FR2842812A1 - Solubilizing hydrophobic peptides, useful e.g. in vaccines against infectious microbes or tumors, by attachment of at least three lysine residues - Google Patents

Abstract

Solubilizing, or improving solubility of, a peptide (I) in aqueous medium comprising covalent attachment of at least 3 residues of Lys, in L or D form, distributed over the N and/or C termini in the form of a linear or branched chain, is new. Independent claims are also included for: (1) peptide ligand (Ia) of MHC (major histocompatibility complex) modified by attachment of at least 3 residues of Lys, in L or D form, distributed over the N and/or C termini in the form of a linear or branched chain; and (2) vaccine containing at least one peptide that contains at least one hydrophobic epitope derived from an antigenic protein of a microbial pathogen or tumor and modified by the new method or is (Ia). ACTIVITY : Virucide; Antibacterial; Parasiticide; Fungicide; Cytostatic. The modified influenza matrix M1 peptide (4K-FluM1) of sequence (Arg) 4-Glu-Ile-Leu-Gly-Phe-Val-Phe-Thr-Leu was administered (twice, 100 Microg plus 300 Microg of the P40 adjuvant) subcutaneously to mice. Ten days later, spleen cells were recovered and cultured in presence of interleukin-2 and of fixed antigen-presenting cells loaded with FluM1. The effector cells were restimulated after 7 days, then after a further 5 days they were tested in a chromium release assay against EL4 A2/kb cells loaded with peptide. At an effector:target ratio 100, the specific lysis was about 75%. MECHANISM OF ACTION : Vaccine; Gene Therapy.

Description

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 The subject of the present invention is a general process for the solubilization in an aqueous medium of a hydrophobic peptide, in particular of a peptide compound of pharmaceutical interest, such as a hydrophobic major histocompatibility complex (MHC) ligand soluble in solvents such as DMSO. The invention also relates to such MHC ligands thus modified in particular as an anti-infective or antitumor drug and their use for the preparation of a pharmaceutical composition intended to generate an immune response mediated by cytotoxic T lymphocytes. The invention also relates to the use of such ligands mixed or coupled to a carrier protein for the preparation of a vaccine composition for the prevention of infectious diseases or the treatment of cancers.

 Vaccination is an effective way to prevent or reduce viral or bacterial infections. The success of vaccination campaigns in this area has helped to expand the concept of vaccine in the field of autoimmune diseases and cancer.

 In the context of anti-viral or anticancer therapy, the generation of CTL and auxillary T responses is of any importance (Bachmann et al., Eur J.

Immunol., 1994, 24, 2128-2136; Borrow P., J. Viral Hepat., 1997, 4, 16-24, Rivoltini et al., Crit. Rev. Immunol., 1998, 18, 55-63), as evidenced by numerous studies showing in vivo the protective role of responses directed against viral epitopes (Arvin AM., J. Dis.D., 1992, 166, S35- S41, Koszinowski et al., Immunol Lett., 1987, 16, 185-192).

 Cytotoxic T lymphocytes recognize peptides derived from the degradation of intracellular proteins that are presented by the class # 5 major histocompatibility complex (MHC) molecules on the cell surface.

After processing the proteins, the CTL peptides are selected from the pool of cytosolic peptides on the basis of their length and their amino acid sequence and transported to the endoplasmic reticulum and then associated with the class I MHC molecules. Several studies have shown that these peptides naturally presented by the MHC class I are generally 91 amino acids in length and contain specific anchoring residues which have particular properties in terms of hydrophobicity and charge (Falk K. et al., Nature 1991, 351: 290-296, Hunt DF et al., Science, 1992, 255: 1261-1263). The quality and position of these anchor residues are dictated by the pockets of the antigen presenting groove specific to

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 each MHC class I allele (Matsumara M. et al., Science, 1992, 257: 927-934) suggesting that anchor residues are primarily involved in MHC class I binding. This hypothesis seems to be confirmed by the fact that that any alteration (deletion replacement) of the anchoring residues of these CTL peptides abrogates their attachment to the MHC class I, and that any mutation of the MHC class # molecules at the level of the fixation pockets modifies the peptide repertoire (Ruppert J. et al., Cell., 1993, 74: 929-937; Rohren EM et al., J. Exp. Med., 1993, 177: 1713-1721).

 However, several studies show that longer peptides can accommodate an association with HLA-A2 molecules (EJ Collins et al., Nature, 1994, 371, 666-629, Y. Chen et al., J. Immunol., 1994, 152, 2874- 2881) or other HLA class I molecules (RG Urban et al., PNAS, USA, 1994, 91, 1534-1538).

 It has also been shown in the literature that modifications to CTL epitopes at the N- or C-terminal of the peptide could either be able to maintain the CTL activity of the peptide while increasing its stability (Brinckerhoff LH et al., 1999). J. Cancer, 83, 326-334, Loing E. et al., 2000, J. Immunol., 164, 900-907), or lead to a complete loss of peptide activity (Beck et al. 2001, J.

 Pep. Res. 57,528-538; Widmann et al., 1991, J. Immunol., 147, 3745-3751).

 Many MHC ligands (classes 1 and II) and especially CTL epitope peptides have been identified (HG Rammensee et al., Immunogenetics, 1999, 50 (3-4), 213-219, Sette et al., Immunogenetics, 1999). , 50, 201-212). The interest of these MHC ligands is confirmed by the increasing number of clinical studies in humans of these compounds as vaccine candidates against various pathologies, especially against melanoma (Jager E. et al., J. Int. Cancer, 2000, 86, 538-547, ML Salgaller et al., Cancer Research, 1996, 56, 4749-4757, SA Rosenberg et al., 1998 Nature Medecine, 4, 3, 321-327, SR Reynolds et al. 1998, J. Immunol., 161, 6970-6976), against HBV (Livingston et al., J. Immunol., 1999, 162, 3088-3095) and other antigens (reviewed by CJM Melief and WM Kast Immunological Reviews, 1995, 146, 167-177). However, the difficulty of these studies lies both in the fact that these peptides are very often not very immunogenic (Melief et al., 1992, Adv Cancer Res., 58, 143-175, Nanda and Sercarz, 1995, Cell, 82, 13-17) therefore require the presence of a carrier and an adjuvant, but also in that they are very often hydrophobic and therefore require the use of solvents such as DMSO for their solubilization before their administration to patients . The presence of DMSO is very often detrimental to the carrier protein which can precipitate, posing problems of

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 reproducibility of injectable preparations, which is not tolerable for pharmaceutical use.

 In addition, the use of DMSO remains problematic especially during repeated injections during anticancer therapies. Indeed, DMSO is rarely associated with an injectable form because of its significant pharmacological and toxicological activities. It is included in only two idoxuridine-based specialties (C. Heintz and C. Boymond STP PHARMA 5, 1989, 548-560).

 Our study aimed at solubilizing hydrophobic peptides was carried out on two model CTL peptides described in the literature as being very hydrophobic: FluM1 peptide 58-66 of the M1 protein of the influenza virus and the peptide ELA 26-35 of the Melan / Mart-1 protein, an antigen described as being associated with tumor cells of melanoma. These two peptides are described below.

 Peptide FluM1 56-68 of sequence TKGILGFVFTLTV (SEQ ID No. 1) has been described as HLA-A2 restricted CTL epitope by Gotch et al. (Nature, 1987, 326, 881-882). This sequence was first optimized by Gotch et al. (J. Exp.

Med. 1988, 168, 2045-2057), which clearly showed that the FluM1 57-68, 58-68,59-68 peptides had the same CTL activity as the 56-68 FluM1 peptide. Bednarek et al. (J. Immunol 1991, 147, 4047-4053) also optimized this peptide and demonstrated that the FluM1 peptide 58-66 GILGFVFTL (SEQ ID No. 2) gave a CTL 100 response 1000 times greater than the peptide 57 -68.

 Since this peptide is highly hydrophobic, a study to render this peptide water-soluble has already been conducted (Bednarek et al J. Immunol Meth, 1991, 139, 41-47). The authors of this article have carried out this study on the peptide FluM1 57-68 which is less active than the peptide FluM1 58-66, substituting residues 58, 59, 60 as well as residues 65, 67 and 68 by amino acids that are less hydrophobic. making this peptide more water soluble.

 The dominant CTL epitopes derived from the HLA-A2 restricted Mart-1 melanoma associated antigen of AAGIGILTV (SEQ ID No. 45) and EAAGIGILTV (SEQ ID No. 46) sequences were identified by Y. Kawakami et al. (J. Exp Med., 1994, 180, 347-352).

 The ELA peptide of ELAGIGILTV sequence (SEQ ID No. 47) corresponding to the Melan26-35 peptide analogous to the EAAGIGILTV parent decamer (SEQ ID No. 46), has been described by Valmori D. et al. (International Immunology, Vol 11 N 12, pp1971-1979) as an excellent HLA-A2 ligand and capable of inducing a CTL response in A2-Kb mice greater than that obtained with the parent peptide.

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 This highly hydrophobic peptide is solubilized and stored in DMSO for bioassays. It appears that no studies to improve its solubility in aqueous media have been conducted.

 The ELA and FluM1 peptides have already been injected into humans solubilized in 20% DMSO (Jager E. et al., Int J Cancer, 2000, 86, 538-547) or in pure DMSO for ELA (Wang F. et al., Clinical Cancer Research, 1999,5, 2756-2765) in clinical studies in patients with melanoma.

 Thus, it remains desirable to have a general method for making soluble or improve the solubility in aqueous medium, especially in a medium for injection in humans, a peptide compound hydrophobic nature. Preferably said method must allow, if necessary, to retain at least a sufficient part of a biological activity of interest of said hydrophobic compound.

 More particularly, it remains desirable to have a method which makes it possible, on the one hand, to render soluble or to improve the solubility in aqueous medium injectable to humans, of a class I or II MHC hydrophobic peptide ligand, in particular a peptide ligand whose CTL epitope fragment is hydrophobic in nature, while retaining its ability to bind to the MHC molecules and / or its ability to induce a CTL response.

 This is precisely the object of the present invention.

 The inventors have indeed found that it is possible to very significantly improve the solubility in an aqueous medium, especially in an aqueous medium injectable to humans, peptide compounds having a desired activity such as those having a pharmaceutical interest such as MHC ligands comprising a CTL epitope of hydrophobic nature, this by adding to them at least a total of 3 lysine residues distributed at the N-terminal and / or C-terminal end of these hydrophobic ligands while retaining at least partly this biological activity sought when these peptide compounds are used in an aqueous medium.

 The inventors have demonstrated, quite surprisingly, that the addition of lysine residues at the N-terminal and / or C-terminal end of peptide, such as the MHC FluM1 ligand of SEQ ID No. 2 sequence. or the MHC ligand ELA of sequence SEQ ID No. 47 or the MTER ligands derived from telomerase hTERT 572 Y1 of sequence SEQ ID No. 66, hTERT 988 of sequence SEQ ID No. 67 and hTERT 988 Y1 of sequence SEQ ID No. 68, allowed to obtain an increased solubility of these peptides thus modified in aqueous medium while retaining for those tested their ability to induce a CTL response in an aqueous medium.

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 The inventors have for example demonstrated that the addition of 3 aspartic acid residues to the N-terminus or C-terminus of the MHC FluM1 ligand of SEQ ID No. 2 sequence if it does not interfere with the binding capacity of this peptide with HLA-A2 nevertheless causes a total loss of its ability to induce a CTL response when it is tested under the same conditions as the FluM1 peptide to which Lysine residues are added in N- or C-terminal, thus showing the additional difficulty of obtain both a better solubility for a peptide, while maintaining the biological activity sought for this peptide.

 Thus, in a first aspect, the present invention relates to a method for rendering soluble or improving the solubility in an aqueous medium of a peptide, characterized in that it comprises a step in which said peptide is modified by covalent addition of at least a total of 3 lysine residues, of L or D form, distributed at the N-terminal and / or C-terminal end of said peptide, these at least 3 total residues lysine may constitute a chain linear or connected.

 For the sake of clarity, in the present description, especially in the set of claims, it will be understood that, in the absence of additional precision by peptide or ligand of the MHC, the peptide or ligand of the MHC not yet modified by the method of the invention, in contrast to the term respectively modified peptide (or peptide thus modified) or modified MHC ligand (or MHC ligand thus modified).

 Preferably, the process of the invention is characterized in that the aqueous solulibility obtained for the peptide thus modified is sufficient so that at least a part of the biological activity of said unmodified peptide that is sought to be exerted or conserved by said peptide thus modified by the process of the invention when the latter is used in an aqueous medium.

 By biological activity for a peptide or for a peptide compound, is meant any biological activity such as, for example, and without limitation, an antigenic activity, in particular capable of inducing an immune response of humoral or cellular type, such as that the ability to bind to the MHC (MHC ligand), especially class # or II and, where appropriate, to generate helper T cells and / or CTL-type lymphocytes, or antibody activity, ligand receptor, receptor, inhibitor, enzymatic, hormonal, signal mediator, inducer, substrate for a biochemical reaction, etc.

 By at least a part of the biological activity for a modified peptide or for a modified peptide compound according to the method of the invention, it is intended to designate

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 any biological activity exerted by the unmodified peptide and which is at least partially exerted in an aqueous medium by the peptide or the modified peptide compound.

 It is also understood that also part of the invention is a process according to the invention in which a residue other than a lysine residue is also added to the N-terminus or C-terminus of said peptide compound, this furthermore said at least 3 lysine residues, if this other added residue does not significantly reduce the increase in solubility and, where appropriate, the biological activity observed in an aqueous medium for the modified peptide by adding only the lysine residues.

 In the present description, the term "peptide compound" will be understood to denote any compound comprising a peptide consisting of a sequence of natural amino acids or not, of L or D form, said peptide compound possibly being chosen especially from peptides, peptides, nucleic acids, lipopeptides or glycopeptides.

 In the present invention, the term "peptide" will also be understood to mean proteins or polypeptides, terms which will be used interchangeably here.

 By aqueous medium is meant very particularly an aqueous solution such as a buffer solution, including physiological, such as a saline buffer such as a phosphate buffered saline (PBS) or any aqueous solution for injection into humans.

 In a also preferred embodiment, the subject of the present invention is a process according to the invention, characterized in that the total number of lysine residues distributed at the N-terminal and / or C-terminal end of said peptide is between 3 and and 6, inclusive terminals, preferably between 3 and 5 inclusive.

 In a also preferred embodiment, said at least 3 lysine residues in total constitute a linear amino acid chain.

 In a also preferred embodiment, said at least a total of 3 lysine residues are added either to the N-terminal free end or to the C-terminal free end of said peptide.

 In a also preferred embodiment, said at least a total of 3 lysine residues added are of L-form.

 In a also preferred embodiment, said peptide (not yet modified by the process according to the invention) comprises at least 6 amino acids, preferably at least 7 or 8 amino acids.

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 In a also preferred embodiment, said peptide (not yet modified) comprises at most 50 amino acids, preferably at most 45.40 or 35 amino acids, more preferably at most 30.25, 20 or 15 amino acids.

 In a also preferred embodiment, the covalent addition of said at least 3 total lysine residues to the N-terminal and / or terminal free end of said peptide is carried out by chemical synthesis, or else by genetic fusion when the structure of said peptide thus modified allows it.

 In a also preferred embodiment, the modified peptide obtained by the process according to the invention is soluble in an aqueous medium, in particular in a PBS buffer, at least 5 mg / ml, preferably at least 6 or 7 mg / ml. ml, at least 7 mg / ml being the most preferred solubility minimum.

 In another preferred aspect, said peptide is a peptide useful for the preparation of a pharmaceutical or cosmetic composition.

 Preferably, said peptide is an active ingredient capable of exerting a therapeutic action (preventive or curative).

 More preferably, said peptide is a peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature.

 By antigenic epitope is meant here a peptide which when administered in an animal or man, alone or in the presence of adjuvant immunity or in combination with a carrier molecule, is capable of inducing a a humoral or cell-specific immune response directed specifically against this epitope, preferably capable of generating CTL lymphocytes directed specifically against this epitope.

 In another particularly preferred aspect, said peptide is a ligand, natural or synthetic, of MHC, in particular of class 1 or II.

 Preferably, said peptide is chosen from antigenic epitopes derived from an antigenic microorganism protein, preferably pathogenic for a mammal such as man, these microorganisms possibly being viruses, bacteria, yeasts, fungi or parasites, or derivatives thereof. of an antigenic protein associated with a tumor.

 Preferably, said peptide is a ligand, natural or synthetic, of MHC, characterized in that it can also be chosen from multiepitopic polypeptide constructs, pseudopeptides, retro-inverso peptide peptides, peptoids, peptides and peptides. -mimetics, or chosen from peptides

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 included in peptide compounds such as acid nucleic peptides, lipopeptides or glycopeptides.

 In the present invention, the term "multi-epitopic polypeptide" will be understood to mean a peptide construct comprising, on the same block, a set of several epitopes of peptide nature.

 In the present invention, the term "pseudopeptide" will be understood to mean any peptide, or analogous molecule, containing one or more non-peptide bonds between the amino acids.

X-X2-.........-X~-X"-COOH, un peptide de n acides aminés de séquence H2N-XnXn~i- X2-X1-COOH, où les acides aminés de la série L sont remplacés par des acides aminés de la série D, où H2N-Xi (ou H2N-Xn t) et Xn-COOH (ou X1-COOH) représentent respectivement le résidu d'acide aminé en position N-terminale et le résidu d'acide aminé en position C-terminale dudit peptide. In the present invention, the term "peptide retroinverso" will be used for a given peptide of n amino acids and of general sequence H2N-

X-X2-X'-X "-COOH, a peptide of n amino acids of the sequence H2N-XnXn-i-X2-X1-COOH, where the amino acids of the L-series are replaced by amino acids of the series D, where H2N-Xi (or H2N-Xn t) and Xn-COOH (or X1-COOH) represent respectively the amino acid residue in the N-terminal position and the residue of amino acid at the C-terminal position of said peptide.

 In the present invention, the term "peptoid" will be understood to mean oligomers obtained from N-substituted glycine derivatives.

 In the present invention, the term "peptidomimetic" will be understood to mean any molecule having at least one functional property specific to peptides and, more particularly, any molecule in which the amide linkages are completely or partially replaced by non-peptide bonds while retaining the amino acid chains essential for the activity in a defined spatial position and / or any molecule in which the CH are replaced by a nitrogen atom and / or any molecule in which the side chains of the amino acids are modified.

 Preferably, said unmodified peptide is a ligand, natural or synthetic, of MHC for the generation of cytotoxic T lymphocytes, characterized in that it is chosen from CTL epitopes.

 Preferably, said unmodified peptide is a ligand, natural or synthetic, of MHC for the generation of cytotoxic T lymphocytes, characterized in that it is chosen from CTL epitopes in the form of octapeptide, nonapeptide, decapeptide hydrophobic, preferably in the form of a peptide of at most 15 amino acids, or in the form of a grouping of hydrophobic epitopes.

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 Preferably, said peptide is a MHC ligand allowing the generation of cytotoxic T lymphocytes, characterized in that said MHC ligand comprises a peptide chosen from peptides of sequence SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47.

 Preferably, said peptide is a MHC ligand, characterized in that said MHC ligand comprises a peptide selected from the following peptides: a) a peptide whose amino acid sequence is the sequence of the retroinverso form of a peptide SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; and b) a peptide whose sequence has an identity level after optimal alignment of at least 70%, preferably at least 80%, 85%, 90% or 95% with the sequence of a sequence peptide SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47, and characterized in that said MHC ligand is capable of generating T lymphocytes of cytotoxic and / or T helper type, specific for said MHC ligand, and / or capable of generating a significant increase in the level of CD8 + and / or CD4 + T cells producing INF-γ and, if appropriate, TNF-α, when said MHC ligand is put in the presence of T lymphocytes of subjects having presented the antigens from which said ligand is derived.

notamment de classe 1 ou II, de nature peptidique, modifié par l'adjonction d'au moins au total 3 résidus lysine, répartis à son extrémité N-terminale et/ou C-terminale, ces 3 résidus lysine pouvant constituer une chaîne linéaire ou branchée, de préférence le ligand du CMH avant modification étant de nature hydrophobe. In another aspect, the present invention relates to a MHC ligand,

in particular of class 1 or II, of peptide nature, modified by the addition of at least 3 total lysine residues, distributed at its N-terminal and / or C-terminal end, these 3 lysine residues being able to constitute a linear chain or branched, preferably the MHC ligand before modification being hydrophobic in nature.

 By the hydrophobic term in the present description, it is meant for a peptidic MHC peptide or ligand, a peptide or ligand MHC not soluble in an aqueous medium, or insufficiently soluble to be able to exploit in this aqueous medium the together or at least one of the biological activities that can be exerted by the same peptide or MHC ligand in another medium (especially in a medium containing DMSO).

 Preferably, said MHC ligand according to the invention is characterized in that the total number of lysine residues distributed at the N-terminal and / or C-terminal end of said ligand is between 3 and 6 inclusive, preferably between 3 and 5, terminals included.

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 More preferably, said at least a total of 3 lysine residues constitute a linear amino acid chain.

 More preferably, said at least a total of 3 lysine residues are added either to the N-terminal free end or to the C-terminal free end of said MHC ligand.

 More preferably, all the lysine residues added is of L-form.

 More preferably, said MHC ligand (unmodified) comprises at most 50 amino acids, preferably at most 45.40 or 35 amino acids, more preferably at most 30.25, 20 or 15 amino acids.

 Of these MHC ligands (unmodified), MHC ligands of up to 15 amino acids are preferred.

 Preferably, said unmodified peptide-like MHC ligand is capable of generating cytotoxic T lymphocytes and / or helper T lymphocytes, in particular specific for said MHC ligand and / or capable of generating a significant increase in the level of CD8 + T lymphocytes and / or CD4 + producing INF-γ and, if appropriate, TNF-α, when said MHC ligand is brought into the presence of T cells of subjects having presented the antigens from which said ligand is derived; and, is characterized in that the solubility in an aqueous medium of said peptide MHC ligand before modification is significantly lower than that under the same conditions of said MHC ligand thus modified according to the invention.

 Preferably, said unmodified MHC ligand is not soluble in aqueous medium, especially in phosphate buffered saline (PBS), at a concentration greater than 1 mg / ml.

 Preferably, the solubility difference evaluated in PBS buffer between said unmodified MHC ligand and said modified MHC ligand is greater than or equal to 2, preferably 3 and 4 mg / ml.

 As solubility calculation method, we can refer in particular to the method as described in Example 2.

 Preferably said modified MHC ligand is characterized in that it is soluble in an aqueous medium, especially in phosphate buffered saline (PBS), at least 5 mg / ml, preferably at least 6 or 7 mg / ml, 7 mg / ml being the most preferred minimum solubility.

 Preferably, said MHC ligand thus modified is capable in aqueous solution of generating cytotoxic T lymphocytes and / or T helper, in particular specific for said MHC ligand, and / or capable of generating an increase

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 of the level of CD8 + and / or CD4 + T lymphocytes producing INF-γ and, if appropriate, TNF-α, when said MHC ligand is placed in the presence of T cells of subjects having presented antigens including said ligand of the CMH is issued.

 More preferably, said MHC ligand according to the invention is characterized in that it is selected from CTL epitopes.

 More preferably, said MHC ligand according to the invention is characterized in that it is chosen from CTL epitopes in the form of octapeptide, nonapeptide or decapeptide or a grouping of hydrophobic epitopes.

 More preferably, said peptide MHC ligand before modification comprises one or consists of a peptide chosen from the peptides of the following sequences: a) SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; b) a peptide whose amino acid sequence is the sequence of the retroinverso form of a peptide of sequence SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; and c) a peptide whose sequence has an identity level after optimal alignment of at least 70%, preferably at least 80%, 85%, 90% or 95% with the sequence of a sequence peptide SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47.

 Among said MHC ligands thus modified according to the invention, peptide MHC ligands chosen from the peptides of sequence SEQ ID Nos. 5.6, 7, 43 and 44, and the peptides of sequence SEQ ID Nos. 50,51, 52,53 and 54, these peptides possibly being able to comprise a point mutation in the sequence of the unmodified MHC ligand from which it is derived, in particular a substitution of one amino acid by another natural amino acid. or not natural.

 In another aspect, the present invention relates to a peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature, said peptide having been modified by a method according to the invention, or a MHC ligand according to the invention as a medicament .

 In another aspect, the subject of the present invention is a pharmaceutical composition comprising a compound chosen from: a peptide modified by a process according to the invention, or a modified MHC ligand according to the invention, where appropriate mixed or coupled with a carrier protein or one of its fragments;

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 a nucleic construct containing a DNA encoding a peptide modified by a method according to the invention, or for a modified MHC ligand according to the invention, where appropriate further containing a DNA coding for a carrier protein or one of its fragments.

 In another aspect, the present invention relates to the use of a peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature derived from an antigenic protein of pathogenic microorganism, said peptide having been modified by a method according to US Pat. invention, or a modified MHC ligand according to the invention comprising such antigenic epitopes, for the preparation of a vaccine for the prophylactic or therapeutic treatment of infections by this microorganism, in particular viral, bacterial, parasitic or fungal infections .

 In another aspect, the present invention relates to the use of a peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature derived from a tumor-associated antigenic protein, said peptide having been modified by a method according to the invention, or a modified MHC ligand according to the invention comprising such antigenic epitopes, for the preparation of a vaccine intended for the prophylactic or therapeutic treatment of cancers associated with this antigen, preferably to inhibit the growth of associated tumors to this antigen.

 In another aspect, the present invention relates to a vaccine, characterized in that it comprises at least one peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature derived from an antigenic protein of pathogenic microorganism or derived from an antigenic protein associated with a tumor, said peptide having been modified by a method according to the invention, or in that it comprises a MHC ligand modified according to the invention comprising at least one such antigenic epitope.

 The present invention also relates to a vaccine according to the invention, characterized in that it further comprises an adjuvant, preferably selected from the following adjuvants: type A outer membrane proteins, called Enterobacterium OmpA, TT, PLGA, ISCOM, IFA, Montanide, ISA 720, ISA 51, lipid quaternary ammonium, MPL-A, Quil-A and other saponins (QS21) CpG.

 The present invention also relates to a vaccine according to the invention characterized in that it further comprises a mixed or coupled carrier compound.

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 said modified peptide comprising one or more antigenic epitopes or said modified MHC ligand.

 The present invention also relates to a vaccine according to the invention characterized in that said modified peptide comprising one or more antigenic epitopes or said modified MHC ligand, optionally associated with a carrier compound, is incorporated into vectors chosen from liposomes, virosomes, nanospheres, microspheres or microcapsules.

 The subject of the invention is also a vaccine according to the invention, characterized in that said modified peptide comprising one or more antigenic epitopes or said modified MHC ligand, optionally associated with a carrier compound, is transported in a form which makes it possible to improve its stability and / or immunogenicity.

 Among the vehicles making it possible to improve the stability and / or the immunogenicity of said peptides, liposomes, PLGAs or other polymers as well as viral or bacterial vectors containing a nucleic construct coding for said peptide and optionally also said protein, are particularly preferred. carrier, or one of its fragments.

 Preferably, said carrier compound is a carrier protein selected from the TT protein (Tetanus toxoid) or its derivatives, the DT protein (Diphtheria toxoid) or its derivatives, KLH (Key Limpet Haemocyanin), heat shock proteins or HSP, Omp-type bacterial membrane proteins, in particular enterobacteria, or their fragments.

 In the present invention, the term Omp (for Outer Protein Membrane), the proteins of the outer membrane and OmpA will be referred to as those of type A.

 By fragment or compounds derived from a carrier protein is meant in particular any amino acid sequence fragment included in the amino acid sequence of the carrier protein which, when associated with an immunogen, is capable of generate or increase an immune response, in particular a humoral and / or CTL-like response, directed against said immunogen and comprising at least 8 consecutive amino acids, preferably at least 10 amino acids or more preferably at least 15,20, 30 or 50 amino acid sequence of the amino acid sequence of said carrier protein, their derivative compounds comprising at least one of its fragments.

 In a particular embodiment, the invention comprises a vaccine according to the invention in which the carrier Omp carrier protein of enterobacteria or one of its

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 fragments, is obtained by a method of extraction from a culture of said enterobacterium or recombinantly.

 The methods for extracting bacterial membrane proteins are known to those skilled in the art and will not be developed in the present description.

For example, but not limited to the extraction method described by Haeuw J.F. et al. (Eur J. Biochem, 255, 466-454, 1998).

 Recombinant protein preparation methods are also now well known to those skilled in the art. Among the cells that can be used for the production of these recombinant proteins, mention may be made, for example, of bacterial cells (Olins PO and Lee SC, 1993, Curr Op Op Biotechnology, 4: 520-525), but also yeast cells (Buckholz). RG, 1993, Curr Op., Biotechnology, 4: 538-542), as well as animal cells, particularly mammalian cell cultures (Edwards CP and Aruffo A., 1993, Curr Op Op Biotechnology, 4: 558-563) but also insect cells in which methods using, for example, baculoviruses can be used (Luckow VA, 1993, Curr Op.

Biotechnology, 4: 564-572).

 Most preferably, said carrier protein is an OmpA protein of Klebsiella pneumoniae, in particular the P40 protein of sequence SEQ ID No. 72, one of its fragments of at least 8 amino acids, preferably at least 10 amino acids. , 15, 20, 30 or 50 amino acids, or a peptide whose sequence has an identity level after optimal alignment of at least 80%, preferably 85%, 90% or 95% with the sequence of P40 or one of these fragments.

 The carrier protein named P40, derived from the outer membrane type A Klebsiella pneumoniae has been described in particular in patent applications WO 95/27787 and WO 96/14415 as to increase the immune response, including humoral and when it was associated with an immunogen.

 In another aspect, the invention relates to a vaccine according to the invention, characterized in that said vaccine contains a vector comprising a nucleic construct coding for the hybrid peptide resulting from the coupling between the modified peptide comprising one or more antigenic epitopes or for said MHC ligand and said carrier protein, or a fragment thereof, or contains a host cell transformed by said vector capable of expressing said hybrid peptide.

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 In another aspect, the subject of the present invention is a pharmaceutical composition or a vaccine according to the invention that can be administered systemically, in particular by injectable, mucosal or transdermal route.

 The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope.

Legends of the Figures: FIGS. 1A to 1D: Secretion of IFNγ, induced by the peptides FluM1 (FIG. 1A), 4K-FluM1 (FIG. 1C), FluM1-4K (FIG. 1D), and 4k-FluM1 (FIG. 1B) , solubilized in PBS, in a HLA-A2 positive subject and sensitized by the influenza virus.

FIGS. 2A to 2D: IFNγ secretion, induced by the peptides FluM1 (FIG. 2A), 4K-FluM1 (FIG. 2C), FluM1-4K (FIG. 2D), and 4k-FluM1 (FIG. 2B), solubilized in DMSO, in a HLA-A2 negative subject.

 FIGS. 3A to 3C: of the CTL response induced by the 4K-FluM1 peptides (FIG. 3A), FluM1-4K (FIG. 3B) and 4k-FluM1 (FIG. 3C) solubilized at 2 mg / ml in PBS in the presence of P40 after immunization of the HLA-A2 / Kb mouse.

 FIGS. 4A to 4C: of the CTL response induced by the 4K-ELA peptides (FIG. 4A), ELA-4K (FIG. 4B) and 4k-ELA (FIG. 4C) solubilized at 2 mg / ml in PBS in the presence of P40 after immunization of the HLA-A2 / Kb mouse.

FIG. 5: Study of the CTL response induced by the solubilized 5K-ELA peptide at 2 mg / ml in PBS in the presence of P40 after immunization of the HLA-A2 / Kb mouse.

Figures 7A à 7E : Sécrétion d'IFNy, induite par les peptides FluMl, 4K-FluM1, FluM1- 4K et 4k-FluM1 solubilisés dans le DMSO, chez un clone T CD8+ humain, spécifique du peptide FluMl. FIG. 6: Study of the CTL response induced by the 3D-FluM1 peptide solubilized at 2 mg / ml in PBS in the presence of P40 after immunization of the HLA-A2 / Kb mouse.

FIGS. 7A to 7E: IFNγ secretion, induced by the FluM1, 4K-FluM1, FluM1-4K and 4k-FluM1 peptides solubilized in DMSO, in a human CD8 + T clone, specific for the FluM1 peptide.

FIGS. 8A to 8D: Secretion of IFNγ, induced by the peptides 4K-FluM1, FluM1-4K and 4k-FluM1 solubilized in PBS, in a human CD8 + T clone, specific for the FluM1 peptide.

EXAMPLES Example 1 Synthesis, Purification and Characterization of Peptides
The 62 peptides FluM1 and ELA and derivatives as well as the 6 HLA-A2 CTL epitope peptides and derivatives belonging to telomerase, were synthesized using a solid-phase peptide synthesizer (Advanced ChemTech, model 348 omega) using Fmoc chemistry at the 50 μmol scale. Resins of types

<Desc / Clms Page number 16>

 Preloaded Wang-or 2-chlorotrityl bearing the COOH-terminal residue of the peptides to be synthesized were introduced into the wells of a reactor having 96 wells. The α-NH 2 function of the amino acids used for synthesis was protected by a group (Fmoc) and the amino acid side chain functions were protected by groups compatible with Fmoc chemistry [Cys (Trt); Arg (Pmc); His (Trt); Trp (Boc); Asn (Trt); Gln (Trt); Lilies (Boc); Ser (tBu); Thr (tBu); Tyr (tBu); Asp (OtBu); Glu (OtBu)].

Lavages préliminaires : DCM 1500 pi/puits 3 fois 5 min
DMF 1500 l/puits 3 fois 10 min 1. Déprotections : a. DMF 1125 l/puits pipéridine 375 l/puits 1 fois 2 min rinçage: DMF 1500 l/puits vidange b. DMF 1125 pi/puits pipéridine 375 l/puits 1 fois 10 min 2. Lavages : DMF 1500 l/puits 6 fois 2 min 3. Couplages : a. aa/HOBt 0. 5M 500 l/puits
HBTU 0. 5M 500 l/puits
DIEA 2M 250 l/puits 1 fois 20 min Lavages DMF 1500 l/puits 2 fois 2 min b. Aa/HOBt 0. 5M 500 l/puits
HATU 0. 5M 500 l/puits
DIEA 2M 250 l/puits 1 fois 20 min 4. Lavages DMF 1500 l/puits 4 fois 2 min
DCM 1500 l/puits 3 fois 1 min 5. Acétylation mélange
1500 l/puits 1 fois 10 min I. Summary protocol

Preliminary Washes: DCM 1500 ft / well 3 times 5 min
DMF 1500 l / well 3 times 10 min 1. Deprotections: a. DMF 1125 l / piperidine well 375 l / well 1 time 2 min rinse: DMF 1500 l / well drain b. DMF 1125 pi / piperidine well 375 l / well 1 time 10 min 2. Washings: DMF 1500 l / well 6 times 2 min 3. Couplings: a. aa / HOBt 0. 5M 500 l / well
HBTU 0. 5M 500 l / well
DIEA 2M 250 l / well 1 time 20 min Washing DMF 1500 l / well 2 times 2 min b. Aa / HOBt 0. 5M 500 l / well
HATU 0. 5M 500 l / well
DIEA 2M 250 l / well 1 time 20 min 4. Wash DMF 1500 l / well 4 times 2 min
DCM 1500 l / well 3 times 1 min 5. Acetylation mixture
1500 l / well 1 time 10 min

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DMF 1500 /-tl/puits 4 fois 2 min ≈Mélange utilisé : Anhydride acétique 10%, DIEA 5%, dans le DCM. 6 .Wash DCM 1500 l / well 3 times 1 min

DMF 1500 / -tl / well 4 times 2 min ≈Mixture used: Acetic anhydride 10%, DIEA 5%, in DCM.

 The cycle comprising steps 1 to 6 is repeated n times until the desired sequences are obtained. After the last coupling cycle, the α-NH 2 function of peptidylresin is deprotected with piperidine as described in step 1.

At the end of the synthesis, the peptidylresins are washed as follows: Washing DMF 1500 l / well 4 times 2 min
DCM 1500 l / well 4 times 3 min before being dried for 3 hours under nitrogen.

II. Cleavage of peptidylresins and recovery of peptides
The peptides are then cleaved from the resin and the functions of the side chains of the amino acids are deprotected by reaction for 3 hours at room temperature of peptidylresin with a cleavage mixture whose composition is as follows for 100 mg of peptidylresin, 1.5 ml. of the mixture: TFA 82.5%; H20 5%; 2.5%; Thioanisol 5% and phenol 5%.

 The resin is separated from the peptide by simple filtration. The peptide is then precipitated by adding diethyl ether at 4 ° C. The crude peptide is recovered by centrifugation at 3000 rpm for 3 min. After washing with ether three times, the peptide is taken up with the aid of an aqueous acetic acid solution and then lyophilized.

III. Purification of peptides
The peptides are taken up in a mixture of acetic acid and water and purified by RP-HPLC (reverse-phase high performance liquid chromatography) semipréparative on a C4 or C18 column. The elution system being an H 2 O and acetonitrile gradient in the presence of 0.1% TFA.

IV. Peptide analyzes
The purity of the peptide is verified by RP-HPLC in reverse phase. The identity of the peptide is confirmed by ES-MS mass spectrometry.

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Characteristics of the synthesized nonapeptides: purity of the peptides: 85% (RP-HPLC) identity of the peptides: agreement with the expected mass (ES-MS) Abbreviations: Aa: Fmoc amino acid; Boc: Tertiobutyloxycarbonyl; DCM: dichloromethane; DIEA: Diisopropylethylamine; DMF: dimethylformamide; EDT: Ethanedithiol; ES-MS: Electrospray Mass Spectrometry; Fmoc: 9-Fluorenylmethoxycarbonyl; HATU: O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU: 2- (1H-Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt: Hydroxybenzotriazole; NMP: N-methyl-2-pyrrolidone; Pmc: 2,2,5,7,8-Pentamethylchroman-6-sulfonyl; RP-HPLC: Reverse Phase - High Performance Liquid Chromatography; TBu (OtBu): Tertiobutyl; TFA: Trifluoroacetic acid; Trt: Trityl.

Table 1: Sequence of the 43 FLUM1 peptides and synthesized derivatives

<Tb>
<tb> Peptide <SEP> Sequence <SEP> SEQ <SEP> ID <SEP> No.
<Tb>

FluM1 <SEP> GILGFVFTL <SEP> 2 <SEP>
<tb> 1K-FluM1 <SEP> KGILGFVFTL <SEP> 3 <SEP>
<tb> 2K-FluM1 <SEP> KKGILGFVFTL <SEP> 4 <SEP>
<tb> 3K-FluM1 <SEP> KKKGILGFVFTL <SEP> 5
<tb> 4K-FluM1 <SEP> KKKKGILGFVFTL <SEP> 6 <SEP>
<tb> 5K-FluM1 <SEP> KKKKKGILGFVFTL <SEP> 7 <SEP>
<tb> 1H-FluM1 <SEP> HGILGFVFTL <SEP> 8 <SEP>
<tb> 2H-FluM1 <SEP> HHGILGFVFTL <SEP> 9 <SEP>
<tb> 3H-FluM1 <SEP> HHHGILGFVFTL <SEP> 10
<tb> 4H-FluM1 <SEP> HHHHGILGFVFTL <SEP> 11 <SEP>
<tb> 5H-FluM1 <SEP> HHHHHGILGFVFTL <SEP> 12
<tb> 1D-FluM1 <SEP> DGILGFVFTL <SEP> 13 <SEP>
<tb> 2D-FluM1 <SEP> DDGILGFVFTL <SEP> 14
<tb> 3D-FluM1 <SEP> DDDGILGFVFTL <SEP> 15
<tb> 4D-FluM1 <SEP> DDDDGILGFVFTL <SEP> 16
<tb> 5D-FluM1 <SEP> DDDDDGILGFVFTL <SEP> 17
<tb> 1E-FluM1 <SEP> EGILGFVFTL <SEP> 18 <SEP>
<Tb>

<Desc / Clms Page number 19>

<Tb>
<tb> 2E-FluM1 <SEP> EEGILGFVFTL <SEP> 19
<tb> 3E-FluM1 <SEP> EEEGILGFVFTL <SEP> 20
<tb> 4E-FluM1 <SEP> EEEEGILGFVFTL <SEP> 21
<tb> 5E-FluM1 <SEP> EEEEEGILGFVFTL <SEP> 22
<tb> 1S-FluM1 <SEP> SGILGFVFTL <SEP> 23
<tb> 2S-FluM1 <SEP> SSGILGFVFTL <SEP> 24
<tb> 3S-FluM1 <SEP> SSSGILGFVFTL <SEP> 25
<tb> 4S-FluM1 <SEP> SSSSGILGFVFTL <SEP> 26
<tb> 5S-FluM1 <SEP> SSSSSGILGFVFTL <SEP> 27
<tb> 1T-FluM1 <SEP> TGILGFVFTL <SEP> 28
<tb> 2T-FluM1 <SEP> TTGILGFVFTL <SEP> 29
<tb> 3T-FluM1 <SEP> TTTGILGFVFTL <SEP> 30
<tb> 4T-FluM1 <SEP> TTTTGILGFVFTL <SEP> 31
<tb> 5T-FluM1 <SEP> TTTTTGILGFVFTL <SEP> 32
<tb> 1N-FluM1 <SEP> NGILGFVFTL <SEP> 33
<tb> 2N-FluM1 <SEP> NNGILGFVFTL <SEP> 34
<tb> 3N-FluM1 <SEP> NNNGILGFVFTL <SEP> 35
<tb> 4N-FluM1 <SEP> NNNNGILGFVFTL <SEP> 36
<tb> 5N-FluM1 <SEP> NNNNNGILGFVFTL <SEP> 37
<tb> 1Q-FluM1 <SEP> QGILGFVFTL <SEP> 38 <SEP>
<tb> 2Q-FluM1 <SEP> QQGILGFVFTL <SEP> 39
<tb> 3Q-FluM1 <SEP> QQQGILGFVFTL <SEP> 40
<tb> 4Q-FluM1 <SEP> QQQQGILGFVFTL <SEP> 41
<tb> 5Q-FluM1 <SEP> QQQQQGILGFVFTL <SEP> 42
<tb> FluM1-4K <SEP> GILGFVFTLKKKK <SEP> 43
<tb> 4k * -FluM1 <SEP> kkkkGILGFVFTL <SEP> 44
<Tb>
* k = D-Lysine

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Table II: Sequence of the 19 ELA peptides and synthesized derivatives

<Tb>
<tb> Peptide <SEP> Sequence <SEP> SEQ <SEP> ID <SEP> No.
<Tb>

ELA <SEP> ELAGIGILTV <SEP> 47
<tb> K-ELA <SEP> KELAGIGILTV <SEP> 48
<tb> 2K-ELA <SEP> KKELAGIGILTV <SEP> 49
<tb> 3K-ELA <SEP> KKKELAGIGILTV <SEP> 50
<tb> 4K-ELA <SEP> KKKKELAGIGILTV <SEP> 51
<tb> 5K-ELA <SEP> KKKKKELAGIGILTV <SEP> 52
<tb> ELA-4K <SEP> kkkkELAGIGILTV <SEP> 53
<tb> 4k * -ELA <SEP> ELAGIGILTVKKKK <SEP> 54
<tb> D-ELA <SEP> DELAGIGILTV <SEP> 55
<tb> 2D-ELA <SEP> DDELAGIGILTV <SEP> 56
<tb> 3D-ELA <SEP> DDDELAGIGILTV <SEP> 57
<tb> 4D-ELA <SEP> DDDDELAGIGILTV <SEP> 58
<tb> 5D-ELA <SEP> DDDDDELAGIGILTV <SEP> 59
<tb> 4H-ELA <SEP> HHHHELAGIGILTV <SEP> 60
<tb> 4T-ELA <SEP> TTTTELAGIGILTV <SEP> 61
<tb> 4N-ELA <SEP> NNNNELAGIGILTV <SEP> 62
<tb> 4Q-ELA <SEP> QQQQELAGIGILTV <SEP> 63
<tb> 4E-ELA <SEP> EEEEELAGIGILTV <SEP> 64
<tb> 4S-ELA <SEP> SSSSELAGIGILTV <SEP> 65
<Tb>
* k = D-lysine Table III: Sequence of the synthesized 6 synthesized telomerase-derived CTL HLA-A2 peptides

<Tb>
<tb> Peptide <SEP> Sequence <SEP> SEQ <SEP> ID <SEP> No.
<tb> hTERT <SEP> 572 <SEP> Y1 <SEP> YLFFYRLSV <SEP> 66
<tb> hTERT <SEP> 988 <SEP> DLQVNSLQVT <SEP> 67
<tb> hTERT <SEP> 988 <SEP> Y1 <SEP> YLQVNSLQVT <SEP> 68
<tb> 4K <SEP> hTERT <SEP> 572 <SEQ> Y1 <SEP> KKKKYLFFYRLSV <SEP> 69
<tb> 4K <SEP> hTERT <SEP> 988 <SEP> KKKKDLQVNSLQVT <SEP> 70
<tb> 4K <SEP> hTERT <SEP> 988 <SEP> Y1 <SEP> KKKKYLQVNSLQVT <SEP> 71
<Tb>

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EXAMPLE 2 Identification of peptides soluble in PBS, especially at 7 mg / ml 1. General results obtained for all synthesized peptides
It emerges from the results obtained that, whatever the nature of the basic peptide to which the process is applied, the coupling with amino acids other than lysine, and in particular with 4 of said amino acids other than lysine, only gives levels. solubility below about 2 mg / ml, which is not exploitable for the desired applications.

He. Identification of peptides soluble in PBS at 7 mg / ml
Each peptide (6 different weighings) is taken up at 1 mg / ml in DMSO (3 samples) as well as in PBS at 10 mg / ml (3 samples).

 The solutions of each peptide solubilized in DMSO or PBS are vortexed and placed on a vibrating shaker for 1 hour at room temperature. The solutions are then placed for 5 minutes in an ultrasonic bath and then centrifuged at 10,000 rpm for 5 minutes.

 The supernatants are analyzed by RP-HPLC (pure or diluted 1 / 5th or 1 / 10th) on a C18 column (250 mm × 2.1 mm × 5 m). The eluents are as follows: A = H 2 O + 0.05% TFA and B = CH 3 CN / H 2 O 80/20 + 0.05% TFA. The gradient used is from 20% to 100% of eluent B with a slope of 2% eluent B / min, the flow rate is 0.25 ml / min.

 The concentration obtained is the result of the calculation of the ratio average: area of the HPLC peak obtained with the peptide solubilized in PBS (at 10 mg / ml) / area of the HPLC peak obtained with the peptide solubilized in DMSO (at 1 mg / ml ) on 3 different samples.

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Table IV: Sequences of FLUM1 / ELA peptides and PBS-soluble derivatives at a concentration of at least 7 mg / ml

<Tb>
<tb> Peptide <SEP> Sequence <SEP> Solubility <SEP> (mg / ml)
<tb> 3K-FluM1 <SEP> KKKGILGFVFTL <SEP> 11.4 +/- 0.4
<Tb>

4K-FIuM1 KKKKGILGFVFTL 9.4 +I- 0.3

4K-FIUM1 KKKKGILGFVFTL 9.4 + I- 0.3

<Tb>
<tb> 5K-FluM1 <SEP> KKKKKKGILGFVFTL <SEP> 9.0 +/- 1.4
<tb> FluM1-4K <SEP> GILGFVFTLKKKK <SEP> 7.3 <SEP> +/- <SEP> 0.3 <SEP>
<tb> 4k-FluM1 <SEP> kkkkGILGFVFTL <SEP> 8.8 <SEP> +/- <SEP> 0.2 <SEP>
<tb> 4K-ELA <SEP> KKKKELAGIGILTV <SEP> 11.5 +/- 0.3
<Tb>

5K-ELA KKKKKELAGIGILTV 9.0 +/- 1.0 ELA-4K ELAGIGILTVKKKK 10.9+/-0.5 4k-ELA kkkkELAGIGIL TV 11 +/- 2
Les peptides insolubles, modifiés par 3,4 ou 5 résidus L- ou D- Lysine en N- ou C-terminal sont solubles dans PBS à au moins 7 mg/ml (Tableau IV) et donc, a fortiori, ont une solubilité supérieure à la limite exploitable dans les applications visées, à savoir 2 mg/ml.

5K-ELA KKKKKELAGIGILTV 9.0 +/- 1.0 ELA-4K ELAGIGILTVKKKK 10.9 +/- 0.5 4k-ELA kkkkELAGIGIL TV 11 +/- 2
The insoluble peptides, modified with 3,4 or 5 residues L- or D-Lysine in N- or C-terminal are soluble in PBS at least 7 mg / ml (Table IV) and thus, a fortiori, have a higher solubility at the exploitable limit in the targeted applications, namely 2 mg / ml.

Table V: Sequences of HLA-A2 CTL Peptides and HTERT-derivatives Soluble in PBS at a Concentration of at Least 7 mg / ml

<Tb>
<tb> Peptide <SEP> Sequence <SEP> Solubility <SEP> (mg / ml)
<tb> 4K <SEP> hTERT <SEP> 572 <SEQ> Y1 <SEP> KKKKYLFFYRLSV <SEP> 11.7 +/- 1
<tb> 4K <SEP> hTERT <SEP> 988 <SEP> KKKKDLQVNSLQVT <SEP> 12.6 +/- 0.2
<tb> 4K <SEP> hTERT <SEP> 988 <SEP> Y1 <SEP> KKKKYLQVNSLQVT <SEP> 8.5 <SEP> +/- <SEP> 0.2 <SEP>
<Tb>

Of the 6 peptides synthesized, only those which have been modified by the addition of 4 lysine residues (shown in this table) show an increase in solubility in aqueous medium sufficient for the intended applications, namely a solubility greater than about 2 mg / ml. ml.

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Example 3 Identification of Peptides Affecting the HLA-A2 Molecules
In order to rapidly screen the 60 peptides derived from synthesized ELA and FluM1, the latter were evaluated, initially, by studying their ability to bind to the HLA-A2 complex, a property necessary for any initiation of an immune response of cytotoxic type. To do this the T2 cell line was used.

 The T2 line is a human lymphocyte line corresponding to a hybrid T cells / B cells, constitutively expressing HLA-A2.

 These cells, deficient in TAP transporter, express few HLA-A2 molecules on their surface. In the absence of peptide, the HLA-A2-J32 microglobulin complex is continuously recycled into the cell and a basal level of complex is observed on the surface of the cell. The presence of an exogenous peptide that binds to the HLA-A2 molecules stabilizes the complex for a longer or shorter time, depending on its affinity, preventing the reinternalisation of the HLA-A2 molecules and thus inducing an increase in the number of HLA-A2 molecules to the surface of the T2 cell.

 The 60 peptides derived from ELA and FluM1 were tested for their ability to induce a more or less significant stabilization according to the peptide of HLAA2 molecules on the surface of T2 cells solubilized in DMSO, whereas only peptides soluble at least 7 mg / ml were tested in PBS (Table IV). To do this, T2 cells were incubated overnight at 37 C with 100 g / ml each of the peptides in OptiMEM medium without FBS and supplemented with 100 ng / ml ss2 microglobulin. After 4 washes in order to remove the excess of unbound peptide, the cells were resuspended with 1 ml of RPMI 1640.10% FCS medium containing 10 g / ml of Brefeldin A capable of blocking the surface expression of the cells. newly synthesized HLA-A2 molecules. After one hour of incubation at 37 ° C. and 3 washes, the cells were again incubated at 37 ° C. for 0.2, 4 or 6 hours before being labeled with the MA2 monoclonal antibody. 1 to evaluate the surface expression of HLA-A2 molecules in FACS (Fluorescens Activated Cells Sorter).

 The results (Table VI) show that 40 peptides derived from FluM1 solubilized in DMSO as the parent peptide FluM1, are capable, among the synthesized 42 to induce an increase in the expression of HLA-A2 on the surface of T2 cells. Peptides unable to induce an increase in HLA-A2 expression on the T2 cell surface are: 5Q-FluM1 and 4k-FluM1.

 The results (Table VII) also show that 6 peptides derived from ELA solubilized in DMSO, among the 18 synthesized are capable of inducing a

<Desc / Clms Page number 24>

 increased expression of HLA-A2 on the surface of T2 cells. Peptides unable to induce an increase in HLA-A2 expression on the surface of T2 cells are: 4k-ELA; 1D-, 2D-, 3D-, 4D- and 5D-ELA; 4H-, 4T-, 4N-, 4Q-, 4E- and 4S-ELA.

 The results (Tables VI and VII) show that all peptides derived from ELA and FluM1 soluble in PBS at at least 7 mg / ml are capable of inducing an increase in the expression of HLA-A2 on the surface of T2 cells. with the exception of 4k-FluM1 and 4k-ELA peptides.

TABLE VI Fixation of Peptides Derived from FluM1 Solubilized in DMSO or PBS on HLA-A2 Molecules of T2 Cells

<Tb>
<tb> Peptides <SEP> DMSO <SEP> PBS
<tb> FluM1 <SEP> ++++
<tb> 1K-FluM1 <SEP> +++ <SEP> nd
<tb> 2K-FluM1 <SEP> +++ <SEP> nd
<tb> 3K-FluM1 <SEP> +++ <SEP> ++
<tb> 4K-FluM1 <SEP> ++ <SEP> ++
<tb> 5K-FluM1 <SEP> ++ <SEP> +
<tb> FluM1-4K <SEP> + <SEP> +
<tb> 4k-FluM1
<tb> 1 <SEP> H-FluM1 <SEP> +++ <SEP> nd
<tb> 2H-FluM1 <SEP> ++ <SEP> nd
<tb> 3H-FluM1 <SEP> ++ <SEP> nd
<tb> 4H-FluM1 <SEP> + <SEP> nd
<tb> 5H-FluM1 <SEP> + <SEP> nd
<tb> 1D-FluM1 <SEP> ++ <SEP> nd
<tb> 2D-FluM1 <SEP> ++ <SEP> nd
<tb> 3D-FluM1 <SEP> ++ <SEP> nd
<tb> 4D-FluM1 <SEP> ++ <SEP> nd
<tb> 5D-FluM1 <SEP> + <SEP> nd
<tb> 1E-FluM1 <SEP> ++ <SEP> nd
<tb> 2E-FluM1 <SEP> +++ <SEP> nd
<tb> 3E-FluM1 <SEP> +++ <SEP> nd
<Tb>

<Desc / Clms Page number 25>

<Tb>
<tb> 4E-FluM1 <SEP> ++ <SEP> nd
<tb> 5E-FluM1 <SEP> ++ <SEP> nd
<tb> 1S-FluM1 <SEP> ++ <SEP> nd <SEP>
<tb> 2S-FluM1 <SEP> ++ <SEP> nd
<tb> 3S-FluM1 <SEP> ++ <SEP> nd
<tb> 4S-FluM1 <SEP> ++ <SEP> nd
<tb> 5S-FluM1 <SEP> ++ <SEP> nd
<Tb>

1T-FluMl + nd

1T-FluMl + nd

<Tb>
<tb> 2T-FluM1 <SEP> + <SEP> nd
<tb> 3T-FluM1 <SEP> ++ <SEP> nd
<tb> 4T-FluM1 <SEP> + <SEP> nd
<tb> 5T-FluM1 <SEP> + <SEP> nd
<tb> 1 <SEP> N-FluM1 <SEP> ++ <SEP> nd
<tb> 2N-FluM1 <SEP> + <SEP> nd
<tb> 3N-FluM1 <SEP> ++ <SEP> nd
<tb> 4N-FluM1 <SEP> + <SEP> nd
<tb> 5N-FluM1 <SEP> + <SEP> nd
<Tb>

1Q-FluMl ++ nd

1Q-FluMl ++ nd

<Tb>
<tb> 2Q-FluM1 <SEP> +++ <SEP> nd
<tb> 3Q-FluM1 <SEP> ++++ <SEP> nd
<tb> 4Q-FluM1 <SEP> ++ <SEP> nd
<tb> 5Q-FluM1 <SEP> - <SEP> nd
<Tb>
nd: not determined (because not soluble or not sufficiently soluble in PBS to be tested)

<Desc / Clms Page number 26>

TABLE VII Fixation of Solubilized ELA Peptides in DMSO or PBS on HLA-A2 Molecules of T2 Cells

<Tb>
<tb> Peptides <SEP> DMSO <SEP> PBS
<tb> ELA <SEP> ++++ <SEP> -
<tb> 1 <SEP> K-ELA <SEP> +++ <SEP> N / A
<tb> 2K-ELA <SEP> +++ <SEP> nd
<tb> 3K-ELA <SEP> +++ <SEP> nd
<tb> 4K-ELA <SEP> ++ <SEP> ++
<tb> 5K-ELA <SEP> +++ <SEP> +++
<tb> ELA-4K <SEP> + <SEP> +
<tb> 4k-ELA-
<tb> 1 <SEP> D-ELA <SEP> - <SEP> nd <SEP>
<tb> 2D-ELA- <SEP> nd
<tb> 3D-ELA- <SEP> nd
<tb> 4D-ELA- <SEP> nd
<tb> 5D-ELA <SEP> - <SEP> nd
<tb> 4H-ELA <SEP> - <SEP> nd <SEP>
<tb> 4T-ELA <SEP> - <SEP> nd <SEP>
<tb> 4S-ELA <SEP> - <SEP> nd <SEP>
<tb> 4E-ELA <SEP> - <SEP> nd <SEP>
<tb> 4N-ELA <SEP> - <SEP> nd <SEP>
<tb> 4Q-ELA- <SEP> nd
<Tb>
nd: not determined (because not soluble or not sufficiently soluble in PBS to be tested) Example 4: Measurement of the secretion of IFNy by human T lymphocytes, induced by the peptides 4K-FluM1, FluM1-4K and 4k-FluM1
The peptides derived from FluM1 4K-FluM1 and FluM1-4K, positive in the T2 cell binding assay, as well as the negative 4k-FluM1 derivative in this same test, were solubilized in PBS tested on CD8 + cytotoxic T cells of a HLA-A2 positive subject and sensitized by the influenza virus, for their ability to induce secretion of interferon y by these cells.

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 Briefly, 106 peripheral blood cells obtained after Ficoll are taken up in RPMI-10% FCS in 15 ml polypropylene tubes and placed in the presence of 100 l of test peptide, at a concentration of 10 g / ml (stock solution). at 4 mg / ml) for 2 hours at 37 ° C in an incubator. 800 μl of RPMI-10% FCS containing 12.5 g / ml of Brefeldin A, blocking the secretion of cytokines, are added and the tubes are then placed back in the incubator for 4 hours at 37 ° C.

 At the end of the incubation period, 100 l of cold 20 mM PBS-EDTA are added and after 5 minutes, the cells are centrifuged and resuspended in 100 l of PBS-1% BSA-0.01% NaN 3 ( BSA for bovine serum albumin), containing 0.7 l of anti-CD8 antibody coupled to allophycocyanin (APC).

 After transferring to a 96-well plate with conical bottoms, the cells are incubated for 20 minutes at 4 ° C. and then fixed for 10 minutes, in the dark, at room temperature with 100 l of 2% paraformaldehyde (PFA) in PBS. After two washes in PBS, 50 l of 1 mM Saponin-1% PBS-Hepes, containing 0.4 l of anti-IFNγ antibody coupled to FITC (stock solution at 0.5 mg / ml) are added to the cell pellets, in each well. After 30 minutes of incubation at 4 ° C., the cells are washed twice and then transferred to Micronic tubes for flow cytometric analysis.

 Flow cytometric analysis of the IFNγ secretion by CD8 + T lymphocytes, shown in FIG. 1, shows that in the presence of the 4K-FluM1 and FluM1-4K peptides, 0.16% and 0.14% respectively of the CD8 + T cells secrete IFNγ whereas in the presence of the 4k-FluM1 peptide, 0.03% of the CD8 + Ts are positive for secretion of IFNy.

 Under these conditions, the peptide FluM1 is not soluble and does not induce secretion of IFNy by CD8 + T cells (0.06% of positive cells).

 Figure 2 shows that regardless of the FluM1-derived peptide tested in an HLA-A2 negative individual, there is no secretion of IFNy by CD8 + T cells.

EXAMPLE 5 Verification of the CTL Activity of Peptides Derived from FluM1 and ELA Soluble in PBS (Table IV) in a HLA A2 / Kb Transgenic Mouse Model
In addition to interferon-γ measurements in the HLA-A2 positive subject sensitized with the influenza virus, each of the PBS-soluble peptides described above in Table IV as well as the 10 mg non-soluble 3D-FluM1 peptide / ml in PBS but soluble to 2 mg / ml in PBS concentration sufficient to allow CTL assay, were tested for induction of a type response

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 cytotoxic (CTL) in HLA A2 / kb mice. To do this, these peptides were administered individually, subcutaneously. An injection consisting of 50 g of peptide derived from ELA, or two injections one week apart consisting of 100 g of peptide derived from FluM1 + 300 g of P40 were made. Ten days after the last injection, the spleen cells are cultured in the presence of IL2 and presenting cells fixed and loaded with the peptide ELA or FluM1. Seven days after this first stimulation, a second stimulation of the effector cells is performed followed, after 5 days of evaluation of the cytotoxic activity by a 51 Cr release test. This test consists in incubating peptide-loaded EL4 A2 / kb cells with different ratios of effector cells resulting from culturing and evaluating the cytotoxic activity of these effector cells by calculating the amount of 51Cr released by the cells. targets when lysed by effector cells according to the formula: (Lysis of charged target cells - spontaneous lysis) / (total lysis of charged target cells - spontaneous lysis) x 100.

 In this formula, the lysis of the target cells corresponds for each ratio to the contacting of the target cells and the effector cells; spontaneous lysis corresponds to the spontaneous release of 51 Cr of the target cells in the absence of any other cell and the total lysis corresponds to the maximum release of 51 Cr when lysis of all the target cells by addition of Triton X100.

 In order to determine the specificity of the reaction, a control is carried out by replacing the target cells loaded with the peptide with uncharged target cells which therefore do not have to be recognized by the effector cells and therefore must not be lysed by the latter. .

 FIGS. 3A to 3C and 4A to 4C show that among the peptides tested, the derivatives having 4 Lysine residues on the N-terminal side or 4 Lysine residues on the C-terminal side of the FluM1 peptide (FIGS. 3A to 3C) and the ELA peptide (FIGS. at 4C) are able to generate a significant CTL response in HLA A2 Kb mice.

The particular importance of the 4K-FluM1, 5K-FluM1, 4K-ELA and FluM1-4K peptides, ELA-4K, lies in fact in the observation that effector cells from mice immunized with these modified peptides are capable of lysing target cells loaded with the parental peptides FluM1 or ELA respectively (FIGS. 3A and 3B, 4A and 4B and 5).

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 On the other hand, the substitution of L-Lysine residues by D-Lysine residues seems to have a negative effect on the induction of the cytotoxic response despite an equivalent aqueous solubility. In fact, the results obtained with the 4k-FluM1 and 4k-ELA peptides (FIGS. 3C and 4C) show the absence of any induction of CTL response when these peptides are used to immunize HLA-A2 / Kb mice. The results obtained in the CTL tests are in perfect agreement with those observed in the subject tested in Example 4 of secretion of IFNy.

 It is also important to note that the 3D-FluM1 peptide, which stabilizes HLA-A2 molecules on the surface of T2 cells in a manner similar to the 4K-FluM1 peptide, is not capable of generating a cytotoxic response (FIG. It is tested under conditions identical to those of 4K-FluM1.

EXAMPLE 6 Measurement of the IFNγ Secretion Induced by the 4K-FluM1, FluM1-4K and 4k-FluM1 Peptides, in a Human CD8 + T Clone, Specific for the FluM1 Peptide
The peptides derived from FluM1, 4K-FluM1 and FluM1-4K, positive in the T2 cell binding assay, as well as the negative 4k-FluM1 derivative in the same test were solubilized in DMSO or PBS and tested on cells. from a human CD8 + cytotoxic clone, specific for FluM1, for their ability to induce secretion of IFNy by these cells.

 Briefly, 5.10 cells of the FluM1 clone are incubated, in a tube, for 1 hour at room temperature, with slow stirring, in the presence of empty dimers or dimers loaded the day before at 37 ° C. in the presence of an excess of the peptide to be tested. The cells are then washed twice with 0.2% BSA PBS and labeled before passing through the FACS.

 FIGS. 7C to 7E and 8B to 8D respectively show that among the modified FluM1 peptides 4K-FluM1, 4k-FluM1 and FluM1-4K, solubilized in DMSO or in PBS, only the 4K-FluM1 and FluM1-4K peptides are capable of when presented via class I MHC dimers, to induce secretion of interferon γ in the whole clonal population tested, a secretion comparable to that induced by the native peptide FluM1 (see FIG. 7B). As expected, when the experiment is performed in the presence of uncharged dimers, no production of interferon is observed (see Figures 7A and 8A).

Claims (40)

A method for solubilizing or improving the solubility in an aqueous medium of a peptide, characterized in that it comprises a step in which said peptide is modified by the addition by covalent bond of at least a total of 3 residues lysine, of L or D form, distributed at the N-terminal and / or C-terminal of said peptide, these at least a total of 3 lysine residues may constitute a linear or branched chain.  2. Method according to claim 1, characterized in that the total number of lysine residues distributed at the N-terminal and / or C-terminal end of said modified peptide is between 3 and 6 inclusive, preferably between 3 and 5. , terminals included.  3. Method according to claim 1 or 2, characterized in that said at least a total of 3 lysine residues are added either to the N-terminal free end or to the C-terminal free end of said peptide.  4. Method according to one of claims 1 to 3, characterized in that all the lysine residues added is form L.  5. Method according to one of claims 1 to 4, characterized in that the covalent addition of said at least a total of 3 lysine residues at the N-terminal and / or C-terminal free end of said peptide is carried out by chemical synthesis, or by genetic fusion when the structure of said peptide thus modified allows it.  6. Method according to one of claims 1 to 5, characterized in that the modified peptide is soluble in aqueous medium at least 5 mg / ml.  7. Method according to one of claims 1 to 6, characterized in that said modified peptide is soluble in aqueous medium at least 7 mg / ml.  8. Method according to one of claims 1 to 7, characterized in that said peptide is an active ingredient capable of exerting a therapeutic action.  9. Method according to one of claims 1 to 8, characterized in that said peptide is a peptide whose sequence comprises one or more antigenic epitopes hydrophobic nature.  10. The method of claim 9, characterized in that said peptide is capable of inducing a humoral or cell type immune response directed specifically against this epitope.  11. Process according to claim 10, characterized in that said peptide is chosen from antigenic epitopes derived from an antigenic protein of <Desc / Clms Page number 31>  pathogenic microorganism for a mammal, or derived from a tumor-associated antigenic protein.  12. The method of claim 10 or 11, characterized in that said peptide is capable of generating CTL lymphocytes directed specifically against this epitope.  13. Method according to one of claims 10 to 12, characterized in that said peptide is a ligand, natural or synthetic, MHC, including class 1 or II.  14. Method according to one of claims 10 to 13, characterized in that said peptide is a ligand, natural or synthetic, MHC for the generation of cytotoxic T lymphocytes selected from CTL epitopes.  15. Method according to one of claims 10 to 14, characterized in that said peptide is selected from CTL epitopes in the form of octapeptide, nonapeptide, decapeptide hydrophobic, a hydrophobic peptide of at most 15 amino acids, or in the form of a grouping of hydrophobic epitopes.  16. Method according to one of claims 10 to 15, characterized in that: - said peptide is a MHC ligand capable of generating cytotoxic T lymphocytes and / or T helper, in particular specific for said MHC ligand, and / or capable of generating a significant increase in the level of CD8 + and / or CD4 + T lymphocytes producing INF-γ and, if appropriate, TNF-α, when said MHC ligand is placed in the presence of T lymphocytes of subjects having presented antigens from which said MHC ligand is derived; and characterized in that: said modified MHC ligand is capable in aqueous solution of performing at least one of the activities mentioned in a).  17. Method according to one of claims 12 to 16, characterized in that said peptide is a MHC ligand for the generation of cytotoxic T lymphocytes, comprising or consisting of a peptide selected from the peptides SEQ ID No. 2 sequences , SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47.  18. Method according to one of claims 12 to 16, characterized in that said peptide is a MHC ligand for the generation of cytotoxic T lymphocytes, comprising or consisting of a peptide selected from the peptides of the following sequences: a) the sequence of the retroinverso form of a peptide of SEQ ID No. 2 sequence, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; and <Desc / Clms Page number 32>  b) a peptide whose sequence has an identity level after optimal alignment of at least 70%, preferably at least 80%, 85%, 90% or 95% with the sequence of a SEQ sequence peptide ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; characterized in that: the modified MHC ligand is capable of generating cytotoxic T lymphocytes and / or T helper, in particular specific for said MHC ligand, and / or capable of generating a significant increase in the level of CD8 + T lymphocytes and / or CD4 + producing INF-γ and, if appropriate, TNF-α, when said modified MHC ligand is brought into the presence of T cells of subjects having presented the antigens from which said MHC ligand is derived.  19. Ligand MHC peptide modified by the addition of at least a total of 3 lysine residues, distributed at its N-terminal and / or C-terminal, these 3 lysine residues may be a linear or branched chain.  20. Modified MHC ligand according to claim 19, characterized in that the total number of lysine residues distributed at the N-terminus and / or C-terminus of said modified ligand is between 3 and 6 inclusive, preferably between 3 and 5, terminals included.  21. A modified MHC ligand according to claim 19 or 20, characterized in that at least 3 lysine residues have been added either to the N-terminal free end or to the C-terminal free end of said MHC ligand.  22. Modified MHC ligand according to one of claims 19 to 21, characterized in that all the lysine residues added is L-shaped.  23. Modified MHC ligand according to one of claims 19 to 22, characterized in that said MHC ligand before modification consists of at most 15 amino acids.  24. Modified MHC ligand according to one of claims 19 to 23, characterized in that said unmodified MHC ligand is a peptide selected from antigenic epitopes derived from an antigenic protein of pathogenic microorganism for a mammal, or derivatives thereof. of an antigenic protein associated with a tumor.  25. Modified MHC ligand according to one of claims 19 to 24, characterized in that said unmodified MHC ligand is a peptide selected from CTL epitopes.  26. Modified MHC ligand according to one of claims 19 to 25, characterized in that said unmodified MHC ligand is a peptide chosen from the <Desc / Clms Page number 33>  CTL epitopes in octapeptide, nonapeptide or decapeptide form or a grouping of hydrophobic epitopes.  27. Modified MHC ligand according to one of claims 19 to 26, characterized in that said MHC ligand before modification is capable of generating cytotoxic T lymphocytes and / or T helper, in particular specific for said MHC ligand and / or capable of generating a significant increase in the level of CD8 + and / or CD4 + T lymphocytes producing INF-γ and, if appropriate, TNF-α, when said MHC ligand is placed in the presence of T lymphocytes of subjects having presented the antigens from which said ligand is derived; and characterized in that the aqueous solubility of said MHC ligand before modification is less than 1 mg / ml.  28. Modified MHC ligand according to one of claims 19 to 27, characterized in that said modified MHC ligand is soluble in aqueous medium at least 5 mg / ml, preferably at least 7 mg / ml.  29. Modified MHC ligand according to one of claims 19 to 28, characterized in that said modified MHC ligand is capable in aqueous solution of generating cytotoxic T lymphocytes and / or T helper, in particular specific for said MHC ligand, and / or capable of generating a significant increase in the level of CD8 + and / or CD4 + T cells producing INF-γ and, if appropriate, TNF-α, when said MHC ligand is brought into contact with subjects having presented the antigens of which said MHC ligand is derived.  30. Modified MHC ligand according to one of claims 19 to 29, characterized in that said MHC ligand before modification comprises one or consists of a peptide chosen from the peptides of the following sequences: a) SEQ ID No. 2 , SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; b) a peptide whose amino acid sequence is the sequence of the retroinverso form of a peptide of sequence SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47; and c) a peptide whose sequence has an identity level after optimal alignment of at least 70%, preferably at least 80%, 85%, 90% or 95% with the sequence of a sequence peptide SEQ ID No. 2, SEQ ID No. 45, SEQ ID No. 46 or SEQ ID No. 47.  31. Modified MHC ligand according to one of claims 19 to 30, characterized in that said modified MHC ligand is chosen from peptides of sequence SEQ ID Nos. 5.6, 7, 43 and 44, and the peptides of sequence SEQ ID Nos. 50 <Desc / Clms Page number 34>  51,52, 53 and 54, these peptides possibly being able to comprise a point mutation in the sequence of the unmodified MHC ligand from which it is derived, in particular a substitution of an amino acid by another natural amino acid or not natural.  32. Peptide modified by a method according to one of claims 9 to 18, or a MHC ligand according to one of claims 19 to 31, as a medicament.  33. A pharmaceutical composition comprising a compound chosen from: a peptide modified by a process according to one of claims 9 to 18, or a MHC ligand according to one of claims 19 to 31, where appropriate mixed or coupled with a carrier protein or one of its fragments; a nucleic construct containing a DNA encoding a peptide modified by a method according to one of claims 9 to 18, or a MHC ligand according to one of claims 19 to 31, if appropriate further containing a DNA coding for a carrier protein or one of its fragments.  34. Use of a peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature derived from a pathogenic microorganism antigenic protein, said peptide having been modified by a method according to one of claims 11 to 16, or of a modified MHC ligand according to one of claims 19 to 30 comprising one or more of these antigenic epitopes, for the preparation of a vaccine for the prophylactic or therapeutic treatment of infections with this microorganism, in particular viral, bacterial, parasitic infections or fungal.  35. Use of a peptide whose sequence comprises one or more hydrophobic antigenic epitopes derived from a tumor-associated antigenic protein, said peptide having been modified by a method according to one of claims 11 to 18, or of A modified MHC ligand according to one of claims 19 to 32 comprising one or more of these antigenic epitopes, for the preparation of a vaccine for the prophylactic or therapeutic treatment of cancers associated with this antigen, preferentially to inhibit the growth of tumors associated with this antigen.  36. Vaccine, characterized in that it comprises at least one peptide whose sequence comprises one or more antigenic epitopes of hydrophobic nature derived from a pathogenic or tumor-associated microorganism antigenic protein, said peptide having been modified by a method according to one of the claims <Desc / Clms Page number 35>  11 to 18, or a modified MHC ligand according to one of claims 19 to 32 comprising one or more of these antigenic epitopes.  37. Pharmaceutical composition according to claim 33 or vaccine according to claim 36, characterized in that it further comprises an adjuvant.  38. Pharmaceutical composition according to claim 33 or 37 or vaccine according to claim 36 or 37, characterized in that it further comprises a carrier compound mixed or coupled to said modified peptide or said modified MHC ligand.  39. Pharmaceutical composition according to one of claims 33, 37 and 38 or vaccine according to one of claims 36, 37 and 38, characterized in that it is incorporated into vectors selected from liposomes, virosomes, nanospheres. , microspheres or microcapsules.  40. Pharmaceutical composition according to one of claims 33,37 to 39 or vaccine according to one of claims 36,37 to 39, characterized in that it is administrable systemically, especially by injection, mucosal or transdermal route.