Immunogenic complexes for inducing immune responses directed against interleukin-6

Abstract

The present invention provides an immunogenic conjugate for enhancing the production of anti-IL-6 antibodies in active immunotherapy. The present invention provides an immunogenic complex comprising a transport protein and at least one polypeptide having at most 100 amino acids, the polypeptide comprising a 5-50 amino acid sequence of interleukin 6 (IL-6) or interleukin 6 receptor (IL-6R), or a variant sequence exhibiting at least 75% identity with the 5-50 amino acid sequence of IL-6 or IL-6R, covalently linked to the transport protein, wherein the transport protein is a non-toxic mutant diphtheria toxin.

Description

[Technical Field]

[0001] The present invention is directed to immunogenic complexes and pharmaceutical compositions, in particular vaccine compositions, comprising the immunogenic complexes and their use in methods for the prevention or treatment of diseases associated with the overexpression or overproduction of interleukin-6. [Background technology]

[0002] Interleukin-6 (IL-6) is an essential cytokine involved in regulating the proliferation and differentiation of immune cells, among other things.

[0003] IL-6 is strongly overproduced during inflammatory processes, and its overproduction is observed in numerous diseases such as, for example, infectious diseases, acute or chronic inflammatory diseases, as well as cancer.

[0004] IL-6 therefore appears as a triggering signal in inflammatory diseases, and preclinical studies in animal models of human disease have shown that inhibiting IL-6 activity alleviates symptoms or completely prevents the onset of disease.

[0005] The IL-6 signaling pathway offers multiple options for therapeutic intervention, ranging from biologics that inhibit the cytokine or its receptor outside the cell to small chemical molecules that target kinases and transcription factors contained within the cell. It is interesting to note that antibodies targeting IL-6 can be used in patients in significantly lower doses than antibodies that inhibit the IL-6 receptor (IL-6R). Indeed, soluble IL-6 receptors (sIL-6R) are present in high concentrations in serum, and because these soluble receptors also capture neutralizing IL-6R antibodies, a large number of neutralizing antibodies are needed to block the membrane receptor IL-6R on the cell surface and thus prevent IL-6 signaling.

[0006] The concentration of IL-6 is relatively low in healthy individuals (a few picograms per milliliter of serum), and antibodies directly targeting the cytokine IL-6 need only capture newly synthesized and liberated IL-6 molecules for activity. Direct blockade of IL-6 does not interfere with other cytokines that may signal through the IL-6R, thus providing specific inhibition of IL-6.

[0007] Over the years, a class of therapeutic agents targeting components of the IL-6 signaling pathway have been developed. The efficacy of therapeutic interventions that neutralize IL-6 or interfere with signaling has demonstrated the primary deleterious function of IL-6 in several diseases.

[0008] Thus, the first monoclonal antibody developed against the IL-6 pathway, the IL-6R blocker tricizumab, is today approved for the treatment of moderate to severe active rheumatoid polyarthritis (PR) in adults. Other monoclonal antibodies targeting the IL-6R are in development: sarilumab is approved for rheumatoid polyarthritis, NI-1201 is currently in preclinical trials, and bovalilizumab is currently in Phase II clinical trials for the treatment of rheumatoid polyarthritis and systemic lupus erythematosus.

[0009] Several monoclonal antibodies have also been developed that directly target IL-6, such as sirukumab (Phase II) for the treatment of rheumatoid polyarthritis, depression, and lupus nephritis, olokizumab for the treatment of rheumatoid polyarthritis and Crohn's disease, clazakizumab for the treatment of organ transplant rejection, and siltuximab, which is indicated for the treatment of Castleman's disease, on the other hand, for the treatment of multiple myeloma.

[0010] However, all these passive, i.e. immunotherapeutic treatments involving the administration of antibodies, especially monoclonal antibodies, require too frequent and prolonged administration, which poses significant limitations in terms of both patient comfort and cost.

[0011] To overcome these disadvantages, a method for preventing and treating IL-6-associated diseases based on active (or vaccination) immunotherapy has been developed, which is based on inducing a humoral immune response using immunogenic polypeptides derived from IL-6, leading to the patient's own production of anti-IL-6 antibodies.

[0012] Desallais et al. (2016) Sci. Rep. 6:19549 thus described the production of human anti-IL-6 neutralizing antibodies in cynomolgus monkeys administered with IL-6-derived polypeptides. However, the antibody ratios obtained remained modest. The object of the present invention is therefore to improve the production of anti-IL-6 antibodies in active immunotherapy. Summary of the Invention

[0013] The present invention arose from the unexpected discovery by the inventors that binding of an IL-6-derived polypeptide to the transport protein CRM197 can increase the production of anti-IL-6 antibodies in individuals to whom the conjugate is administered, compared to other transport proteins.

[0014] Therefore, the present invention provides: -transport proteins, - at least one polypeptide having at most 100 amino acids comprising a sequence of 5 to 50 amino acids of interleukin 6 (IL-6) or of the receptor for interleukin 6 (IL-6R), or a variant sequence exhibiting at least 75% identity with a sequence of 5 to 50 amino acids of IL-6 or IL-6R; and an immunogenic complex comprising: In the complex, at least one polypeptide is covalently linked to a transport protein, which is a non-toxic mutant diphtheria toxin. In certain embodiments of the immunogenic complex as defined above, the polypeptide is linked to the transport protein via a non-peptide linking agent.

[0015] The present invention also relates to an immunogenic complex as defined above for use in a method of therapeutic treatment, for use in a method of prevention or treatment of diseases associated with the overproduction or overexpression of IL-6, or for use in a method of vaccinating an individual against IL-6 or IL-6R or a method of inducing an immune response against IL-6 or IL-6R.

[0016] The present invention also relates to a method for preventing or treating diseases associated with overexpression or overproduction of IL-6 in an individual, which comprises administering to the individual a prophylactically or therapeutically effective amount of an immunogenic complex as defined above.The present invention also relates to a method for administering a vaccine against IL-6 or IL-6R or a method for inducing an immune response against IL-6 or IL-6R in an individual, which comprises administering to the individual an effective amount of an immunogenic complex as defined above.

[0017] The present invention also relates to pharmaceutical compositions, in particular vaccine compositions, comprising as active substance at least one immunogenic complex as defined above, optionally in combination with at least one pharmaceutically acceptable vehicle and / or excipient.

[0018] In a particular embodiment of the present invention, the pharmaceutical composition, in particular the vaccine composition, as defined above further comprises at least one adjuvant.

[0019] The present invention also relates to a pharmaceutical composition, in particular a vaccine composition, as defined above for use in a method for preventing or treating a disease associated with overexpression or overproduction of IL-6, or for use in a method for administering a vaccine against IL-6 or IL-6R or a method for inducing an immune response against IL-6 or IL-6R in an individual.

[0020] The present invention also relates to a method for the prevention or treatment of diseases associated with the overexpression of IL-6 in an individual, comprising administering to said individual a prophylactically or therapeutically effective amount of a pharmaceutical composition as defined above.

[0021] The present invention also relates to a method for administering a vaccine against IL-6 or IL-6R or a method for inducing an immune response against IL-6 or IL-6R in an individual, which comprises administering to the individual an effective amount of the pharmaceutical composition as defined above.

[0022] The present invention also relates to a method for preparing an immunogenic complex as defined above, comprising a step of covalently binding at least one polypeptide having at most 100 amino acids, comprising a sequence of 5 to 50 amino acids of interleukin 6 (IL-6) or of the receptor for IL-6 (IL-6R), or a variant sequence exhibiting at least 75% identity with a sequence of 5 to 50 amino acids of IL-6 or IL-6R, to a transport protein which is a non-toxic mutated diphtheria toxin. [Brief explanation of the drawings]

[0023] [Figure 1] Mean human anti-IL-6 antibody titers (vertical axis, arbitrary units) of five rabbits from six groups immunized on days 0, 13, 43, and 73 (arrows) with 5 μg (group 1, small circles), 7 μg (group 2, squares), 10 μg (group 3, up-pointing triangles), 15 μg (group 4, down-pointing triangles), or 50 μg (group 5, diamonds) of polypeptide or with the transport protein CRM197 (group 6, large circles) as a function of time (horizontal axis, days). DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description As a preamble, it should be noted that the expression "consisting of" means "consisting of," i.e., when an object consists of one element or more elements, the object may not include elements other than the mentioned elements. In contrast, the term "comprise" means "to include," "to contain," or "to include." That is, when an object "comprises" one element or more elements, elements other than the mentioned elements may be contained within the object. In other words, when an object "comprises" one element or more elements, it is composed of the one element or more elements, possibly other elements.

[0025] Polypeptides definition Interleukin 6 (IL-6), sometimes also called B lymphocyte stimulating factor 2 (B cell stimulating factor 2, BSF-2), cytotoxic T lymphocyte differentiation factor (CTL differentiation factor, CDF), hybridoma growth factor or interferon beta-2 (IFN-β-2), is well known to those skilled in the art. Numerous sequences of IL-6 from various animal species are available in sequence databases. By way of example, human IL-6 is listed in the UniProt / Swissprot database under the reference number P05231 (SEQ ID NO: 1). Also by way of example, the alpha subunit of the receptor for IL-6 is listed in the UniProt / Swissprot database under the reference number P08887 (SEQ ID NO: 2).

[0026] As used herein, the terms "peptide" and "polypeptide" are used in their broadest sense to refer to a molecule of two or more amino acid residues. The amino acid residues are joined by peptide bonds or, in variants, by other bonds such as ester bonds, ether bonds, etc. However, the amino acid residues are preferably joined between them by peptide bonds.

[0027] As used herein, the expressions "amino acid" and "amino acid residue" encompass natural, unnatural, or synthetic amino acids, including D- and L-forms, and amino acid analogs. An "amino acid analog" is to be understood as an unnatural amino acid that differs from the corresponding native natural amino acid by one or more atoms. For example, an amino acid analog of cysteine ​​may be homocysteine. However, the amino acid or amino acid residue is preferably natural.

[0028] The polypeptides according to the present invention are such that they are capable of eliciting an immune response directed against IL-6 or IL-6R, i.e., such that administration of such polypeptides, e.g., associated with a transport protein such as protein CRM-197 or KLH, to an animal such as a mouse, rat, or rabbit results in the production of antibodies directed against IL-6 or IL-6R. Thus, the polypeptides according to the present invention are immunogenic and contain one or more epitopes of IL-6 or IL-6R. Those skilled in the art are familiar with how to determine whether an antibody is directed against IL-6 or IL-6R, in particular by performing an ELISA test.

[0029] As used herein, the percentage identity between two peptide sequences can be determined by performing an optimal alignment over the entire length of the sequences, determining the number of aligned positions where amino acids are identical in each sequence, and dividing that number by the total number of amino acids in the longest of the two sequences. The optimal alignment is the one that gives the highest percentage identity between the two sequences.

[0030] As used herein, the terms "overproduction" and "overexpression" of IL-6 are considered equivalent and mean that IL-6 is present in the organism of an individual to be treated at a concentration above normal values ​​or at a concentration that is not physiological or pathological.

[0031] length Polypeptides according to the present invention preferably comprise at most 100, 90, 80, 70, 60, 50, 40, 30, 25, 24, 23, 22, 21 or 20 amino acids. Preferably, polypeptides according to the present invention comprise at least 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 40, 50, 60, 70, 80 or 90 amino acids. Preferably, polypeptides according to the present invention comprise 10 to 40 amino acids, more preferably 15 to 35 amino acids.

[0032] IL-6 Preferably, the IL-6 according to the present invention is selected from the group consisting of human IL-6 (hIL-6), murine IL-6, monkey, particularly macaque IL-6, equine IL-6, canine IL-6, feline IL-6, or rabbit IL-6. Particularly preferably, the IL-6 according to the present invention is human IL-6.

[0033] array Preferably, the polypeptide according to the present invention comprises or consists of a sequence of 5, 6, 7, 8, 9, 10, 11, or 12 amino acids of IL-6 or IL-6R. Preferably, the polypeptide according to the present invention comprises or consists of a sequence of at most 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids of IL-6 or IL-6R. Preferably, the polypeptide according to the present invention comprises or consists of a sequence of 6 to 40, 7 to 35, or 8 to 30 amino acids of IL-6 or IL-6R.

[0034] Preferably, the polypeptide according to the present invention comprises or consists of a sequence of 7 to 35 amino acids of IL-6 or IL-6R, or a variant sequence that exhibits at least 90% identity to the sequence of 7 to 35 amino acids of IL-6 or IL-6R.

[0035] Polypeptides according to the invention are in particular those described in WO 2013 / 021284, which is incorporated herein by reference.

[0036] Preferably, the amino acid sequence of IL-6 or IL-6R according to the present invention comprises at least 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, or all of them, in the IL-6 sequence 58 to 78, 73 to 94, 96 to 111, 122 to 141, or 172 to 189. Preferably, the amino acid sequence of IL-6 or IL-6R according to the present invention consists of 5, 6, 7, 8, 9, 10, 11, or 12 amino acids, or all of them, in the IL-6 sequence 58 to 78, 73 to 94, 96 to 111, 122 to 141, or 172 to 189.

[0037] Preferably, the IL-6 or IL-6 amino acid sequence according to the present invention comprises at most 21, 20, 19, 18, 17, 16, 15, 14 or 13 amino acids of the IL-6 sequence 58-78, 73-94, 96-111, 122-141, or 172-189.

[0038] The numbering below is based on the reference sequences, including the peptide signals, shown as examples for human IL-6 and murine IL-6. Human IL-6 (hIL-6): Sequences 58-78: RYIIDGISALRKETCNKSNMC (SEQ ID NO: 3) Sequences 73-94: NKSNMCESSKEALAENNLNLPK (SEQ ID NO: 4) Sequences 96-111: AEKDGCFQSGFNEETC (SEQ ID NO: 5) Sequences 122-141: FEVYLEYLQNRFESSEEQAR (SEQ ID NO: 6) Sequences 172-189: NASLLTKLQAQNQWLQDM (Sequence ID No. 7) Sequences 196-212: RSFKEFLQSSLRALRQM (Sequence ID No. 8) Murine IL-6 (mIL-6) Sequences 58-78: VLWEIVEMRKELCNGNSDCMN (SEQ ID NO: 9) Sequence 73-94; NSDCMNNDDALAENNLKLPEIG (SEQ ID NO: 10) Sequences 96-111: NDGCYQTGYNQEICLL (SEQ ID NO: 11) Sequences 122-141: SYLEYMKNNLKDNKKDKARV (SEQ ID NO: 12) Sequences 172-189: ALLTDKLESQKEWLRTKT (SEQ ID NO: 13) Sequence 196-211: SLEEFLKVTLRSTRQT (SEQ ID NO: 14).

[0039] Thus, preferably, the polypeptide as defined above has the sequence: ·RYIIDGISALRKETCNKSNMC, ·NKSNMCESSKEALAENNLNLPK, ·AEKDGCFQSGFNEETC, ·FEVYLEYLQNRFESSEEQAR, ·NASLLTKLQAQNQWLQDM, and ·RSFKEFLQSSLRALRQM The IL6 sequence may comprise at least 5, 6, 7, 8, 9, 10, 11 or 12 amino acids, or all of them, of an IL6 sequence selected from the group consisting of:

[0040] Alternatively, the polypeptide according to the invention may comprise multiple repeats, for example 2, 3, 4, 5, 10 or 20 repeats, of the sequence of IL-6 or IL-6R, respectively, or of the variant sequence defined above.

[0041] Mutant sequences A variant sequence according to the present invention having at least 75% identity with the sequence of IL-6 or IL-6R according to the present invention will preferably have at least 80%, 85%, 90%, 95% or 98% identity with the sequence of IL-6 or IL-6R according to the present invention, and more preferably will have 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 or more amino acids.

[0042] cyclization Preferably, the polypeptides according to the invention are cyclized.

[0043] The polypeptides according to the invention may be cyclized, ie the entire polypeptide forms a ring, or a part of the polypeptide forms a ring, according to any type of method known to those skilled in the art.

[0044] Depending on the functional groups present in the polypeptide, the cyclization can be carried out in several different ways, such as from the C-terminus to the N-terminus, from the N-terminus to a side chain, from a side chain to the C-terminus, or even between two side chains. The set of side chains involved in cyclization are in particular the functional groups -NH, -COOH, and -SH.

[0045] Among the various modes of polypeptide cyclization, mention may be made of the formation of a peptide bond between the two ends of the polypeptide, the N-terminus and the C-terminus, lactamization, lactonization, or the formation of a disulfide bridge between two cysteines (C) of the polypeptide. In particular, when a disulfide bridge is formed between cysteines, i.e., between the -SH radicals of two cysteines, cysteines may already be present in the sequence of IL6 or IL-6R, or cysteines may be added within these sequences or at the N-terminus and / or C-terminus of one or more of them to form a variant sequence.

[0046] qualification The polypeptides according to the invention may comprise post-translational modifications such as glycosylation, methylation, acylation, in particular by fatty acids or by acetyl groups, amidation, or phosphorylation. For example, the N-terminus of the polypeptides according to the invention may be acetylated or the C-terminus may be modified by amidation.

[0047] Additional sequences Preferably, the polypeptide according to the present invention comprises one or more additional sequences in addition to the sequence of IL-6 or IL-6R according to the present invention.

[0048] These additional sequences according to the invention may in particular confer physicochemical properties that allow the polypeptides according to the invention to have an improved structural antigen presentation or to have improved solubility compared to similar polypeptides that do not contain these additional sequences. Additional sequences according to the invention may in particular comprise one or more peptide bonds or peptide linkages ("peptide linkers") that are particularly useful for binding to transport proteins. Peptide linkages. Such sequences usually contain 1 to 10, in particular 1 to 6, and in particular 2 to 5 amino acids.

[0049] A particularly preferred binding sequence according to the invention is the sequence EGEZ (SEQ ID NO: 15), where Z is an amino acid that allows binding to transport proteins, in particular selected from the group consisting of cysteine ​​(C), tyrosine (Y) and lysine (K), particularly preferably selected from the group consisting of cysteine ​​(C) and tyrosine (Y). Particularly preferably, Z is tyrosine (Y). Advantageously, the sequence EGEZ improves solubility while at the same time maintaining a good immune response, as shown in Examples 4, 5 and 6.

[0050] Another preferred binding sequence is the amino acid Z, which allows binding to the transport protein, in particular selected from the group consisting of cysteine ​​(C), tyrosine (Y) and lysine (K), particularly preferably selected from the group consisting of cysteine ​​(C) and tyrosine (Y). More preferably, Z is tyrosine (Y).

[0051] On the other hand, the additional sequence(s) may contain epitopes that belong to proteins other than IL-6 or IL-6R and that make it possible to elicit or raise an immune response directed against those other proteins.

[0052] Furthermore, additional sequences according to the invention also comprise sequences of exogenous or pan-T cell epitope(s), which advantageously can enhance the immunogenicity of the polypeptide according to the invention.

[0053] preparation The polypeptides according to the invention can be prepared by any method known in the art, in particular by chemical synthesis, as well as by recombinant methods in eukaryotic or prokaryotic cells.

[0054] transport proteins The transport protein according to the invention is a non-toxic mutant diphtheria toxin, i.e. diphtheria anatoxin, obtained by mutagenesis. Advantageously, the mutant diphtheria toxin is pharmaceutically acceptable.

[0055] The transport protein according to the invention is preferably selected from the group consisting of CRM197, CRM176, CRM228, CRM45, CRM9, CRM102, CRM103, and CRM107.

[0056] Particularly preferably, the transport protein according to the invention is CRM197.

[0057] CRM197 is a genetically attenuated form of diphtheria toxin. This form has a single mutation at position 52, substituting glutamic acid (E) for glycine (G), which results in the loss of ADP-ribosyltransferase activity. This form instead retains all lysine (K) amino radicals available for binding. CRM197 has 535 amino acids (58.4 kDa) and is composed of two subunits linked by a disulfide bridge. This form is described in GenBank accession number 1007216A (SEQ ID NO: 16). This form can be produced recombinantly in corynebacteria as well as other bacteria such as Pseudomonas fluorescens or Escherichia coli.

[0058] Binder Preferably, binding agents according to the present invention are non-peptidic.

[0059] The binding agent can be heterobifunctional, such as N-γ-maleimidobutyryloxysuccinimide (GMBS) ester and sulfo-GMBS derivatives, m-maleimidobenzoyl-n-hydroxysuccinimide (MBS) ester and sulfo-MBS derivatives, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), carbodiimides, bisdiazonium benzidine (BDB), or glutaraldehyde. The use of binding agents is particularly described in the reference book "Protein Protocol Handbook" (2002), in the chapter "Preparation of Antisera Using Peptide Conjugates," which is incorporated herein by reference.

[0060] When GMBS, MBS, SMCC or sulfo-SMCC are used, they are preferably anchored on a cysteine ​​(C), which, if not present in the sequence of the peptide, can be added, in particular to its N-terminus or C-terminus.

[0061] Complex Preferably, the polypeptide of the immunogenic complex according to the invention comprises or consists of the sequence SSKEALAENNLNLPK (SEQ ID NO: 17), more particularly the sequence ESSKEALAENNLNLPK (SEQ ID NO: 18), even more particularly the sequence ESSKEALAENNLNLPKC (SEQ ID NO: 19), the sequence AESSKEALAENNLNLPKC (SEQ ID NO: 20) or the sequence CESSKEALAENNLNLPKC (SEQ ID NO: 21), optionally supplemented C-terminally with the additional sequence EGEZ or Z for a peptide bond as defined above. Preferably, the polypeptide is likewise conjugated to the transport protein CRM197 as defined above by a binding agent as defined above.

[0062] Preferably, therefore, the immunogenic conjugate as defined above is of formula (I): [(AESSKEALAENNLNLPKC)-GMB]-CRM197 (I) During the ceremony, the polypeptide consists of the sequence AESSKEALAENNLNLPKC, The transport protein is CRM197, The polypeptide is covalently attached to the transport protein via the coupling agent N-γ-maleimidobutyryl-(GMB), The parentheses indicate that the polypeptide is cyclized by forming a peptide bond between the C-terminus cysteine ​​(C) and the N-terminus alanine (A); · Square brackets indicate that at least one polypeptide is bound to a transport protein.

[0063] Preferably as well, the immunogenic complex as defined above is of formula (II): [Acetyl-(CESSKEALAENNLNLPKC)-(X) i -Z-GMB]-CRM197 (II) During the ceremony, The polypeptide has the sequence CESSKEALAENNLNLPKC-(X) i -Z, The transport protein is CRM197, The polypeptide is covalently attached to the transport protein via the coupling agent N-γ-maleimidobutyryl-(GMB), The parentheses indicate that the portion of the polypeptide sequence CESSKEALAENNLNLPKC is cyclized by forming a disulfide bridge between the C-terminal cysteine ​​(C) and the N-terminal cysteine ​​(C), ·Acetyl- indicates that the free radical -NH2 at the N-terminus of cysteine ​​(C) is protected by an acetyl group by forming an amide bond; (X) represents any peptide sequence of 1 to 5 amino groups, in particular EGE; i is a value of 0 or 1, Z represents tyrosine (Y) or cysteine ​​(C), · Square brackets indicate that at least one polypeptide is bound to a transport protein.

[0064] Preferably, the immunogenic conjugate as defined above is of formula (III): [Acetyl-(CESSKEALAENNLNLPKC)-(X) i -Y-GMB]-CRM197 (III) During the ceremony, The peptide has the sequence CESSKEALAENNLNLPKC)-(X) i -Y, The transport protein is CRM197, The polypeptide is covalently attached to the transport protein via the coupling agent N-γ-maleimidobutyryl-(GMB), The parentheses indicate that the portion of the polypeptide sequence CESSKEALAENNLNLPKC is cyclized by forming a disulfide bridge between the C-terminal cysteine ​​(C) and the N-terminal cysteine ​​(C), ·Acetyl- indicates that the free radical -NH2 at the N-terminus of cysteine ​​(C) is protected by an acetyl group by forming an amide bond; (X) represents any peptide sequence of 1 to 5 amino groups, in particular EGE; i is 0 or 1, in particular 0, · Square brackets indicate that at least one polypeptide is bound to a transport protein.

[0065] Preferably as well, the immunogenic conjugate as defined above is of formula (IV): [Acetyl-(CESSKEALAENNLNLPKC)-(X) i -C-GMB]-CRM197 (IV) During the ceremony, The peptide has the sequence CESSKEALAENNLNLPKC)-(X) i -C, The transport protein is CRM197, The polypeptide is covalently attached to the transport protein via the coupling agent N-γ-maleimidobutyryl-(GMB), The parentheses indicate that the portion of the polypeptide sequence CESSKEALAENNLNLPKC is cyclized by forming a disulfide bridge between the C-terminal cysteine ​​(C) and the N-terminal cysteine ​​(C), ·Acetyl- indicates that the free radical -NH2 at the N-terminus of cysteine ​​(C) is protected by an acetyl group by forming an amide bond; (X) represents any peptide sequence of 1 to 5 amino groups, in particular EGE; i is a value of 0 or 1, · Square brackets indicate that at least one polypeptide is bound to a transport protein.

[0066] Pharmaceutical compositions, particularly vaccine compositions Among the adjuvants that may be administered in combination with the immunogenic complexes defined above or that may be present in the pharmaceutical compositions, in particular vaccine compositions, defined above, mention may be made of alum (aluminum hydroxide), Montanide® ISA51VG, Montanide® ISA720VG, any water-in-oil suspension or any oil-in-water suspension, as well as all adjuvants generally known in the scientific literature.

[0067] Likewise, immunomodulatory agents such as MP40 may be administered in combination with the immunogenic complexes of the invention or added to pharmaceutical compositions, particularly vaccine compositions.

[0068] therapeutic use The immunogenic complexes according to the present invention or pharmaceutical compositions, in particular vaccine compositions, comprising said immunogenic complexes are intended for active immunization and can be administered to treat all diseases associated with the overproduction or overexpression of the inflammatory cytokine IL-6 by inducing the production of antibodies related to the overproduced or overexpressed IL-6 to prevent its effects.

[0069] Preferably, the IL-6 overproduction or overexpression associated disease according to the present invention is selected from the group consisting of: - chronic inflammatory diseases of the small intestine, such as Crohn's disease, hemorrhagic proctocolitis or ulcerative colitis, -Arthritis such as rheumatoid polyarthritis, juvenile arthritis, psoriatic arthritis, arthropathy, refractory rheumatoid polyarthritis, non-rheumatic chronic arthritis, ankylosing spondylitis, - chronic inflammatory bone diseases associated with IL-6, in particular osteoporosis or any bone resorption disease, IL-6-associated inflammatory diseases, whether chronic or associated with an infection, such as septic shock, endotoxic shock, sepsis, hepatitis C, malaria, AIDS or other HIV-related infections, chronic or IL-6-associated inflammatory diseases of the cardiovascular system, such as arteriosclerosis, damage associated with reperfusion following ischemia, coronary artery disease, vasculitis such as Behçet's disease or Wegener's granulomatosis, autoimmune diseases such as scleroderma, systemic sclerosis, lupus erythematosus, in particular disseminated lupus erythematosus, sclerosis, multiple sclerosis or psoriasis, Transplant-related diseases, especially the reaction of the transplanted tissue to the host, transplant rejection, and traumatic injury; - skin diseases associated with allergies, especially allergic asthma, and delayed hypersensitivity reactions, -Immune deficiency, such as idiopathic immune deficiency syndrome (CVID), chronic or respiratory IL-6-associated inflammatory diseases, in particular respiratory distress syndrome or pulmonary fibrosis, Cancers with a chronic or IL-6-associated inflammatory component, such as plasmacytoma, colorectal cancer, uterine cancer, lymphoproliferative syndromes, multiple myeloma, especially refractory multiple myeloma, or myeloproliferative syndromes, -Diabetes, especially juvenile diabetes, -Amyloidosis, especially Alzheimer's disease, - uveitis, especially recurrent forms of uveitis, - cachexia, -Endometriosis.

[0070] Administration Preferably, the amount of the immunogenic complex according to the present invention administered by administration, i.e., the unit amount of the immunogenic complex administered, is 3 ng to 3 g, more preferably 300 ng to 900 μg, even more preferably 15 μg to 450 μg, and most preferably 30 μg to 300 μg. Each of these unit amounts of the complex is preferably 1 ng to 1 g, more preferably 100 ng to 300 μg, even more preferably 5 μg to 150 μg, and most preferably 10 μg to 100 μg of the polypeptide unit amount according to the present invention.

[0071] Preferably, the pharmaceutical composition, in particular the vaccine composition, according to the invention comprises the immunogenic complex according to the invention in an amount of 3 ng to 3 g, more preferably 300 ng to 900 μg, even more preferably 15 μg to 450 μg, and most preferably 30 μg to 300 μg.Similarly, preferably, the pharmaceutical composition, in particular the vaccine composition, according to the invention comprises the peptide according to the invention in an amount of 1 ng to 1 g, more preferably 100 ng to 300 μg, even more preferably 5 μg to 150 μg, and most preferably 10 μg to 100 μg.

[0072] Advantageously, the dosage according to the invention can stimulate the production of antibodies directed against the polypeptide relative to antibodies directed against the transport protein, i.e., a ratio of antibody titers directed against the polypeptide to antibody titers directed against the transport protein of greater than 1. Without wishing to be bound by any particular theory, the inventors believe that if too many antibodies are produced against the transport protein, they will inhibit the immune response when a subsequent immunization is carried out with the immunogenic complex according to the invention, and therefore it is advantageous to keep the antibody titer of the response against the transport protein sufficiently low compared to the titer of antibodies induced against the polypeptide.

[0073] The administration of the immunogenic complexes can be via the intravenous, intradermal, intramuscular, mucosal, especially intranasal, or intraperitoneal route.

[0074] Dosage regimens or therapeutic regimens may range from administration every 15 days to once a year for priming until a good anti-IL-6 or anti-IL-6R antibody response is achieved, followed by revaccination at intervals, for example, once every 2 months to once a year, to allow the antibody to achieve sufficient beneficial activity.

[0075] Preferably, the individual according to the present invention is an animal, particularly a mammal, especially a human. Preferably, the individual according to the present invention overproduces or overexpresses IL-6 or is at risk of overproducing or overexpressing IL-6. Preferably, the individual according to the present invention is 50 years of age or older, 60 years of age or older, 70 years of age or older, 80 years of age or older, or 90 years of age or older.

[0076] The invention will be further clarified by the following non-limiting examples. [Example]

[0077] Example 1 Chemically synthesized peptides ( CESSKEALAENNLNLPK C) Y (SEQ ID NO: 22) is bound to the transport protein CRM197 (Phenex, USA) on the one hand and to the transport protein KLH (Keyhole Limpet Hemocyanin, Sigma) on the other hand.

[0078] The peptide corresponds to the 78-93 region of human IL-6 (underlined above) and contains an additional C-terminal cysteine ​​and tyrosine. The cysteine ​​allows cyclization via a disulfide bridge with the cysteine ​​present at the N-terminus of the peptide (between the parentheses). Conjugation to transport proteins is carried out via the tyrosine using bis-diazobenzidine (BDB, Polypeptide Research Institute, Strasbourg, France) according to standard methods.

[0079] Preparation of these conjugates typically results in a typical mass ratio of peptide to transport protein of approximately 1:2 for KLH and CRM197, as determined by amino acid analysis (AAA), a method for quantifying amino acids. In other words, for a total mass of 300 micrograms of peptide bound to the transport protein, there are 200 micrograms of KLH and 100 micrograms of peptide, and for the CRM197 conjugate, there are 200 micrograms of CRM197 and 100 micrograms of peptide.

[0080] These conjugates were tested in immunization experiments in Swiss mice (Charles River Laboratories, Écully, France) using Montanide™ ISA51VG (or ISA51 for short) (SEPPIC, France) as an adjuvant, with a mixture containing 50 microliters of conjugate and 50 microliters of Montanide™ ISA51VG administered intramuscularly, for an injection volume of 300 micrograms of conjugate for each immunization. Two groups of eight mice were immunized with the peptide conjugated to KLH and the peptide conjugated to CRM197 on days 0, 14, 28, and 42. Mice were sacrificed on day 54, their blood was collected, and the prepared sera were evaluated for their human anti-IL-6 antibody ratios, as measured by ELISA (Desallais et al., 2016, Sci. Rep. 6:19549). On the day of sacrifice, the mice were in good condition (good glossy coat, weight retention, normal defecation), indicating that immunization with the immunogenic complex had no toxic effects at the doses used.

[0081] The anti-IL-6 antibody titers obtained for the two groups KLH and CRM197 are summarized in the table below. TIFF2026041716000002.tif27170

[0082] It can be seen that the anti-IL-6 antibody titer is clearly higher in the CRM197 conjugate than in the KLH conjugate at the same dose of the conjugate.

[0083] Example 2 The same composition as in Example 1 is tested in macaques subcutaneously immunized with Montanide™ ISA51VG as an adjuvant with a mixture of 250 microliters of Montanide™ ISA51VG and 250 microliters of the conjugate on days 1, 15, 30, and 45. 300 micrograms of the KLH conjugate and 300 micrograms of the CRM197 conjugate are administered to two groups of four animals each at each immunization.

[0084] Blood sampling and serum preparation are performed on day 57, and antibody titers directed against human IL-6 are measured by ELISA. Throughout the experiment, the monkeys are evaluated for their general condition and are in good physical condition (normal behavior, good appetite, hair retention, normal defecation), indicating that immunization with the immunogenic complex has no toxic effects at the doses used.

[0085] The anti-IL-6 antibody titers obtained for the two groups KLH and CRM197 are summarized in the table below. TIFF2026041716000003.tif27170

[0086] Again, the titers obtained after immunization with the CRM197 conjugate are clearly higher in mean and median than those obtained with KLH.

[0087] Example 3 Chemically synthesized cyclic peptides ( LTKLQAQNQWLQDM C) (SEQ ID NO: 23) The peptides were conjugated to the transport proteins CRM197 or KLH via the -SH group of the cysteine. Conjugation was performed using the bifunctional conjugation agent N-γ-maleimidobutyryl succinimide (GMBS, Thermo Fisher Scientific 22309) according to standard procedures ("GMBS and Sulfo-GMBS User Guide," Thermo Fisher Scientific). The peptides contained the human IL-6 sequence 176-189 (underlined above), to which a cysteine ​​was added at the C-terminus and cyclized by lactamization (see bracketed above).

[0088] The preparation of these conjugates results in a typical mass ratio of peptide to transport protein of 1:2 for KLH and 1:2 for CRM197. In other words, for a total mass of 300 milligrams of conjugated peptide, there are measured to be 200 micrograms of KLH and 100 micrograms of peptide in the KLH conjugate, and similarly for the CRM197 conjugate (200 micrograms of CRM197 and 100 micrograms of peptide).

[0089] These conjugates are tested in immunization experiments in mice intramuscularly, using Montanide™ ISA51VG as adjuvant (50 microliters of conjugate per 50 microliters of Montanide™ ISA51VG) at a conjugate dose of 450 micrograms for each immunization. Two groups of eight mice are immunized with peptides conjugated to KLH and with peptides conjugated to CRM197 on days 0, 14, 28, and 42. The mice are sacrificed on day 54, their blood is collected, and the serum so prepared is evaluated for human anti-IL-6 antibody ratios measured by ELISA. On the day of sacrifice, the mice are in good physical condition (good, lustrous coat, weight retention, and normal bowel movements), indicating that immunization with the immunogenic conjugates is not toxic at the doses used.

[0090] The anti-hIL-6 antibody titers obtained for the two groups KLH and CRM197 are summarized in the table below. TIFF2026041716000004.tif27170

[0091] It can be seen that for the same dose of conjugate, antibody titers are once again significantly higher for the CMR197 conjugate compared to the KLH conjugate.

[0092] Example 4 Chemically synthesized peptides (C IDKQIRYIIDGISALRKET C) EGEC (SEQ ID NO: 24) The peptides are conjugated to CM197 on the one hand or to KLH on the other. They contain the sequence 53-71 of human IL-6 (underlined above) flanked by two cysteines that allow cyclization of the peptide (between the parentheses above) by the formation of disulfide bridges. The conjugation is carried out via the C-terminal cysteine ​​using the bifunctional coupling agent N-γ-maleimidobutyryl succinimide (GMBS). A linker peptide of the sequence EGEC is present.

[0093] The preparation of these conjugates results in a typical mass ratio of peptide to transport protein of 1:2 for KLH and 1:2 for CRM197. In other words, for a total mass of 300 milligrams of conjugated peptide, there are measured to be 200 micrograms of KLH and 100 micrograms of peptide in the KLH conjugate, and similarly for the CRM197 conjugate (200 micrograms of CRM197 and 100 micrograms of peptide).

[0094] The conjugates were tested by immunizing two groups of six New Zealand White (NZW) rabbits (Charles River) subcutaneously with Montanide™ ISA51VG adjuvant on days 1, 15, and 45. Rabbits were immunized with 450 micrograms of conjugate (peptide-KLH or peptide-CRM197) at each immunization. The injection volume of Montanide™ ISA51VG was 400 microliters (200 microliters of conjugate and 200 microliters of Montanide™ ISA51VG).

[0095] Blood sampling and preparation of serum from these samples is performed on day 60. Antibody titers directed against human IL-6 are measured by ELISA. On the day of sacrifice, the rabbits were in good physical condition (good glossy coat, weight retention, normal defecation), indicating that immunization with the immunogenic complex had no toxic effects at the doses used.

[0096] The titers obtained for the two groups KLH and CRM197 are summarized in the table below. TIFF2026041716000005.tif24170

[0097] Again, the titers obtained after immunization against the CRM197 conjugate are clearly higher in mean and median than those obtained against KLH.

[0098] Example 5 Chemically synthesized peptides (A)(C LQAQNQWLQDM C) Y (SEQ ID NO: 25) and (B)(C LQAQNQWLQDM C) EGEY (SEQ ID NO: 26) is conjugated to the transport protein CRM197. The peptide contains the sequence spanning residues 179 to 189 of human IL-6 (underlined above), with cysteines added to the N- and C-termini for cyclization. The peptides are cyclized by the formation of disulfide bridges between the cysteines (underlined), and the binding of the peptides to CRM197 is achieved via the terminal tyrosines. The bifunctional linker used is BDB. Peptide (B) is identical to peptide (A) except for the linker sequence, which consists of a tripeptide of the sequence EGE.

[0099] The preparation of these complexes results in a typical mass ratio of peptide to transport protein of 1:2. In other words, for a total complex weight of 300 micrograms, 200 micrograms of CMR197 and 100 micrograms of peptide are measured.

[0100] The compounds are tested by subcutaneously immunizing two groups of six mice with Montanide™ ISA51VG as an adjuvant on days 1, 15, and 45. Mice are immunized with 300 micrograms of the compound at each immunization. The injection volume of Montanide™ ISA51VG is 200 microliters (100 microliters of the compound and 100 microliters of Montanide™ ISA51VG).

[0101] Blood sampling and preparation of serum from these samples is carried out on day 54. Antibody titers directed against human IL-6 are measured by ELISA. On the day of sacrifice, the mice are in good physical condition (good glossy coat, weight retention, normal defecation), indicating that immunization with the immunogenic complexes has no toxic effects at the doses used.

[0102] The titers obtained for the two groups are summarized in the table below. TIFF2026041716000006.tif26170

[0103] It can be seen that the titers obtained after immunization against peptides with the attached peptide EGE are clearly higher in mean and median values ​​than those obtained against peptides without the attached peptide.

[0104] Example 6 Chemically synthesized peptides (A)( CFQSGFNEETC ) Y (SEQ ID NO: 27) and (B)( CFQSGFNEETC )EGEY (SEQ ID NO: 28) are conjugated to the transport protein CRM197. The peptides contain the sequence 101-111 of human IL-6 (underlined above, the cysteine ​​(C) is naturally occurring). The peptides are cyclized by the formation of a disulfide bridge between the two cysteines. The peptides are conjugated to CRM197 via the terminal tyrosine using the bifunctional coupling agent BDB.

[0105] The preparation of these complexes results in a typical mass ratio of peptide to transport protein of 1:2. In other words, for a total complex weight of 300 micrograms, 200 micrograms of CMR197 and 100 micrograms of peptide are measured.

[0106] These compounds are tested by subcutaneously immunizing two groups of six mice with Montanide™ ISA51VG as an adjuvant on days 1, 15, and 45. Mice are immunized with 300 micrograms of the compound at each immunization. The injection volume of Montanide™ ISA51VG is 200 microliters (100 microliters of the compound and 100 microliters of Montanide™ ISA51VG).

[0107] Blood sampling and preparation of serum from these samples is performed on day 54. Antibody titers directed against human IL-6 are measured by ELISA. On the day of sacrifice, the mice were in good physical condition (good glossy coat, weight retention, normal defecation), indicating that immunization with the immunogenic complex had no toxic effects at the doses used.

[0108] The titers obtained for the two groups are summarized in the table below. TIFF2026041716000007.tif26170

[0109] Again, it can be seen that the titers obtained after immunization against peptides with the attached peptide EGE are clearly higher in mean and median than those obtained against peptides without the attached peptide.

[0110] Example 7 Chemically synthesized peptides ( CESSKEALAENNLNLPK C) Y (SEQ ID NO: 22) is conjugated to the transport protein CRM197 (Phenex, USA) as previously described in Example 1. The peptide is tested at three doses: 600 micrograms of conjugate (i.e., 200 μg of peptide), 300 micrograms of conjugate (i.e., 100 μg of peptide), and 150 micrograms of conjugate (i.e., 50 μg of peptide), with subcutaneous immunization of three groups of 10 macaques using Montanide® ISA51VG as adjuvant, with a mixture of 250 microliters of Montanide® ISA51VG and 250 microliters of conjugate, on days 0, 15, 30, 45, 60, and 75.

[0111] Blood sampling and serum preparation are performed on days 30, 45, 60, 75 and 90, and antibody titers directed against human IL-6 and against CRM197 are measured by ELISA. In the following table, the anti-IL-6 and anti-CRM197 titers, as well as the ratio (anti-IL-6 / anti-CRM197), are shown for the three groups. TIFF2026041716000008.tif159170

[0112] Surprisingly, the group immunized with 150 μg of complex (i.e., 50 μg of peptide present in the complex), i.e., the lowest dose, showed anti-IL-6 titers that were as good as those of the groups immunized with 600 μg or 300 μg of complex, and in particular the ratio (anti-IL-6 / anti-CRM197) was superior in that group at all times to the group immunized with 600 μg of complex (corresponding to 200 μg of peptide present in the complex).

[0113] Example 8 Chemically synthesized peptides ( CESSKEALAENNLNLPK C) Y (SEQ ID NO: 22) is conjugated to the transport protein CRM197 (Phenex, USA) as previously described in Example 1. The peptide is tested at five different doses during subcutaneous immunization in groups of five immunized rabbits: 5 micrograms of peptide (i.e., 15 μg of conjugate), 7 μg of peptide (i.e., 21 μg of conjugate), 10 μg of peptide (i.e., 30 μg of conjugate), 15 μg of peptide (i.e., 45 μg of conjugate), and 50 μg of peptide (i.e., 150 μg of conjugate). Rabbits were immunized on days 0, 13, 43, and 73 by subcutaneous injection of a mixture of 250 microliters of Montanide™ ISA51VG and 250 microliters of conjugate.

[0114] Sera were collected on days 0, 13, 30, 43, 60, 73, 90, and 104, and antibody responses to IL-6 were measured by ELISA. The results of the immunizations are given in the graph in Figure 1, where each point represents the average titer obtained for five rabbits in a group.

[0115] CRM197 alone does not induce the production of anti-IL-6 antibodies, and all groups immunized with peptides conjugated to CRM197 produce anti-IL-6 antibodies. Surprisingly, we find that a dose of 10 μg of peptide (i.e., 30 μg of conjugate) gives equivalent antibody titers to a dose of 50 μg of peptide (i.e., 150 μg of conjugate).

[0116] TIFF2026041716000009.tif168170

[0117] [Sequence table] SEQUENCE LISTING <110> PEPTINOV SAS CONSERVATOIRE NATIONAL DES ARTS ET METIERS <120> CONJUGUE IMMUNOGENE DESTINE A INDUIRE UNE REPONSE IMMUNITAIRE DIRIGEE CONTRE L'INTERLEUKINE-6 <130> B000112WO <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 212 <212> PRT <213> Homo sapiens <400> 1 Met Asn Ser Phe Ser Thr Ser Ala Phe Gly Pro Val Ala Phe Ser Leu 1 5 10 15 Gly Leu Leu Leu Val Leu Pro Ala Ala Phe Pro Ala Pro Val Pro Pro 20 25 30 Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg Gln Pro Leu Thr 35 40 45 Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile 50 55 60 Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys Glu Ser 65 70 75 80 Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala 85 90 95 Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Leu 100 105 110 Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr 115 120 125 Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala Arg Ala Val Gln 130 135 140 Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys Lys Ala Lys Asn 145 150 155 160 Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn Ala Ser Leu Leu 165 170 175 Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Thr Thr His 180 185 190 Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala 195 200 205 Leu Arg Gln Met 210 <210> 2 <211> 468 <212> PRT <213> Homo sapiens <400> 2 Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro 1 5 10 15 Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg 20 25 30 Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro 35 40 45 Gly Val Glu Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys 50 55 60 Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg 65 70 75 80 Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys 85 90 95 Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val 100 105 110 Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser 115 120 125 Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr 130 135 140 Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp 145 150 155 160 Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175 Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met 180 185 190 Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe 195 200 205 Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val 210 215 220 Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp 225 230 235 240 Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg 245 250 255 Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp 260 265 270 Leu Gln His His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His 275 280 285 Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300 Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser 305 310 315 320 Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr 325 330 335 Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr 340 345 350 Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pro Leu Pro Thr Phe Leu 355 360 365 Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu Leu Cys Ile Ala Ile 370 375 380 Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Arg Ala Leu Lys Glu Gly 385 390 395 400 Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gly Gln Leu Val Pro Glu 405 410 415 Arg Pro Arg Pro Thr Pro Val Leu Val Pro Leu Ile Ser Pro Pro Val 420 425 430 Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro 435 440 445 Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr Asp Tyr 450 455 460 Phe Phe Pro Arg 465 <210> 3 <211> 21 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 3 Arg Tyr Ile Ile Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn 1 5 10 15 Lys Ser Asn Met Cys 20 <210> 4 <211> 22 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 4 Asn Lys Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn 1 5 10 15 Asn Leu Asn Leu Pro Lys 20 <210> 5 <211> 16 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 5 Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys 1 5 10 15 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 6 Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu 1 5 10 15 Glu Gln Ala Arg 20 <210> 7 <211> 18 <212> PRT <213> Artificial sequence <220> <223> IL-6 Sequence <400> 7 Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln 1 5 10 15 Asp Met <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> IL-6 sequence <400> 8 Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln 1 5 10 15 Met <210> 9 <211> 21 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 9 Val Leu Trp Glu Ile Val Glu Met Arg Lys Glu Leu Cys Asn Gly Asn 1 5 10 15 Ser Asp Cys Met Asn 20 <210> 10 <211> 22 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 10 Asn Ser Asp Cys Met Asn Asn Asp Asp Ala Leu Ala Glu Asn Asn Leu 1 5 10 15 Lys Leu Pro Glu Ile Gly 20 <210> 11 <211> 16 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 11 Asn Asp Gly Cys Tyr Gln Thr Gly Tyr Asn Gln Glu Ile Cys Leu Leu 1 5 10 15 <210> 12 <211> 20 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 12 Ser Tyr Leu Glu Tyr Met Lys Asn Asn Leu Lys Asp Asn Lys Lys Asp 1 5 10 15 Lys Ala Arg Val 20 <210> 13 <211> 18 <212> PRT <213> Artificial sequence <220> <223> S quence d'IL-6 <400> 13 Ala Leu Leu Thr Asp Lys Leu Glu Ser Gln Lys Glu Trp Leu Arg Thr 1 5 10 15 Lys Thr <210> 14 <211> 16 <212> PRT <213> Artificial sequence <220> <223> IL-6 sequence <400> 14 Ser Leu Glu Glu Phe Leu Lys Val Thr Leu Arg Ser Thr Arg Gln Thr 1 5 10 15 <210> 15 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Peptide link <220> <221> MISC_FEATURE <222> (4)..(4) <223> Cys (C), Tyr (Y), Lys (K) <400> 15 Glu Gly Glu Xaa 1 <210> 16 <211> 535 <212> PRT <213> Corynebacterium diphtheriae <400> 16 Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 1 5 10 15 Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 20 25 30 Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp 35 40 45 Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly 50 55 60 Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val 65 70 75 80 Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val 85 90 95 Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110 Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125 Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140 Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 145 150 155 160 Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 165 170 175 Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg 180 185 190 Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val 195 200 205 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly 210 215 220 Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu 225 230 235 240 Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255 His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270 Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280 285 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 290 295 300 Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 305 310 315 320 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 325 330 335 Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 340 345 350 Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360 365 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr 385 390 395 400 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 405 410 415 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val 420 425 430 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr 435 440 445 His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile 450 455 460 Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly 465 470 475 480 Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser 485 490 495 Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510 Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520 525 Leu Phe Phe Glu Ile Lys Ser 530 535 <210> 17 <211> 15 <212> PRT <213> Artificial sequence <220> <223> IL-6 sequence variant <400> 17 Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial sequence <220> <223> IL-6 sequence variant <400> 18 Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys 1 5 10 15 <210> 19 <211> 17 <212> PRT <213> Artificial sequence <220> <223> IL-6 sequence variant <400> 19 Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys 1 5 10 15 Cys <210> 20 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Variant de s quence d'IL-6 <400> 20 Ala Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro 1 5 10 15 Lys Cys <210> 21 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Variant de s quence d'IL-6 <400> 21 Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro 1 5 10 15 Lys Cys <210> 22 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 22 Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro 1 5 10 15 Lys Cys Tyr <210> 23 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 23 Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Cys 1 5 10 15 <210> 24 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 24 Cys Ile Asp Lys Gln Ile Arg Tyr Ile Ile Asp Gly Ile Ser Ala Leu 1 5 10 15 Arg Lys Glu Thr Cys Glu Gly Glu Cys 20 25 <210> 25 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 25 Cys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Cys Tyr 1 5 10 <210> 26 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 26 Cys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met Cys Glu Gly Glu 1 5 10 15 Tyr <210> 27 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 27 Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys 1 5 10 <210> 28 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Polypeptide selon l'invention <400> 28 Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr Cys Glu Gly Glu Tyr 1 5 10 15

Claims

1. - transport proteins, at least one polypeptide having at most 100 amino acids comprising a sequence of 5 to 50 amino acids of interleukin 6 (IL-6) or of the receptor for interleukin 6 (IL-6R), or a variant sequence exhibiting at least 75% identity with said sequence of 5 to 50 amino acids of IL-6 or IL-6R; An immunogenic conjugate comprising: An immunogenic conjugate wherein said polypeptide is covalently linked to said transport protein, said transport protein being a non-toxic mutant diphtheria toxin.

2. The immunogenic conjugate of claim 1, wherein the transport protein is CRM197.

3. 3. The immunogenic complex of claim 1, wherein the polypeptide comprises a sequence of 7 to 35 amino acids of IL-6 or IL-6R, or a variant sequence exhibiting at least 90% identity with the sequence of 7 to 35 amino acids of IL-6 or IL-6R.

4. The immunogenic conjugate of any one of claims 1 to 3, wherein the polypeptide is cyclized.

5. The polypeptide has the sequence: ・RYIIDGISALRKETCNKSNMC, ・NKSNMCESSKEALAENNLNLPK, ・AEKDGCFQSGFNEETC, ・FEVYLEYLQNRFESSEEQAR, NASLLTKLQAQNQWLQDM, and ・RSFKEFLQSSLRALRQM The immunogenic conjugate of any one of claims 1 to 4, comprising at least 5 amino acids of a sequence selected from the group consisting of:

6. The immunogenic complex of any one of claims 1 to 5, wherein the polypeptide is conjugated to the transport protein via a non-peptide binding agent.

7. The following formula (I): [(AESSKEALAENNLNLPKC)-GMB]-CRM197 (I) an immunogenic conjugate of During the ceremony, - the polypeptide consists of the sequence AESSKEALAENNLNLPKC, - the transport protein is CRM197, - the polypeptide is covalently attached to a transport protein via the linker N-γ-maleimidobutyryl- (GMB), - the parentheses indicate that the polypeptide is cyclized by forming a peptide bond between the cysteine ​​(C) C-terminus and the alanine (A) N-terminus; - square brackets indicate that at least one polypeptide is bound to said transport protein; The immunogenic complex according to any one of claims 1 to 6.

8. The following formula (II): [Acetyl-(CESSKEALAENNLNLPKC)-(X)i-Z-GMB]-CRM197 (II) an immunogenic conjugate of During the ceremony, - the polypeptide consists of the sequence CESSKEALAENNLNLPKC-(X)i-Z, - the transport protein is CRM197, - the polypeptide is covalently attached to the transport protein via the linker N-γ-maleimidobutyryl- (GMB), The parentheses indicate that the portion of the sequence of the polypeptide, CESSKEALAENNLNLPKC, is cyclized by forming a disulfide bridge between the C-terminal cysteine ​​(C) and the N-terminal cysteine ​​(C), Acetyl- is the free radical -NH at the N-terminus of the cysteine ​​(C) ２ is protected by an acetyl group by forming an amide bond, (X) represents any peptide sequence of 1 to 5 amino groups, i is a value of 0 or 1, Z represents tyrosine (Y) or cysteine ​​(C), - square brackets indicate that the at least one polypeptide is bound to a transport protein; The immunogenic complex according to any one of claims 1 to 6.

9. An immunogenic complex according to any one of claims 1 to 8 for use in the therapeutic treatment or in a method for the prevention or treatment of a disease associated with the overproduction of IL-6 in an individual.

10. The immunogenic complex according to any one of claims 1 to 8 for use in a method for administering a vaccine against IL-6 or IL-6R to an individual, or for use in a method for inducing an immune response against IL-6 or IL-6R.

11. A pharmaceutical composition, in particular a vaccine composition, comprising as active substance at least one immunogenic complex according to any one of claims 1 to 8, optionally in combination with at least one pharmaceutically acceptable vehicle and / or excipient.

12. 12. A pharmaceutical composition, in particular a vaccine composition, according to claim 11, further comprising at least one adjuvant.

13. A pharmaceutical composition, in particular a vaccine composition, according to claim 11 or 12 for use in a method for the therapeutic treatment or in a method for the prevention or treatment of a disease associated with the overproduction of IL-6 in an individual.

14. 13. A pharmaceutical composition, particularly a vaccine composition, according to claim 11 or 12, for use in a method for administering a vaccine against IL-6 or IL-6R to an individual, or for use in a method for inducing an immune response against IL-6 or IL-6R.

15. 9. A method for preparing an immunogenic complex according to any one of claims 1 to 8, comprising a step of covalently binding at least one polypeptide having at most 100 amino acids, comprising a sequence of 5 to 50 amino acids of interleukin-6 (IL-6) or of the receptor for IL-6 (IL-6R), or a variant sequence exhibiting at least 75% identity with said sequence of 5 to 50 amino acids of IL-6 or IL-6R, to a transport protein which is a non-toxic mutated diphtheria toxin.

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