrlp Confers Resistance to Neutrophil Killing and Is a Potential Vaccine Target. The Journal of Infectious Diseases 201(11): 1718-1728.

[0258] Mogavero, Selene, Frank M. Sauer, Sascha Brunke, et al. 2021, Candidalysin Delivery to the Invasion Pocket Is Critical for Host Epithelial Damage Induced by Candida Albicans. Cellular Microbiology 23(10): el3378.

[0259] Moyes, David L., Duncan Wilson, Jonathan P. Richardson, et al. 2016, Candidalysin Is a Fungal Peptide Toxin Critical for Mucosal Infection. Nature 532(7597): 64-68.

[0260] Revankar, Sanjay G., and Jack D. Sobel. 2011, Mucosal Candidiasis. In Candida and Candidiasis Pp. 417-427. John Wiley & Sons, Ltd.

[0261] Richard, M.L., Plaine, M. 2007, Comprehensive analysis of glycosylphosphatidylinositol- anchored proteins in Candida albicans. Eukaryot Cell. 2007 Feb;6(2):l 19-33.

[0262] Richardson, J. P., H. M. E. Willems, and D. L. Moyes. 2018, Candidalysin Drives Epithelial Signaling, Neutrophil Recruitment, and Immunopathology at the Vaginal Mucosa. Infect Immun. 2018 Jan 22;86(2):e00645-17

[0263] Yano, Junko, Brian M. Peters, Mairi C. Noverr, and Paul L. Fidel. 2018, Novel Mechanism behind the Immunopathogenesis of Vulvovaginal Candidiasis: “Neutrophil Anergy.” Infection and Immunity 86(3).

Claims

Claims 1. An antibody against candidalysin, comprising a candidalysin binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO:2 (CDR1-H1), SEQ ID NO:3 (CDR2-H2) and SEQ ID NO:4 (CDR3-H3), respectively; and the VL comprises SEQ ID NO:5 (CDR1-L1), SEQ ID NO:6 (CDR2-L2) and SEQ ID NO:7 (CDR3-L3), respectively.

2. Antibody according to claim 1, said antibody being a neutralizing antibody.

3. An antibody directed against an IFF family protein, comprising a Hyrl-binding domain, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises SEQ ID NO: 11 (CDR1-H1), SEQ ID NO: 12 (CDR2-H2) and SEQ ID NO: 13 (CDR3-H3), respectively; and the VL comprises SEQ ID NO: 14 (CDR1-L1), SEQ ID NO: 15 (CDR2-L2) and SEQ ID NO: 16 (CDR3-L3), respectively.

4. Antibody according to claim 3, said antibody being capable of binding to the Hyrl protein.

5. Antibody according to claim 3 or 4, said antibody being an opsonizing antibody.

6. Antibody according to any one of claims 1 to 5, said antibody being a human or humanized antibody.

7. Pharmaceutical combination comprising (i) an antibody directed against candidalysin, or a fragment of this antibody, and (ii) an antibody directed against a protein of the IFF family, in particular the Hyrl protein, or a fragment of this antibody.

8. Pharmaceutical combination according to claim 7, wherein the antibody directed against candidalysin is a neutralizing antibody and / or wherein the antibody directed against an IFF protein is an opsonizing antibody.

9. A pharmaceutical combination according to claim 7 or 8, wherein the antibody directed against candidalysin is an antibody according to claim 1, 2 or 6.

10. Pharmaceutical combination according to any one of claims 7 to 9, wherein the antibody directed against a protein of the IFF family is an antibody according to any one of claims 3 to 6.

11. Pharmaceutical combination according to any one of claims 7 to 10, said combination being a kit comprising (i) a first composition comprising the antibody directed against candidalysin, and (ii) a second composition comprising the antibody directed against an IFF protein, in particular against the Hyrl protein.

12. Pharmaceutical combination according to claim 11, the first and second compositions being in the form of a tablet, optionally mucoadhesive.

13. Pharmaceutical composition comprising the antibody according to any one of claims 1 to 6, or the combination according to any one of claims 7 to 10.

14. Pharmaceutical composition according to claim 13, said composition being a tablet, optionally mucoadhesive.

15. Antibody according to any one of claims 1 to 6, pharmaceutical combination according to any one of claims 7 to 12, or pharmaceutical composition according to claim 13 or 14, for use in the treatment of candidiasis, in particular vulvovaginal candidiasis.

16. A process for the preparation of a tablet comprising an antibody, characterized in that it comprises the following steps: (a) preparation of an antibody solution; (b) lyophilization of the antibody solution; and (c) compression of the lyophilized powder.

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