Lipopeptide building blocks and synthetic virus-like particles

Abstract

The present invention relates to a lipopeptide building block, the lipid building block consisting of: (i) a peptide portion, the peptide portion comprising a coiled-coil peptide segment, wherein the coiled-coil peptide segment comprises 3 to 8 repeating units, and wherein the repeating units consist of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid, and wherein preferably the repeating units consist of a sequence selected from IEKKIEG (SEQ ID NO: 59), IEKKIEA (SEQ ID NO: 12) or IEKKIES (SEQ ID NO: 13), and wherein further preferably the repeating units consist of the sequence IEKKIES (SEQ ID NO: 13); (ii) a lipid portion, the lipid portion comprising or preferably consisting of formula LM-I, wherein R 1 and R 2 Independently C 11‑15 Alkyl, wherein preferably R 1 and R 2 Independently C 11 H 23 ,‑C 13 H 27 or ‑C 15 H 31 , and wherein further preferably R 1 and R 2 C 15 H 31 ; and where R 3 is hydrogen or ‑C(O)C 11‑15 alkyl, and wherein preferably R 3 H or ‑C(O)C 15 H 31 and wherein the lipid portion is linked to the peptide portion, wherein the wavy line in formula LM-I indicates the site of attachment to the peptide portion, and wherein preferably the lipid portion is linked to the N-terminus of the peptide portion, and the present invention relates to conjugates comprising the lipopeptide building block to which an antigen is coupled, bundles of such conjugates, synthetic virus-like particles (SVLPs) comprising a bundle of at least one conjugate, and pharmaceutical compositions comprising the conjugates. The present invention further relates to the conjugates, bundles of conjugates, the SVLPs and the pharmaceutical compositions for use as medicaments, as vaccines and in methods for preventing or treating diseases preferably selected from infectious diseases, allergies and cancer and generally for effectively inducing an antigen-specific immune response. #imgabs0#

Description

[0001] The present invention relates to lipopeptide building blocks, conjugates comprising said lipopeptide building blocks coupled to an antigen, bundles of such conjugates, synthetic virus-like particles (SVLPs) comprising a bundle of at least one conjugate, and pharmaceutical compositions comprising said conjugates. The present invention further relates to said conjugates, bundles of conjugates, said SVLPs and said pharmaceutical compositions for use as medicaments, as vaccines and in methods for preventing or treating diseases preferably selected from infectious diseases, allergies and cancer and generally for effectively inducing an antigen-specific immune response. Background Art

[0002] Synthetic virus-like particles (SVLPs) have recently attracted widespread attention, especially vaccine candidates based on SVLPs using synthetic lipopeptide building blocks that are conjugated to antigens and spontaneously self-assemble into highly immunogenic nanoparticles of 20-30 nm and comprise 60-80 or so copies of the lipopeptide building blocks. Because the antigens are displayed on the entire outer surface of the nanoparticles and SVLPs, respectively, a strong immune response to the antigen is induced, especially a B cell response. Importantly, SVLPs do not contain genetic information and cannot replicate in cells. Typically, the lipopeptide building blocks include a coiled-coil peptide sequence and a T helper cell epitope (A. Ghasparian and John A. Robinson, in ..., eds.; A. Ghasparian et al., Chembiochem 2011, 12, 100-109; T. Riedel et al., Chembiochem 2011, 12, 2829; R. Sharma et al., PLoS One 2012, 7, e43248; WO2008 / 068017; WO2015 / 082501). Coiled-coil designs are known to those skilled in the art and have been reviewed (Woolfson, DN, Adv. Prot. Chem., 2005, 70, 79-112; Parry, DAD et al., Adv. Protein Chem., 2005, 70).

[0003] Specially engineered SVLPs and their use as vaccines have been reported recently, and a lipopeptide building block containing a coiled-coil (IEKKIEA)4 known to form a trimeric parallel helical bundle fused to a promiscuous CD4+ T helper epitope (IEKKIAKMEKASSVFNVVNS) identified in the circumsporozoite (CS) protein of the malarial parasite Plasmodium falciparum has been described (A. Ghasparian et al., Chem. Biochem. 2011, 12, 100-109). The T-helper epitope (using two Cys-Ala substitutions) corresponds to residues 379-398 of the CS protein and is recognized by mouse and human T cells in association with a variety of different MHC class II molecules (J. Kilgus et al., J. Immunol. 1991, 146, 307; F. Sinigaglia et al., Nature 1988, 336, 778). The described lipopeptide building block is completed by the addition of KKKC at the C-terminus to allow conjugation of the B-cell epitope via a cysteine ​​thiol group and by the addition of a lipid moiety comprising a phospholipid related to phosphatidylethanolamine, or a Pam2Cys or Pam3Cys moiety at the N-terminus (A. Ghasparian et al., Chem. Biochem. 2011, 12, 100-109). The Pam3Cys moiety, i.e. tripalmitoyl-S-glycerocysteine ​​(N-palmitoyl-S-(2,3-bis-(O-palmitoyloxy)-propyl)-cysteinyl-) and the Pam2Cys moiety, i.e. dipalmitoyl-S-glycerocysteine ​​(2,3-bis-(O-palmitoyloxy)-propyl)-cysteinyl-) are known to those skilled in the art and have been fully described below (Ghielmetti, M. et al., Immunobiology, 2005, 210, 211-215; Reutter, F. et al., J. Pept. Res., 2005, 65, 375-383; Buwitt-Beckmann, U. et al., Eur. J. Immunol., 2005, 35, 1-8).

[0004] Despite recent progress in the development of lipopeptide building blocks and SVLPs, respectively, and in particular SVLP-based vaccine candidates, there is still a need for additional and even more effective lipopeptide building blocks and SVLPs, respectively, in particular to further enhance the quality of the induced immune response. Summary of the Invention

[0005] It has now surprisingly been found that lipopeptide building blocks comprising a Pam2Cys or Pam3Cys moiety with an (R) configuration at the 2-propyl carbon atom and further comprising several units of the sequence IEKKIE-X0 in the form of a coiled-coil peptide segment, wherein preferably X0 is Gly, Ala or Ser, most preferably Ser, show increased affinity for antibodies raised against an antigen linked to the lipopeptide building block of the invention and comprised by the conjugate or SVLP of the invention, respectively.

[0006] Thus, in a first aspect, the present invention provides a lipopeptide building block consisting of:

[0007] (i) a peptide portion comprising a coiled-coil peptide segment, wherein the coiled-coil peptide segment comprises 3 to 8 repeating units, and wherein the repeating units consist of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid, and wherein preferably the repeating units consist of a sequence selected from IEKKIEG (SEQ ID NO: 59), IEKKIEA (SEQ ID NO: 12) or IEKKIES (SEQ ID NO: 13), and wherein further preferably the repeating units consist of the sequence IEKKIES (SEQ ID NO: 13);

[0008] (ii) a lipid portion comprising or preferably consisting of formula LM-I

[0009]

[0010] where R 1 and R 2 Independently C 11-15 Alkyl, wherein preferably R 1 and R 2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 , and wherein further preferably R 1 and R 2 -C 15 H 31 ; and where R 3 is hydrogen or -C(O)C 11-15 alkyl, and wherein preferably R 3 H or -C(O)C 15 H 31 ;

[0011] And wherein the lipid moiety is linked to the peptide moiety, wherein the wavy line in formula LM-I indicates the site of attachment to the peptide moiety, and wherein preferably the lipid moiety is linked to the N-terminus of the peptide moiety.

[0012] In a further aspect, the invention provides a conjugate comprising (a) a lipopeptide building block of the invention and (b) an antigen, wherein the antigen is linked to the lipopeptide building block directly or via a linker.

[0013] In a further aspect, the invention provides a conjugate comprising (a) a lipopeptide building block of the invention and (b) an antigen, wherein the antigen is linked to the lipopeptide building block directly or via a linker.

[0014] In another aspect, the invention provides a bundle of conjugates, said bundle comprising 2, 3, 4, 5, 6 or 7, preferably 2, 3, 4 or 5, more preferably 3, of the conjugates of the invention.

[0015] In another aspect, the present invention provides a synthetic virus-like particle comprising at least one bundle of the conjugate of the present invention.

[0016] In another aspect, the present invention provides a pharmaceutical composition comprising an immunologically effective amount of the conjugate of the present invention or the synthetic virus-like particle of the present invention together with a pharmaceutically acceptable diluent, carrier or excipient, wherein preferably the pharmaceutical composition is a vaccine.

[0017] In yet another aspect, the present invention provides a conjugate of the invention or a synthetic virus-like particle of the invention for use as a medicament.

[0018] In yet another aspect, the present invention provides a conjugate of the invention or a synthetic virus-like particle of the invention for use in a method for preventing a disease or for reducing the risk of a disease, wherein further preferably the disease is an infectious disease, cancer or allergy, and again more preferably wherein the disease is a respiratory syncytial virus (RSV) infection.

[0019] In yet another aspect, the present invention provides a conjugate of the invention or a synthetic virus-like particle of the invention for use in a method for preventing a disease or for reducing the risk of a disease or for treating a disease, wherein further preferably the disease is an infectious disease, cancer or allergy, and again more preferably wherein the disease is respiratory syncytial virus (RSV) infection. In yet another aspect, the present invention provides a conjugate of the invention or a synthetic virus-like particle of the invention for use in a method for preventing an infectious disease or reducing the risk of an infectious disease, preferably for use in a method for preventing an infectious disease associated with or caused by respiratory syncytial virus or reducing the risk of an infectious disease.

[0020] Further aspects and embodiments of the invention will become apparent as this description proceeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are the average avidity indices for conjugates 44, 45, and 46, as determined in the immunogenicity studies described in Example 4. DETAILED DESCRIPTION

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] Throughout the specification and the claims that follow, unless the context requires otherwise, the term "comprise" and variations thereof, such as "comprises" and "comprising," etc., should be understood as non-exhaustive terms and mean the inclusion of the stated features or elements but not the exclusion of any other features or elements. The term "comprises" and variations thereof encompass the term "consisting of." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "reduce," "inhibit," or "reduce" encompass a mere detectable reduction, but also encompass a reduction to zero (a 100% reduction).

[0024] As defined herein, a peptide or peptide portion is a chain of amino acids connected by any peptide bond, regardless of length, secondary and tertiary structure, number of subunits, or post-translational modification. Therefore, the "peptide" of the term "peptide portion" should be understood to encompass the terms "polypeptide," "protein," "amino acid chain," "polypeptide chain." The amino acids included in the peptides of the present invention are proteinogenic amino acids, non-proteinogenic amino acids, and synthetic amino acids. The peptide can be an open linear peptide chain or a cyclic peptide and can include at least one chemical modification, such as lipidation, glycosylation, and phosphorylation. The peptide can be produced by chemical synthesis, RNA translation, and / or recombinant processes.

[0025] As used herein, the term "cyclic peptide" refers to a peptide in which an amino acid chain forms at least one ring structure through covalent bonds. The cyclic peptide of the present invention includes ring structures each formed by a disulfide bond: the side chains of cysteines C4 and C25 are linked to form a first disulfide bond, and the side chains of cysteines C8 and C21 are linked to form a second disulfide bond.

[0026] As used herein, the term "amino acid" refers to an organic compound containing the functional groups amine (-NH2) and carboxylic acid (-COOH) and zwitterions thereof, typically and preferably together with a side chain specific for each amino acid. The term "amino acid" typically and preferably includes naturally occurring amino acids, such as proteinogenic amino acids (produced by RNA translation), non-proteinogenic amino acids (produced by other metabolic mechanisms, e.g., post-translational modifications), standard or typical amino acids (directly encoded by the codons of the genetic code), and non-standard or atypical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include non-eukaryotic amino acids and eukaryotic amino acids.

[0027] As used herein, the term "amino acid" also includes: chemically synthesized non-natural amino acids; amino acids such as α-(α-), β-(β-), γ-(γ-) and δ-(δ-) and mixtures thereof in any ratio; and, if applicable, isomeric forms of amino acids, i.e., their D stereoisomers (marked with lowercase initials) and L stereoisomers (marked with uppercase and lowercase initials) (alternatively addressed by (R) and (S) nomenclature) and mixtures thereof in any ratio, preferably a 1:1 racemic ratio. Unless specifically mentioned in the D configuration, the amino acids in the present invention are preferably in the L configuration. The terms "D stereoisomer", "L stereoisomer", "D amino acid" or "L amino acid" refer to the chiral α carbon of an amino acid. An amino acid may contain one or more modifications and / or attached groups, such as protecting groups used for peptide synthesis, such as Boc, Fmoc or both. The term "deletion" herein refers to a position in an amino acid sequence that is not occupied by an amino acid. In a preferred embodiment of the present invention, the amino acid is a naturally occurring amino acid, wherein said naturally occurring amino acid is in its L configuration, in its D configuration, or a mixture of said L- and D- configurations in any ratio. In another preferred embodiment of the present invention, the amino acid is a naturally occurring amino acid, wherein said naturally occurring amino acid is in its L configuration.

[0028] As used herein, the term "antigen" shall refer to a molecule capable of being bound by an antibody. Antigens may include peptides, proteins, or epitope mimetics having one or more B cell epitopes that are used to elicit an antigen-specific humoral immune response in an animal. Alternatively, antigens may include haptens or carbohydrates. Suitable peptide and protein antigens include up to 150 amino acids and include glycopeptides and glycoproteins. Peptide and protein sequences can be selected to elicit an immune response, for example, against one or more infectious agents. Such antigens are well known in the art. Epitope mimetics are molecules that mimic natural peptide or carbohydrate epitopes and include peptide compounds containing one or more non-natural amino acids, such as D amino acids, β amino acids, γ amino acids, δ amino acids, or ε amino acids, and other substitutes known in the field of epitope mimetics. Preferred are conformationally constrained peptidomimetics that are fixed in a protein-like conformation. A hapten refers to an organic compound with a molecular weight of less than 3,000 that does not elicit a humoral immune response on its own, but will elicit an immune response once attached to a carrier. Exemplary haptens include drugs, hormones, toxins, and carbohydrates.

[0029] As used herein, the term "N-terminus" refers to the end of a peptide having a free (-NH2) or modified amino or amine group. Preferred N-terminal modifications are modifications that protect the N-terminus from proteolytic degradation. N-terminal modifications according to the present invention include, but are not limited to, acetylation of at least one polymer, preferably polyethylene glycol (PEGylation) or poly(lactic acid), attachment of at least one amino acid, preferably at least one D amino acid, or attachment of at least one compound, such as a cell penetrating peptide, a nucleic acid, a carbamate (such as fluorenylmethoxycarbamate or benzyloxycarbamate), an aldehyde, hydrazinonicotinic acid, 4-formylbenzamide, a methyl group, a myristoyl group, a prenyl group, a palmitoyl group, ubiquitin, 7-methoxycoumarinacetic acid (Mca), a dansyl group, a formyl group, a 4-diniphenyl group, a pyroglutamyl group, a urea, a carbamate, a sulfonamide, an alkylamine, a fatty acid (such as palmitic acid), a radioligand, a quencher, fluorescein, or attachment of another dye or label (such as biotin).

[0030] As used herein, the term "C-terminus" refers to the end of a peptide having a free (-COOH) or modified carboxyl group. Preferred C-terminal modifications are modifications that protect the C-terminus from proteolytic degradation. C-terminal modifications according to the present invention include, but are not limited to, amidation or attachment of at least one amino acid, preferably at least one D amino acid, or attachment of at least one compound, such as a cell penetrating peptide, nucleic acid, polyethylene glycol (PEGylation), thiol, ester, aldehyde, sulfonamide, pNA (p-nitroaniline), Amc (7-amino-4-methylcoumarin), hydrazide, hydroxamic acid, chloromethyl ketone, biotin, radioligand, quencher, Abz or other dyes and labels. Here, and by general convention, peptide sequences are written from the N-terminus on the left to the C-terminus on the right (according to the direction of translation).

[0031] As used herein, the term "coiled-coil peptide segment" is a sequence of a peptide chain that is capable of forming a coiled coil (supercoil) with at least one other coiled-coil peptide segment. A coiled-coil is a peptide structure in which at least two coiled-coil peptide segments, each having a preferably alpha-helical secondary structure, associate into a bundle. The coiled-coil peptide segment of the present invention contains a plurality of repeating units, which are typically and preferably continuously linked to each other. The repeating units of the coiled-coil peptide segment may be identical or different, for example, may contain at least one discontinuity within the repeating unit, such as the insertion, deletion, or exchange of at least one, preferably exactly one, two, three, or four amino acids.

[0032] In a first aspect, the present invention provides a lipopeptide building block, the lipopeptide building block consisting of:

[0033] (i) a peptide portion comprising a coiled-coil peptide segment, wherein the coiled-coil peptide segment comprises 3 to 8 repeating units, and wherein the repeating units consist of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid, and wherein preferably the repeating units consist of a sequence selected from IEKKIEG (SEQ ID NO: 59), IEKKIEA (SEQ ID NO: 12) or IEKKIES (SEQ ID NO: 13), and wherein further preferably the repeating units consist of the sequence IEKKIES (SEQ ID NO: 13);

[0034] (ii) a lipid portion comprising or preferably consisting of formula LM-I

[0035]

[0036] where R 1 and R 2 Independently C 11-15 Alkyl, wherein preferably R 1 and R 2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 , and wherein further preferably R 1 and R 2 -C 15 H 31 ; and where R 3 is hydrogen or -C(O)C 11-15 alkyl, and wherein preferably R 3 H or -C(O)C 15 H 31 ;

[0037] And wherein the lipid moiety is linked to the peptide moiety, wherein the wavy line in formula LM-I indicates the site of attachment to the peptide moiety, and wherein preferably the lipid moiety is linked to the N-terminus of the peptide moiety.

[0038] In a preferred embodiment, the coiled-coil peptide segment of the peptide portion consists of 3 to 8 repeating units, including 3, 4, 5, 6, 7, 8 repeating units, and more preferably 4 repeating units. A higher number of repeating units in the peptide portion affects the stability of the coiled-coil. In a preferred embodiment, the coiled-coil peptide segment of the peptide portion includes or preferably consists of 4 repeating units. In a preferred embodiment, the coiled-coil peptide segment of the peptide portion consists of 4 repeating units. In a preferred embodiment, the 4 repeating units are continuously connected to each other.

[0039] The coiled-coil peptide segments of the present invention are based on typical repeating units, typically and preferably based on typical tandem heptad repeats that form right-handed amphipathic α-helices, which then assemble to form helical bundles with left-handed coiled-coils.

[0040] The repeating units of coiled-coil peptide segments have a sequence of a certain number of amino acids, where the positions of the amino acids are traditionally indicated by lower case letters. Design rules are discussed in more detail, for example, in Woolfson, DN, Progress in Protein Chemistry, 2005, 70, 79-112.

[0041] In the present invention, the repeating unit of the coiled-coil peptide segment consists of seven amino acids, wherein the seven amino acid positions are represented by the letters a, b, c, d, e, f, and g. In a preferred embodiment, the heptapeptide motif consists of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid. In a preferred embodiment, the repeating unit consists of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid, with the proviso that X0 is not proline. In another preferred embodiment, the repeating unit consists of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid, wherein the amino acid is a naturally occurring amino acid, wherein the naturally occurring amino acid is in its L configuration, in its D configuration, or in a mixture thereof in any ratio, with the proviso that the amino acid is not proline. In another preferred embodiment, the repeating unit consists of the sequence IEKKIE-X0 (SEQ ID NO: 58), wherein X0 represents an amino acid, wherein the amino acid is a naturally occurring amino acid in its L configuration.

[0042] In a preferred embodiment, the repeat unit consists of a sequence selected from IEKKIEG (SEQ ID NO: 59), IEKKIEA (SEQ ID NO: 12) or IEKKIES (SEQ ID NO: 13). In a preferred embodiment, the repeat unit consists of a sequence selected from IEKKIEA (SEQ ID NO: 12) or IEKKIES (SEQ ID NO: 13). In a preferred embodiment, the repeat unit consists of the sequence IEKKIEG (SEQ ID NO: 59). In a preferred embodiment, the repeat unit consists of the sequence IEKKIEA (SEQ ID NO: 12). In a very preferred embodiment, the repeat unit consists of the sequence IEKKIES (SEQ ID NO: 13).

[0043] In a preferred embodiment, the coiled-coil peptide segment comprises or preferably consists of a sequence selected from the group consisting of (IEKKIEG)4 (SEQ ID NO: 60), (IEKKIEA)4 (SEQ ID NO: 61), or (IEKKIES)4 (SEQ ID NO: 62). In a preferred embodiment, the coiled-coil peptide segment consists of a sequence selected from the group consisting of (IEKKIEG)4 (SEQ ID NO: 60), (IEKKIEA)4 (SEQ ID NO: 61), or (IEKKIES)4 (SEQ ID NO: 62). In a preferred embodiment, the coiled-coil peptide segment comprises or preferably consists of the sequence (IEKKIEG)4 (SEQ ID NO: 60). In a preferred embodiment, the coiled-coil peptide segment comprises or consists of a sequence selected from the group consisting of (IEKKIEG)4 (SEQ ID NO: 60).

[0044] In a preferred embodiment, the coiled-coil peptide segment comprises or preferably consists of the sequence (IEKKIEA)4 (SEQ ID NO: 61). In a preferred embodiment, the coiled-coil peptide segment consists of the sequence (IEKKIEA)4 (SEQ ID NO: 61).

[0045] In a very preferred embodiment, the coiled-coil peptide segment comprises or preferably consists of the sequence (IEKKIES)4 (SEQ ID NO: 62). In a very preferred embodiment, the coiled-coil peptide segment consists of the sequence (IEKKIES)4 (SEQ ID NO: 62).

[0046] In a preferred embodiment, the R 1 and R2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 In a very preferred embodiment, the R 1 and R 2 -C 15 H 31 In a preferred embodiment, the R 3 H or -C(O)C 15 H 31 .

[0047] In a preferred embodiment, the R 1 and R 2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 , and R 3 is hydrogen or -C(O)C 11-15 In a very preferred embodiment, the R 1 and R 2 -C 15 H 31 , and R 3 is hydrogen or -C(O)C 11-15 alkyl.

[0048] In a preferred embodiment, the R 1 and R 2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 , and R 3 H or -C(O)C 15 H 31 In a very preferred embodiment, the R 1 and R 2 -C 15 H 31 , and R 3 H or -C(O)C 15 H 31 .

[0049] In a preferred embodiment, the lipid moiety is linked to the N-terminus of the peptide moiety. This conveniently allows the linkage to be performed on a resin after the peptide chain of the peptide moiety has been assembled by solid phase peptide synthesis. Linking the lipid moiety to the C-terminus of the peptide moiety is also encompassed within the present invention and is possible using linkage chemistries known to those skilled in the art.

[0050] In a preferred embodiment, the peptide portion is 12 to 200 amino acids in length, more preferably 21 to 120 amino acids, even more preferably 21 to 80 amino acids, even more preferably 21 to 70 amino acids, even more preferably 21 to 60 amino acids, even more preferably 21 to 50 amino acids, even more preferably 28 to 48 amino acids in length. Preferably, the peptide portion is a non-human sequence to avoid the risk of autoimmune disorders when used for human vaccination.

[0051] Preferably the lipid moiety is dipalmitoyl-S-glycerocysteinyl (Pam2Cys) or tripalmitoyl-S-glycerocysteinyl (Pam3Cys), which has both an R configuration at the chiral 2-propyl carbon atom and an R configuration at the chiral carbon of the cysteinyl moiety.

[0052] In a preferred embodiment, the lipid moiety is linked to the peptide moiety directly or through a coupling moiety. Preferably, the lipid moiety is linked to the peptide moiety at one end, either the N-terminus or the C-terminus, preferably at or near the N-terminus. In a preferred embodiment, the lipid moiety is linked to the first, second, third, fourth, or fifth amino acid of the peptide moiety, where the amino acids are calculated based on the N-terminus or C-terminus of the peptide moiety. The lipid moiety can be linked directly or through a coupling moiety to the backbone or side chain of one of the amino acids of the peptide moiety, preferably near the end, more preferably the first, second, third, fourth, or fifth amino acid of the peptide moiety.

[0053] The lipid portion is attached to the peptide portion directly or via a coupling portion. If the peptide portion and the lipid portion are directly linked, this is preferably accomplished via an amide bond between a carbonyl function of the lipid portion and an amino function of the peptide portion, such as an N-terminal amino function. It will be apparent to those skilled in the art that there are a variety of suitable coupling portions and coupling strategies, including but not limited to linkers based on dicarboxylic acid derivatives, linkers containing one or more ethylene glycol units, amino acid residues (including α, β, γ, ω amino acids) or sugar (carbohydrate) units, or linkers containing heterocycles.

[0054] In a preferred embodiment, the lipid portion comprises, preferably consists of, the formula LM-I*

[0055]

[0056] where R 3 is hydrogen or -C(O)C 11-15 Alkyl, preferably H or -C(O)C 15 H 31 ; wherein preferably the lipid portion is linked to the N-terminus of the peptide portion.

[0057] In a preferred embodiment, the lipid portion is composed of the formula LM-I*, wherein R 3 is hydrogen or -C(O)C 11-15 In a preferred embodiment, the lipid moiety comprises, preferably consists of, the formula LM-I*, wherein R 3 H or -C(O)C 15 H 31 In a preferred embodiment, the lipid moiety comprises, preferably consists of, the formula LM-I*, wherein R 3 H or -C(O)C 15 H 31 , and wherein the lipid portion is connected to the N-terminus of the peptide portion. In a preferred embodiment, the lipid portion is composed of the formula LM-I*

[0058]

[0059] where R 3 H or -C(O)C 15 H 31 , and wherein preferably the lipid moiety is linked to the N-terminus of the peptide moiety.

[0060] In a very preferred embodiment, the lipid portion comprises, preferably consists of, the formula LM-I*1 or LM-I*2. In a very preferred embodiment, the lipid portion consists of the formula LM-I*1 or LM-I*2.

[0061] In a very preferred embodiment, the lipid portion consists of formula LM-I*1.

[0062]

[0063] In a very preferred embodiment, the lipid portion consists of the formula LM-I*2.

[0064]

[0065] Therefore, very preferred lipid moieties of the present invention are (R,R)-Pam3Cys LM-I*2, i.e. tripalmitoyl-S-glycerocysteine ​​(N-palmitoyl-S-(2,3-bis-(O-palmitoyloxy)-(2R)-propyl)-(R)-cysteinyl-), and (R,R)-Pam2Cys LM-I*1, i.e. dipalmitoyl-S-glycerocysteine ​​(2,3-bis-(O-palmitoyloxy)-(2R)-propyl)-(R)-cysteinyl-).

[0066] Thus, in a further very preferred embodiment, the lipid moiety is N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine ​​or S-[2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine, thus LM-I*1.

[0067] In another preferred embodiment, the peptide portion includes (i) an N-terminal amino acid sequence, wherein the N-terminal amino acid sequence includes or preferably consists of the following: fibroblast stimulating lipopeptide FSL-1 (S-(2,3-bispalmitoyloxypropyl)- or PAM2-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe; SEQ ID NO: 7), FSL-2 (S-(2,3-bispalmitoyloxypropyl)- or PAM2-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Arg; SEQ ID NO: 8), FSL-3 (S-(2,3-bisstearoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe; SEQ ID NO: 9), Mycoplasma fermentans (Mycoplasma fermentans) derived peptide MALP-2 (S-(2,3-dipalmitoyloxypropyl)- or PAM2-Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys; SEQ ID NO: 10) or GG and / or GX, wherein X is Asx or Ser; and / or (ii) a C-terminal amino acid sequence, wherein the C-terminal amino acid sequence comprises a sequence recognized as a cleavage site by an enzyme, or preferably consists of said sequence; wherein preferably the C-terminal amino acid sequence comprises KKKCa (SEQ ID NO: 11) or preferably consists of KKKCa, or wherein preferably the C-terminal amino acid sequence is an amino acid sequence of 5 consecutive amino acids.

[0068] In a preferred embodiment, the lipid portion and the peptide portion are directly linked. In a preferred embodiment, the lipid portion and the peptide portion are linked via a coupling moiety. In a preferred embodiment, the lipid portion and the peptide portion are linked via a coupling moiety, wherein the coupling moiety is an amino acid linker consisting of 2-15 amino acids. Examples herein include amino acid linker sequences comprised by FSL-1, FSL-2, FSL-3, PAM2, or MALP-2 moieties. In a preferred embodiment, the lipid portion and the peptide portion are linked via a coupling moiety, wherein the coupling moiety is an amino acid linker consisting of 2-10 amino acids. In a preferred embodiment, the lipid portion and the peptide portion are linked via a coupling moiety, wherein the coupling moiety is an amino acid linker consisting of 2-5 amino acids. In a preferred embodiment, the lipid portion and the peptide portion are linked via a coupling moiety, wherein the coupling moiety is an amino acid linker, wherein the amino acid linker is a Gly-Gly moiety. In a preferred embodiment, the lipid portion and the peptide portion are linked via a coupling moiety, wherein the coupling moiety is a Gly-Gly moiety.

[0069] In a preferred embodiment, said direct linkage of said lipid moiety and said peptide moiety is via an amide bond between a carbonyl function of said lipid moiety and an amino function of said peptide moiety.

[0070] In a preferred embodiment, said connection of said lipid moiety and said peptide moiety via said coupling moiety is via an amide bond between a carbonyl function of said lipid moiety and an amino function of said coupling moiety.

[0071] In a preferred embodiment, said connection of said lipid moiety and said peptide moiety via said coupling moiety is via an amide bond between a carbonyl function of said lipid moiety and an amino function of said coupling moiety, wherein said coupling moiety is an amino-amino acid linker preferably consisting of 2-15 amino acids, preferably 2-10 amino acids, and wherein said amino function is the N-terminal amino function of said coupling moiety.

[0072] In a preferred embodiment, said connection of said lipid moiety and said peptide moiety via said coupling moiety is via an amide bond between a carbonyl function of said lipid moiety and an amino function of said coupling moiety, wherein said coupling moiety is an amino acid linker preferably consisting of 2-5 amino acids, and wherein said amino function is the N-terminal amino function of said coupling moiety.

[0073] In a preferred embodiment, said direct connection of said lipid moiety and said peptide moiety is via an amide bond between a carbonyl function of said lipid moiety and an amino function of said peptide moiety, wherein said amino function is the N-terminal amino function of said peptide moiety.

[0074] In a preferred embodiment, two Gly residues are included as a linker between the lipid moiety, preferably the (R,R)-Pam2Cys moiety LM-I*1 of the present invention and the start of the coiled-coil heptapeptide repeat sequence, typically and preferably comprising the sequence IEKKIES (SEQ ID NO: 13), preferably consisting of the sequence IEKKIES.

[0075] The introduction of an amino acid linker, and preferably a short amino acid linker consisting of two amino acids, more preferably glycine, can be performed in sequence during peptide synthesis after each amino acid coupling, and a capping step can be performed with acetic anhydride. This has the practical advantage that, after completion of peptide assembly and coupling of a lipid moiety, preferably a lipid moiety consisting of formula LM-I*1 [(R,R)-Pam2Cys moiety] or formula LM-I*2 [(R,R)-Pam3Cys moiety] to the free N-terminus, the HPLC retention time of the peptide is significantly altered by lipidation, thereby greatly facilitating HPLC purification of the desired lipopeptide building blocks of the present invention.

[0076] In a preferred embodiment, the peptide portion further comprises a T helper cell epitope. In a preferred embodiment, the peptide portion further comprises a T helper cell epitope, wherein the T helper cell epitope comprises or preferably consists of a sequence selected from the group consisting of: (i) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87; and (ii) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87, wherein one, two or three amino acids are exchanged or deleted by other amino acids. In a preferred embodiment, the peptide portion further comprises a T helper cell epitope, wherein the T helper cell epitope consists of a sequence selected from the group consisting of: (i) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87; and (ii) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87, wherein one, two or three amino acids are exchanged or deleted by other amino acids. In a preferred embodiment, the peptide portion further comprises a T helper cell epitope, wherein the T helper cell epitope comprises a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87. In a preferred embodiment, the T helper cell epitope consists of a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87.

[0077] In one embodiment, the peptide portion further comprises an amino acid sequence comprising one or more T helper cell epitopes and / or a string of polar residues that promote the solubility of the lipopeptide building block in water. Suitable T-helper cell epitopes are known to those skilled in the art and are described below: for example, Weber et al., Advanced Drug Delivery Reviews, 2009, 61: 11, 965-976; Caro-Aguilar et al., Infect. Immun., 2002, 70: 7, 3479-3492; Mishra et al., Immunology, 1993, 79: 3, 362-367; Kobayashi et al., Cancer Research, 2003, 61: 11, 965-976; Research, 2000, 60:18, 5228-523; Fraser et al., Vaccine, 2014, 32:24, 2896-2903; Grabowska et al., Int. J. Cancer, 2015, 136:1, 212–224 and WO1998 / 023635A1. More preferred T helper cell epitopes comprised in the peptide portion are those listed in WO 2015 / 082501, such as TT830-843, TT1064-1079, TT1084-1099, TT947-968, TT1174-1189, DTD271-290, DTD321-340, DTD331-350, DTD351-370, DTD411-430, DTD431-450, TT632-651, CTMOMP36-60, TraT1, TraT2, TraT3, HbcAg50-69, HbSAg19-33, HA307-319, MA17-31, MVF258-277, MVF288-302, CS.T3, SM. Th, PADRE1 and PADRE2, and variants thereof in which one, two or three amino acids are inserted, substituted by other amino acids, or deleted.

[0078] Preferred T-helper epitopes that can be incorporated into the peptide portion are any one selected from the group listed in Table 1 below and variants thereof in which one, two or three amino acids are substituted by other amino acids or are deleted.

[0079] Table 1:

[0080]

[0081] Therefore, in another preferred embodiment, the T helper cell epitope comprises or preferably consists of a sequence selected from the group consisting of: (i) SEQ ID NO: 1 to SEQ ID NO: 26 and (ii) SEQ ID NO: 1 to SEQ ID NO: 26, wherein one, two or three amino acids are exchanged by other amino acids or deleted.

[0082] In a preferred embodiment, the peptide portion further comprises a T helper cell epitope, wherein the T helper cell epitope comprises or preferably consists of the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the peptide further comprises a T helper cell epitope, wherein the T helper cell epitope consists of the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the T helper cell epitope consists of the sequence of SEQ ID NO: 6.

[0083] In a further very preferred embodiment, the peptide portion comprises or preferably consists of: (SEQ ID NO: 88):

[0084] GGIEKKIESIEKKIESIEKKIESIEKKIESIEKKIAKMEKASSVFNVVNSKKKC. In another very preferred embodiment, the peptide portion consists of (SEQ ID NO: 88).

[0085] In another very preferred embodiment, the lipopeptide building block has the formula LBB-2 or LBB-3, preferably LBB-2

[0086]

[0087] In another very preferred embodiment, the lipopeptide building block has the formula LBB-2. In another very preferred embodiment, the lipopeptide building block has the formula LBB-3.

[0088] In a further aspect, the present invention provides a lipopeptide building block consisting of:

[0089] (i) a peptide portion, said peptide portion comprising a coiled-coil peptide segment, and wherein said coiled-coil peptide segment comprises or preferably consists of the sequence (SEQ ID NO: 62);

[0090] (ii) a lipid portion comprising or preferably consisting of formula LM-I

[0091]

[0092] where R1 and R 2 Independently C 11-15 Alkyl, wherein preferably R 1 and R 2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 , and wherein further preferably R 1 and R 2 -C 15 H 31 ; and where R 3 is hydrogen or -C(O)C 11-15 alkyl, and wherein preferably R 3 H or -C(O)C 15 H 31 ;

[0093] And wherein the lipid moiety is linked to the peptide moiety, wherein the wavy line in formula LM-I indicates the site of attachment to the peptide moiety, and wherein preferably the lipid moiety is linked to the N-terminus of the peptide moiety.

[0094] In a further aspect, the invention provides a conjugate comprising (a) a lipopeptide building block of the invention and (b) an antigen, wherein the antigen is linked to the lipopeptide building block directly or via a linker.

[0095] One or more antigens can be conjugated to the peptide portion, for example, via one or more of the side chains of the amino acids of the peptide portion or via the chain termini of the peptide portion. The antigen typically carries a functional group suitable for conjugation to the functional group in one of the side chains or termini of the peptide portion. In order to elicit an immune response based on antigen-specific antibodies, antigens that are recognized by B cell receptors or haptens are preferred.

[0096] A variety of coupling or conjugation procedures can be used to attach the antigen to the peptide portion, which are well known to those skilled in the art. Thus, free amino groups in the side chains of amino acids in the peptide portion of LBB can be coupled to reactive esters in the antigen (e.g., N-hydroxysuccinimide esters prepared from carboxylic acids); thiols in the peptide portion can be coupled to maleimide groups in the antigen; azides can be incorporated into the side chains of amino acid residues in the peptide portion and coupled to acetylene-containing antigens using a copper-catalyzed cycloaddition reaction; and other nucleophiles in the peptide (e.g., hydrazine, hydroxylamino, vic-aminothiol groups) can be coupled to electrophiles in the antigen (e.g., aldehydes, ketones, active esters). Obviously, it is possible in principle to reverse the positions of the two reactive groups in the peptide chain and antigen to achieve selective coupling.

[0097] In another preferred embodiment, the antigen is selected from a peptide, a protein, an epitope mimetic, a carbohydrate or a hapten.

[0098] In further preferred embodiments, the antigen is derived from a source selected from the group consisting of: (a) viruses; (b) bacteria; (c) parasites, preferably parasitic protozoa; (d) tumors; (e) self molecules; (f) non-peptide hapten molecules; (g) allergens and (h) hormones.

[0099] In another preferred embodiment, the antigen is selected from the group consisting of: (1) antigens suitable for inducing an immune response to cancer cells; (2) antigens suitable for inducing an immune response to infectious diseases; (3) antigens suitable for inducing an immune response to allergens; (4) antigens suitable for inducing a response to self molecules; and (5) antigens suitable for inducing a response to drugs or hormones.

[0100] In another preferred embodiment, the antigen is a tumor antigen, an autoantigen, a peptide of a pathogen, an allergen or a hapten.

[0101] In another preferred embodiment, the antigen is a tumor antigen, an autoantigen, a peptide of a pathogen, an allergen or a hapten.

[0102] In another preferred embodiment, the antigen is derived from the circumsporozoite protein (CS) of the malarial parasite Plasmodium falciparum. In another preferred embodiment, the antigen is antigen 43.

[0103] In another preferred embodiment, the antigen is a proline-rich peptide antigen as described in WO 2015 / 082501, which is incorporated herein by reference in its entirety. In another preferred embodiment, the antigen comprises, preferably consists of, a peptide of SEQ ID NO: 27 to 112 as defined in WO 2015 / 082501 or a peptide of SEQ ID NO: 27 to 112 as defined in WO 2015 / 082501, wherein one, two or three amino acids are replaced by other amino acids. In another preferred embodiment, the antigen comprises (i) SEQ ID NO:27 to 112 peptides as defined in WO 2015 / 082501 or (ii) SEQ ID NO:27 to 112 peptides as defined in WO 2015 / 082501, wherein one, two or three amino acids are replaced by other amino acids, wherein the antigen consists of 8-80 amino acids, and wherein preferably the antigen consists of 8-60 amino acids, and wherein further preferably the antigen consists of 8-50 amino acids. In another preferred embodiment, the antigen comprises (i) SEQ ID NO:27 to 112 peptides as defined in WO 2015 / 082501 or (ii) SEQ ID NO:27 to 112 peptides as defined in WO 2015 / 082501, wherein one, two or three amino acids are replaced by other amino acids, wherein the antigen consists of 8-80 amino acids, and wherein preferably the antigen consists of 8-60 amino acids, and wherein further preferably the antigen consists of 8-50 amino acids. In another preferred embodiment, the antigen comprises (i) a peptide of SEQ ID NO: 27 to 83 as defined in WO 2015 / 082501 or (ii) a peptide of SEQ ID NO: 27 to 83 as defined in WO 2015 / 082501, wherein one, two or three amino acids are replaced by other amino acids, wherein the antigen consists of 8-80 amino acids, and wherein preferably the antigen consists of 8-60 amino acids, and wherein further preferably the antigen consists of 8-50 amino acids. In another preferred embodiment, the antigen comprises (i) a peptide of SEQ ID NO: 27 to 36, 38 to 48, 50 to 55, 61 to 79, 81 to 83 as defined in WO 2015 / 082501, or (ii) a peptide of SEQ ID NO: 27 to 36, 38 to 48, 50 to 55, 61 to 79, 81 to 83 as defined in WO 2015 / 082501, wherein one, two or three amino acids are replaced by other amino acids, wherein the antigen consists of 8-80 amino acids, and wherein preferably the antigen consists of 8-60 amino acids, and wherein further preferably the antigen consists of 8-50 amino acids.

[0104] In another preferred embodiment, the antigen is a cyclic peptide comprising the amino acid sequence (I), wherein the amino acid sequence (I) comprises, preferably consists of, the following amino acid sequence:

[0105] X1-x2- NO:1),

[0106] in

[0107] X1, X2, X3, X5, X6, X7, X9, X10, X11, X22, X23, X24, X26, X27, X28 and X29 are independently amino acids;

[0108] C4, C8, C21 and C25 are independently cysteine;

[0109] P12 is proline;

[0110] I13 is isoleucine;

[0111] T14 is threonine;

[0112] N15 is asparagine;

[0113] D16 is aspartic acid;

[0114] Q17 is glutamine;

[0115] K18 and K19 are independently lysine;

[0116] L20 is leucine; and

[0117] X30 is an amino acid or missing,

[0118] wherein the cysteines C4 and C25 form a first disulfide bond, and the cysteines C8 and C21 form a second disulfide bond.

[0119] The cyclic peptides used in the present invention when the lipid building blocks of the present invention are coupled to the conjugates of the present invention or further and specifically when incorporated into the synthetic virus-like particles (SVLPs) of the present invention can elicit neutralizing and protective antibodies to RSV virus, usually without the administration of an adjuvant. Human respiratory syncytial virus (RSV) is a member of the viral family Pneumoviridae and a highly contagious seasonal respiratory virus that infects the lungs and respiratory tract. It can be spread by droplets that are sneezed or coughed into the air by the infected person. In particular, RSV is a major cause of lower and upper respiratory tract infections and hospital visits, especially during infancy and childhood, but also affects elderly adults and immunocompromised individuals. Due to the high healthcare costs associated with hospitalizations caused by RSV infection, there is a need to prevent RSV infection. One approach is active immunization through RSV vaccines. Several different attempts have been made to generate such vaccines (WO 2006 / 034292, US 2010 / 0239617, WO 2010 / 149745, WO 2014 / 144756, WO 2012 / 048115), but to date no candidate has been approved as a safe and effective vaccine for preventing RSV infection.

[0120] In the cyclic peptide used, specific disulfide bridges between cysteines C4 and C25 and cysteines C8 and C21 stabilize the peptide in a "helical hairpin" or "helix-turn-helix" conformation. Notably, peptides with no, only one, or disulfide bridges at different positions have been found to fail to elicit neutralizing antibodies. Furthermore, the cyclic peptide used is considered a well-tolerated vaccine. The immune response elicited by the cyclic peptide incorporated into SVLPs did not activate VAERD following infection with RSV.

[0121] The cyclic peptide was generated using automated solid phase peptide synthesis, wherein the disulfide bonds between cysteines C4 and C25 and cysteines C8 and C21 were obtained by oxidative refolding, resulting in a favorable steric conformation.

[0122] In another preferred embodiment, the cyclic peptide has a length of at most 80 amino acids, preferably at most 60 amino acids, more preferably at most 40 amino acids, still more preferably at most 30 amino acids.

[0123] In another preferred embodiment, X11 is selected from norleucine, 6-hydroxy-norleucine, norvaline, 5-oxo-norleucine, 2-aminoheptanoic acid, methionine, ethionine, hydroxymethionine, s-oxymethionine, methionine sulfone or methionine sulfoxide, wherein preferably X11 is norleucine.

[0124] In another preferred embodiment, X23 is selected from asparagine, β-hydroxyasparagine, 2,5-diamino-4-hydroxy-5-ketopentanoic acid, glutamine, glutamine hydroxamate, 3-methyl-glutamine, n-methyl-asparagine, n5-methylglutamine, cysteine-S-acetamide; serine, homoserine, allothreonine, 3,3-dihydroxy-alanine, 2-amino-5-hydroxypentanoic acid, 4-hydroxy-1-threonine, threonine, hydroxynorvaline, 6-hydroxy-1-norleucine, or glycine; wherein X23 is preferably asparagine, serine, glutamine, or glycine. In another preferred embodiment, X23 is serine. In another preferred embodiment, X23 is glutamine. In another preferred embodiment, X23 is glycine. In another preferred embodiment, X23 is asparagine.

[0125] In another preferred embodiment, X24 is selected from asparagine, β-hydroxyasparagine, 2,5-diamino-4-hydroxy-5-pentanoic acid, glutamine, glutamine hydroxamate, 3-methyl-glutamine, n-methyl-asparagine, n5-methylglutamine, cysteine-s-acetamide; lysine, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,8-diaminooctanoic acid, ornithine, aminoadipic acid, thiolysine; aspartic acid, 2-amino-6-oxobenzoic acid, 3-methyl-aspartic acid, 1-2-amino-6-methylene- In another preferred embodiment, X24 is selected from asparagine, lysine, ornithine, 2,4-diaminobutyric acid (Dab), glutamine, glycine, or serine. In another preferred embodiment, X24 is glycine, glutamine, or serine. In another preferred embodiment, X24 is serine. In another preferred embodiment, X24 is glutamine.In another preferred embodiment, X24 is glycine.

[0126] In another preferred embodiment, X11 is norleucine, and X24 is selected from asparagine, lysine, ornithine, 2,4-diaminobutyric acid (Dab), glutamine, glycine or serine.

[0127] In another preferred embodiment, X11 is norleucine and X24 is serine. In another preferred embodiment, X11 is norleucine and X24 is glutamine. In another preferred embodiment, X11 is norleucine and X24 is asparagine. In another preferred embodiment, X11 is norleucine and X24 is aspartic acid. In another preferred embodiment, X11 is norleucine and X24 is asparagine. In another preferred embodiment, X11 is norleucine and X24 is ornithine. In another preferred embodiment, X11 is norleucine and X24 is 2,4-diaminobutyric acid. In another preferred embodiment, X11 is norleucine and X24 is lysine.

[0128] In another preferred embodiment, the C-terminal amino acid of the amino acid sequence (I) is selected from alanine, leucine, valine, norleucine, norvaline, isoleucine, homoleucine, vinylglycine, 2-aminobutyric acid, 2-allylglycine, alloleucine, allothreonine, 2-aminoheptanoic acid, serine, glutamine or glycine.

[0129] In another preferred embodiment, X11 is norleucine, X24 is selected from lysine, 2,4-diaminobutyric acid, aspartic acid, asparagine, serine, glutamine or glycine, and the C-terminal amino acid of the amino acid sequence (I) is selected from alanine, leucine, valine, norleucine, norvaline, isoleucine, homoleucine, vinylglycine, 2-aminobutyric acid, 2-allylglycine, alloleucine, alloisoleucine or 2-aminoheptanoic acid. Preferably, the C-terminal amino acid of the amino acid sequence (I) is selected from alanine, leucine, valine, norleucine, norvaline, isoleucine, homoleucine, vinylglycine, 2-aminobutyric acid, 2-allylglycine, isoleucine, alloisoleucine or 2-aminoheptanoic acid, further preferably or alternatively preferably, the C-terminal amino acid of the amino acid sequence (I) is the D stereoisomer.

[0130] In another preferred embodiment, the C-terminal amino acid of the amino acid sequence (I) is selected from the following D stereoisomers: alanine, leucine, valine, norleucine, norvaline, isoleucine, homoleucine, vinylglycine, 2-aminobutyric acid, 2-allylglycine, alloleucine, alloisoleucine or 2-aminoheptanoic acid. In another preferred embodiment, the C-terminal amino acid of the amino acid sequence (I) is alanine, preferably D-alanine.

[0131] In another preferred embodiment, X1 is a polar or hydrophobic amino acid. In another preferred embodiment, X1 is selected from asparagine, β-hydroxyasparagine, 2,5-diamino-4-hydroxy-5-ketopentanoic acid, glutamine, glutamine hydroxamic acid, 3-methyl-l-glutamine, n-methyl-asparagine, n5-methyl-glutamine, cysteine-s-acetamide; leucine, alloleucine, alloisoleucine, homoleucine, isoleucine, 2-aminobutyric acid, norleucine, norvaline, valine; serine, homoserine, allothreonine, 3,3-dihydroxy-alanine, 2-amino-5-hydroxyvaleric acid, 4-hydroxy-1-threonine, threonine, hydroxynorvaline, 6-hydroxy-1-norleucine; or glycine. In another preferred embodiment, X1 is selected from asparagine, glutamine, leucine, serine, or glycine. In another preferred embodiment, X1 is glycine. In another preferred embodiment, the X1 is glutamine. In another preferred embodiment, the X1 is serine.

[0132] In another preferred embodiment, X1, X23 and X24 are each independently selected from the group consisting of ornithine, aspartic acid, lysine, asparagine, 2,4-diaminobutyric acid (Dab), glutamine, leucine, serine and glycine.

[0133] In another preferred embodiment, X1, X23 and X24 are each independently selected from the group consisting of asparagine, 2,4-diaminobutyric acid (Dab), glutamine, leucine, serine and glycine. In another preferred embodiment, X1 is selected from glutamine, serine or glycine; X23 is serine; and X24 is glutamine or serine.

[0134] In another preferred embodiment, X2, X6 and X22 are independently polar amino acids. Preferably, X2, X6 and X22 are independently selected from the group consisting of 2-amino-5-hydroxyvaleric acid, allothreonine, 4-chloro-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxy-leucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, S-(2-hydroxyethyl)-1-cysteine, phosphoserine, serine, 4-hydroxy-1-threonine, threonine, phosphothreonine or hydroxynorvaline. In another preferred embodiment, X2, X6 and X22 are serine.

[0135] In another preferred embodiment, X3 is an amino acid with an acidic or negatively charged side chain at physiological pH (about pH 7). Preferably, X3 is selected from glutamic acid, 5-o-methyl-glutamic acid, (3r)-3-methyl-1-glutamic acid, (3s)-3-methyl-1-glutamic acid, 2s,4r-4-methylglutamic acid, 4-hydroxy-glutamic acid, 2-aminoadipic acid, 1-2-amino-6-methylene-mentholized acid, 2-amino-6-oxomentholized acid; 3-methyl-aspartic acid, 6-carboxylysine, aspartic acid, β-hydroxyaspartic acid, 3,3-dimethylaspartic acid or 2-amino-malonic acid. More preferably, X3 is selected from glutamic acid, 5-o-methyl-glutamic acid, (3r)-3-methyl-1-glutamic acid, (3s)-3-methyl-1-glutamic acid, 2s,4r-4-methylglutamic acid, 4-hydroxy-glutamic acid, 2-aminoadipic acid, 1-2-amino-6-methylene-mentholized acid or 2-amino-6-oxomentholized acid. In another more preferred embodiment, X3 is glutamic acid.

[0136] In another preferred embodiment, X5 and X7 are independently hydrophobic amino acids. Preferably, X5 and X7 are independently selected from leucine, alloleucine, alloisoleucine, homoleucine, isoleucine, 2-aminobutyric acid, norleucine, norvaline, or valine. More preferably, X5 or X7 is leucine. In another more preferred embodiment, X5 and X7 are leucine.

[0137] In another preferred embodiment, X9 and X23 are independently polar amino acids. Preferably, X9 and X23 are independently selected from asparagine, glutamine, serine or glycine. In another more preferred embodiment, X9 or X23 are independently selected from asparagine, glutamine, serine or glycine. In another more preferred embodiment, both X9 and X23 are independently selected from asparagine, glutamine, serine or glycine. In another more preferred embodiment, X9 and X23 are both independently selected from asparagine, glutamine, serine or glycine. In another more preferred embodiment, X9 and X23 are asparagine.

[0138] In another preferred embodiment, X10 is an amino acid with an acidic or negatively charged side chain at physiological pH (approximately pH 7). Preferably, X10 is selected from 3-methyl-aspartic acid, 6-carboxylysine, aspartic acid, β-hydroxyaspartic acid, 3,3-dimethylaspartic acid, or 2-amino-malonic acid. In another more preferred embodiment, X10 is aspartic acid.

[0139] In another preferred embodiment, X26 is a hydrophobic or polar amino acid. Preferably, X26 is selected from leucine, alloleucine, alloisoleucine, homoleucine, isoleucine, 2-aminobutyric acid, norleucine, norvaline, valine; 2,5-diamino-4-hydroxy-5-oxopentanoic acid, glutamine hydroxamate, 3-methyl-1-glutamine, n5-methyl-glutamine, asparagine, 2,5-diamino-4-hydroxy-5-oxopentanoic acid, or n-methyl-asparagine. More preferably, X26 is leucine or glutamine.

[0140] In another preferred embodiment, X27 is a polar or hydrophobic amino acid or an amino acid with an acidic or negatively charged side chain at physiological pH (about pH 7). Preferably, X27 is serine, isoleucine or lysine.

[0141] In another preferred embodiment, X28 is a polar or hydrophobic amino acid. Preferably, X28 is valine or serine.

[0142] In another preferred embodiment, X29 is a hydrophobic amino acid or an amino acid with a negatively charged side chain at physiological pH (about pH 7). Preferably, X29 is D- or L-alanine or D- or L-arginine. In another more preferred embodiment, X29 is D-alanine or D-arginine.

[0143] In another preferred embodiment, X30 is missing or a hydrophobic or polar D- or L-amino acid, preferably X30 is a hydrophobic or polar amino acid D amino acid. Preferably, X30 is missing, or X30 is D- or L-glutamine or D- or L-alanine. In another more preferred embodiment, X30 is D-glutamine or D-alanine. In another even more preferred embodiment, X30 is D-alanine. In a preferred embodiment, X30 is missing, and X29 is alanine, preferably D-alanine. In another preferred embodiment, X30 is alanine, preferably D-alanine, and X29 is arginine.

[0144] In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, and X3 is glutamic acid. In another preferred embodiment, X1 is serine, glycine, glutamine, asparagine, or leucine, X2 is serine, and X3 is glutamic acid. In another preferred embodiment, X5 is leucine, X6 is serine, and X7 is leucine. In another preferred embodiment, X9 is asparagine, X10 is aspartic acid, and X11 is norleucine or methionine, preferably X11 is norleucine.

[0145] In another preferred embodiment, X22 is serine, X23 is asparagine, and X24 is serine, glycine, glutamine, Dab (2,4-diaminobutyric acid), asparagine or lysine, preferably X24 is Dab (2,4-diaminobutyric acid) or lysine. In another preferred embodiment, X26 is glutamine or leucine, X27 is serine, lysine or isoleucine, X28 is valine, X29 is D- or L-arginine or D- or L-alanine, preferably X29 is D- or L-alanine, more preferably X29 is D-alanine, and X30 is deletion, D- or L-alanine or D- or L-glutamine, preferably X30 is D- or L-alanine, more preferably X30 is D-alanine.

[0146] In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, X3 is glutamic acid, X5 is leucine, X6 is serine, and X7 is leucine, and preferably X9 is asparagine, X10 is aspartic acid, and X11 is norleucine or methionine, and further preferably X11 is norleucine. In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, X3 is glutamic acid, X22 is serine, X23 is asparagine, and X24 is Dab (2,4-diaminobutyric acid), asparagine or lysine, preferably X24 is Dab (2,4-diaminobutyric acid) or lysine, and preferably X26 is glutamine or leucine, X27 is serine, lysine or isoleucine, X28 is valine, X29 is D- or L-arginine or D- or L-alanine, further preferably X29 is D- or L-alanine, more preferably X29 is D-alanine, and X30 is deletion, D- or L-alanine or D- or L-glutamine, further preferably X30 is D- or L-alanine, more preferably X30 is D-alanine.

[0147] In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, X3 is glutamic acid, X5 is leucine, X6 is serine, and X7 is leucine. In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, X3 is glutamic acid, X9 is asparagine, X10 is asparagine, and X11 is norleucine or methionine, preferably X11 is norleucine. In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, X3 is glutamic acid, X22 is serine, X23 is asparagine, and X24 is Dab (2,4-diaminobutyric acid), asparagine, or lysine, preferably X24 is Dab (2,4-diaminobutyric acid) or lysine. In another preferred embodiment, X1 is asparagine or leucine, X2 is serine, X3 is glutamate, X26 is glutamine or leucine, X27 is serine, lysine or isoleucine, X28 is valine, X29 is D- or L-arginine or D- or L-alanine, preferably X29 is D- or L-alanine, more preferably X29 is D-alanine, and X30 is deletion, D- or L-alanine or D- or L-glutamine, preferably X30 is D- or L-alanine, more preferably X30 is D-alanine.

[0148] In a preferred embodiment, X2, X3, X5, X6, X7, X9, X10, X11, X22, X23, X24, X26, X27, X28, and X29 are independently L-amino acids. In a preferred embodiment, proline P12, isoleucine I13, threonine T14, asparagine N15, aspartic acid D16, glutamine Q17, lysine K18 and K19, and leucine L20 are independently L-amino acids. In a preferred embodiment, C4, C8, C21, and C25 are independently D-cysteine ​​or L-cysteine, preferably L-cysteine.

[0149] In a preferred embodiment, the cyclic peptide consists of the amino acid sequence (I). In another preferred embodiment, the amino acid sequence (I) consists of the amino acid sequence of SEQ ID NO: 1.

[0150] In another very preferred embodiment, the amino acid sequence of SEQ ID NO: 1 is selected from any one of SEQ ID NO: 2-5, 21-36 or 39-57.

[0151] In another very preferred embodiment, the amino acid sequence of SEQ ID NO: 1 is selected from any one of SEQ ID NO: 39-57.

[0152] In another very preferred embodiment, the amino acid sequence of SEQ ID NO: 1 is selected from any one of SEQ ID NO: 2-5, 21-36.

[0153] In another very preferred embodiment, the amino acid sequence (I) is selected from any one of the following: (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5), (SEQ ID NO: 21), (SEQ ID NO: 22), (SEQ ID NO: 23), (SEQ ID NO: 24), (SEQ ID NO: 25), (SEQ ID NO: 26), (SEQ ID NO: 27), (SEQ ID NO: 28), (SEQ ID NO: 29), (SEQ ID NO: 30), (SEQ ID NO: 31), (SEQ ID NO: 32), (SEQ ID NO: 33), (SEQ ID NO: 34), (SEQ ID NO: 35) or (SEQ ID NO: 36).

[0154] In another very preferred embodiment, the amino acid sequence of SEQ ID NO: 1 is any one of SEQ ID NO: 2, 3, 21, 22 or 23.

[0155] In another very preferred embodiment, the amino acid sequence of SEQ ID NO: 1 is an amino acid selected from SEQ ID NO: 2 or SEQ ID NO: 3.

[0156] In certain embodiments, the amino acid sequence (I) of the cyclic peptide includes (i) an N-terminus selected from a free amino group or an acetylated N-terminus, and / or (ii) a C-terminus selected from a free carboxyl group or an amidated C-terminus.

[0157] In another preferred embodiment, the cyclic peptide further comprises a linker, wherein the linker is attached to the amino acid sequence (I), and wherein the linker comprises (i) at least one attachment moiety, (ii) at least one spacer moiety, (iii) at least one, preferably one, connecting moiety, or (iv) any combination of (i), (ii) and (iii).

[0158] In another preferred embodiment, the at least one attachment moiety comprises or preferably consists of: -O-NH2, -O-NH-(aminooxy moiety), -C(O)-CH2-O-NH2, -C(O)-CH2-O-NH-(aminooxyacetyl moiety), -NH-NH2, -NH-NH-(hydrazine moiety), -E(O)-NH-NH2 or -E(O)-NH-NH-(hydrazine moiety), wherein E is C, S(O) or P. In a further preferred embodiment, the attachment moiety comprises or preferably consists of: -O-NH2, -O-NH-(aminooxy moiety), -C(O)-CH2-O-NH2, -C(O)-CH2-O-NH-(aminooxyacetyl moiety), -NH-NH2, -NH-NH-(hydrazine moiety) or (-C(O)-NH-NH2, -C(O)-NH-NH-(carbohydrazide moiety). In another further preferred embodiment, the attachment moiety comprises or preferably consists of: -O-NH2 or -O-NH-(aminooxy moiety). In another preferred embodiment, the at least one spacer moiety comprises or preferably consists of: NH2-CH2-CH2-(O-CH2-CH2) n -C(O)- or -NH-CH2-CH2-(O-CH2-CH2) n -C(O)-, wherein n is an integer from 1 to 45, preferably from 2 to 20, more preferably from 6 to 8; or NH2-(CH2) m -C(O)- or -NH-(CH2) m-C(O)-, wherein m is an integer from 2 to 45, preferably from 2 to 20, more preferably from 2 to 6. In another preferred embodiment, the at least one linking portion is capable of cross-linking the cyclic peptide with the second peptide. Linking portions capable of cross-linking the cyclic peptide with the second peptide are well known in the art. In one embodiment of the present invention, the linking portion capable of cross-linking the cyclic peptide with the second peptide comprises or consists of an aldehyde portion, such as a glutaraldehyde portion, a suberaldehyde portion, a dialdehyde portion, a succinaldehyde portion; a carbodiimide portion, such as a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride portion; a glyoxal portion; an N-hydroxy-sulfosuccinimide portion, such as an N-hydroxy-sulfosuccinimide portion; a cationic linking portion; a polyethylene glycol portion; a benzoylbenzoic acid portion. Additional suitable linking moieties are listed in Pierce Catalog and Handbook, Pierce Chemical Company, Rockford (1997); Bioconjugate Techniques, Greg T. Hermanson, Pierce Biotechnology, Thermo Fisher Scientific, Rockford (2013); and described in EP 1321466 A1, DE 19821859 A1, US 6875737, US 5456911, US 5612036, US 5965532, WO 2001004135, WO 2001070685, US 20140302001 A1, US 6800728, US 20140171619A1, US8168190, WO 2012 / 166594 A1 and WO 2015 / 082501.

[0159] In one embodiment, the linker is at least one, preferably exactly one attachment moiety. In another embodiment, the linker is at least one, preferably exactly one, two or three spacer moieties, wherein in the case of more than one spacer moiety, they are preferably linked together directly or through an interconnecting group. In another embodiment, the linker is at least one, preferably exactly one spacer moiety. In another embodiment, the linker is at least one, preferably exactly one connecting moiety.

[0160] In another embodiment, the linker comprises at least one attachment moiety and at least one, preferably exactly one, spacer moiety, wherein at least one attachment moiety is attached to the N-terminus of the amino acid sequence (I) and at least one spacer moiety is attached to the attachment moiety. In another embodiment, the linker comprises at least one attachment moiety and at least one, preferably exactly one, spacer moiety, wherein at least one spacer moiety is attached to the N-terminus of the amino acid sequence (I) and at least one attachment moiety is attached to the spacer moiety.

[0161] In one embodiment, the at least one linker comprises at least one attachment moiety and at least one spacer moiety and at least one linking moiety, wherein at least one linking moiety or at least one spacer moiety is attached to the N-terminus of the amino acid sequence (I). In another embodiment, at least one attachment moiety is attached to the N-terminus of the amino acid sequence (I), at least one spacer moiety is attached to the attachment moiety, and at least one linking moiety is attached to the spacer moiety. In another embodiment, at least one spacer moiety is attached to the N-terminus of the amino acid sequence (I), at least one attachment moiety is attached to the spacer moiety, and at least one linker moiety is attached to the attachment moiety.

[0162] In a preferred embodiment, the linker is typically and preferably attached to the amino acid sequence (I), to the N-terminus of the amino acid sequence (I) or to a free amino group of a side chain of an amino acid of the amino acid sequence (I), preferably to the N-terminus of the amino acid sequence (I) via an amide bond. In a preferred embodiment, the linking moiety is capable of cross-linking the cyclic peptide to a sulfhydryl group of a second peptide. In a preferred embodiment, the linking moiety comprises a maleimide moiety.

[0163] In a preferred embodiment, the linker is attached to an amino group contained in the amino acid sequence (I), wherein preferably the linker is attached to a free amino group of: (i) the N-terminus of the amino acid sequence (I), or (ii) the side chain of an amino acid in the amino acid sequence (I). Preferably, the linker is attached to the amino group contained in the amino acid sequence (I) via an amide bond. The side chain preferably comprises the amino acid lysine. In a preferred embodiment, X24 is lysine, and the linker is attached to the free amino group of the side chain of X24.

[0164] In a very preferred embodiment, the linker is selected from the following formula:

[0165]

[0166] wherein n is an integer from 1 to 45, preferably from 6 to 8, and the wavy line indicates the site of attachment to the amino acid sequence (I). Further very preferably, n is 6.

[0167] In the case of the present invention, such as conjugates and compounds comprising the joints thus comprising one or more double bonds, the double bonds may have (E)- or (Z)- configurations or mixtures thereof in any ratio. This is also true for preferred joints comprising oxime moieties. Therefore, preferred joints comprising oxime moieties may comprise joints (and therefore cis-isomers) with the oxime moiety in its cis configuration, joints (and therefore anti-isomers) with the oxime moiety in its anti- configuration, and mixtures thereof in any ratio. Within the chemical formulae presented herein for the double bonds or oxime moieties, this is typically and preferably represented by a wavy line.

[0168] In another very preferred embodiment, the linker is selected from the following formula:

[0169]

[0170] The wavy line indicates the attachment site of the amino acid sequence (I).

[0171] In another very preferred embodiment, the cyclic peptide comprises, preferably is a formula selected from any one of the following formulae:

[0172] In another very preferred embodiment, the cyclic peptide comprises, preferably is, a formula selected from any one of the following formulae: formula (3) (SEQ ID NO: 16), formula (4) (SEQ ID NO: 17), formula (5) (SEQ ID NO: 18), formula (6) (SEQ ID NO: 19), formula (7) (SEQ ID NO: 20) and any one of formulae (19) to (37) as described in the Examples section.

[0173] In another very preferred embodiment, the cyclic peptide comprises, preferably, formula (3) (SEQ ID NO: 16). In another very preferred embodiment, the cyclic peptide comprises, preferably, formula (4) (SEQ ID NO: 17). In another very preferred embodiment, the cyclic peptide comprises, preferably, formula (5) (SEQ ID NO: 18). In another very preferred embodiment, the cyclic peptide comprises, preferably, formula (6) (SEQ ID NO: 19). In another very preferred embodiment, the cyclic peptide comprises, preferably, formula (7) (SEQ ID NO: 20).

[0174] The linking and conjugation procedures that can be used to attach cyclic peptides to lipopeptide building blocks are well known to those skilled in the art (see, for example, Hermanson, GT, Bioconjugation Technology, 2nd ed., Academic Press, 2008). The cyclic peptide can be linked to the lipopeptide building block using any method used to link and conjugate peptides or other antigens to antigen delivery systems, such as carrier proteins, polymers, dendrimers, nanoparticles, or virus-like particles. Free amino groups in the side chains of amino acids in the peptide portion of the lipopeptide building block can be coupled to reactive esters in the cyclic peptide or linker (e.g., N-hydroxysuccinimide esters prepared from carboxylic acids); thiols in the peptide portion can be coupled to maleimide groups in the linker; azides can be incorporated into the side chains of amino acid residues in the peptide portion and coupled to cyclic peptides or linkers containing acetylene groups using a copper-catalyzed cycloaddition reaction; and other nucleophiles in the peptide portion (e.g., hydrazines, hydroxyamino groups, vic-aminothiols) can be coupled to electrophiles in the cyclic peptide or linker (e.g., aldehydes, ketones, active esters). Further, it is envisioned that the positions of the two reactive groups in the peptide portion and the cyclic peptide or linker are reversed to achieve selective coupling.

[0175] All embodiments and preferred and very preferred embodiments of the lipid building blocks, conjugates and all components thereof including antigens and linkers described herein are applicable to all aspects of the invention, even if not all embodiments and preferred and very preferred embodiments do not have to be repeated and reiterated again.

[0176] In another very preferred embodiment, the conjugate is selected from any one of the following formulae:

[0177]

[0178]

[0179]

[0180] In another highly preferred embodiment, the conjugate is (38).

[0181] In another highly preferred embodiment, the conjugate is (39).

[0182] In another highly preferred embodiment, the conjugate is (40).

[0183] In another highly preferred embodiment, the conjugate is (41).

[0184] In another highly preferred embodiment, the conjugate is (42).

[0185] In another highly preferred embodiment, the conjugate is (45).

[0186] In another highly preferred embodiment, the conjugate is (46).

[0187] In another aspect, the present invention provides a bundle of conjugates comprising 2, 3, 4, 5, 6 or 7 of the conjugates of the present invention. In another aspect, the present invention provides a bundle of conjugates comprising 2, 3, 4 or 5 of the conjugates of the present invention. In another very preferred aspect, the present invention provides a bundle of conjugates comprising 3 of the conjugates of the present invention.

[0188] In another aspect, the present invention provides a bundle of conjugates comprising 2, 3, 4, 5, 6 or 7 of the conjugates of the present invention, wherein the conjugates are selected from any one of formula (38), (39), (40), (41), (42), (45) or (46), wherein preferably the conjugates are selected from any one of formula (38), (40), (41), (42) or (46), and wherein further preferably the conjugates are of formula (38) or (46), preferably (38).

[0189] In another very preferred aspect, the present invention provides a bundle of conjugates, said bundle comprising 3, preferably exactly 3, of the conjugates of the present invention, wherein said conjugates are selected from any one of formulae (38), (39), (40), (41), (42), (45) or (46), wherein preferably said conjugates are selected from any one of formulae (38), (40), (41), (42), (46), and wherein further preferably said conjugates are of formula (38) or (46), preferably (38).

[0190] According to a preferred embodiment, in the bundle, the coiled-coil peptide segments of the peptide portion comprised by the conjugate are coiled together, preferably the coiled-coil peptide segments are coiled together helically, more preferably the coiled-coil peptide segments are coiled together α-helically. In a preferred embodiment, the coiled-coil peptide segments of the peptide portion are coiled together in a left-handed or right-handed manner. According to a preferred embodiment, in the bundle, the coiled-coil peptide segments of the peptide portion form an α-helical left-handed helix.

[0191] In a preferred embodiment, the coiled-coil peptide segments have a parallel orientation, i.e. they run in the same direction; or they have an antiparallel orientation, i.e. they run in opposite directions to each other; wherein the first option is preferred. The term "orientation" is based on the orientation of the peptide chain having an N-terminus on one side and a C-terminus on the other side. In a preferred embodiment of the bundle of the present invention, the coiled-coil peptide segments of the peptide portion form a left-handed α-helical coiled coil, wherein the coiled-coil peptide segments have a parallel orientation in the coiled coil. Preferably, the bundle comprises 2 to 7 (e.g., dimers, trimers, tetramers, pentamers, hexamers or heptamers), more preferably 2, 3, 4 or 5, and again more preferably 3 helically twisted coiled-coil peptide segments, wherein the helically twisted coiled-coil peptide segments have a parallel orientation in the coiled coil.

[0192] In another aspect, the present invention provides a synthetic virus-like particle comprising at least one bundle of the conjugate of the present invention.

[0193] In another aspect, the present invention provides a synthetic virus-like particle comprising a bundle of at least one conjugate of the present invention, wherein the conjugate is selected from any one of formula (38), (39), (40), (41), (42), (45) or (46), wherein preferably the conjugate is selected from any one of formula (38), (40), (41), (42) or (46), and wherein further preferably the conjugate is formula (38) or (46), preferably (38).

[0194] The present invention also relates to a method for preparing the synthetic virus-like particles of the present invention. Synthetic virus-like particles (SVLPs) can be produced by a self-assembly process, for example in an aqueous solution. This method can involve dissolving the lipopeptide building blocks in a suitable carrier, preferably an aqueous buffer system (e.g., buffered saline or non-buffered saline). The solvent can be removed after the synthetic virus-like particles are prepared, for example, by lyophilization or spray drying. Conjugates comprising a specific combination of the cyclic peptide of the present invention and the lipopeptide building blocks of the present invention self-assemble into bundles and further assemble into synthetic virus-like particles (SVLPs).

[0195] In another aspect, the present invention provides a pharmaceutical composition comprising an immunologically effective amount of the conjugate of the present invention or the synthetic virus-like particle of the present invention together with a pharmaceutically acceptable diluent, carrier or excipient, wherein preferably the pharmaceutical composition is a vaccine.

[0196] As used herein, the term "effective amount" refers to an amount necessary or sufficient to achieve a desired biological effect. Preferably, the term "effective amount" refers to an amount of an antigen of the invention, a conjugate of the invention, or a synthetic virus-like particle of the invention that (i) treats or prevents a specific disease, medical condition, or disorder, (ii) attenuates, alleviates, or eliminates one or more symptoms of a specific disease, medical condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, medical condition, or disorder as described herein. As understood herein, an effective amount of an immunogen is an amount that is capable of modulating, preferably enhancing, the response of a subject's immune system to an antigen or pathogen.

[0197] The present invention further relates to conjugates or synthetic virus-like particles of the present invention for use as vaccines. As used herein, vaccines are pharmaceutical compositions used to modulate, preferably stimulate, the body's immune system's response to a specific antigen or pathogen. In a preferred embodiment, the pharmaceutical composition or preferably the vaccine is used to prevent or reduce the risk of RSV infection in a subject, preferably a human, more preferably a child or the elderly.

[0198] In yet another aspect, the present invention provides a conjugate of the invention or a synthetic virus-like particle of the invention for use as a medicament, preferably for use in a method for preventing a disease or for reducing the risk of a disease, wherein further preferably the disease is an infectious disease, cancer or allergy, and again more preferably wherein the disease is a respiratory syncytial virus (RSV) infection.

[0199] On the other hand again, the present invention provides a conjugate of the present invention or a synthetic virus-like particle of the present invention for use in a method for preventing a disease or for reducing the risk of a disease or for treating a disease, wherein further preferably the disease is an infectious disease, cancer or allergy, and again more preferably wherein the disease is respiratory syncytial virus (RSV) infection. On the other hand again, the present invention provides a conjugate of the present invention or a synthetic virus-like particle of the present invention for use in a method for preventing an infectious disease or reducing the risk of an infectious disease, preferably for use in a method for preventing an infectious disease associated with or caused by respiratory syncytial virus or reducing the risk of an infectious disease. As used herein, the term treatment refers to therapy and therapeutic treatment.

[0200] The present invention provides a conjugate of the present invention for use as a medicine, preferably for use in a method for preventing an infectious disease or for reducing the risk of an infectious disease, more preferably for use in a method for preventing an infectious disease associated with or caused by respiratory syncytial virus or reducing the risk of an infectious disease, a synthetic virus-like particle of the present invention or a pharmaceutical composition of the present invention. The present invention provides a conjugate of the present invention for use as a medicine, preferably for use in a method for preventing an infection or for reducing the risk of an infection, more preferably for use in a method for preventing an infection caused by respiratory syncytial virus or reducing the risk of an infection, a synthetic virus-like particle of the present invention or a pharmaceutical composition of the present invention. The present invention provides a conjugate of the present invention for use as a medicine, preferably for use in a method for preventing an infection or for reducing the risk of an infection, more preferably for use in a method for preventing RSV infection or reducing the risk of RSV infection, a synthetic virus-like particle of the present invention or a pharmaceutical composition of the present invention.

[0201] The present invention further relates to a method for eliciting or regulating an immune response or to a method for limiting the risk of contracting a disease, preferably an infection, more preferably an infection associated with RSV or caused by RSV, wherein an immunogenic effective amount of a conjugate of the present invention or a synthetic virus-like particle is administered to a subject, preferably a human, more preferably a child or an elderly person. The present invention further relates to a method for treating a disease, preferably an infection, more preferably an infection associated with RSV or caused by RSV, comprising administering an immunogenic effective amount of a conjugate of the present invention or a synthetic virus-like particle to a subject, preferably a human, more preferably a child or an elderly person. In addition, the present invention relates to a conjugate of the present invention or a synthetic virus-like particle for treating a disease, preferably an infection associated with RSV or caused by RSV. Moreover, the present invention relates to a conjugate of the present invention or a synthetic virus-like particle for treating a disease, preferably an infection caused by RSV. In addition, the present invention relates to a cyclic peptide, a conjugate or a synthetic virus-like particle of the present invention for treating a disease, preferably an RSV infection.

[0202] Examples

[0203] Example 1

[0204] Preparation of lipid building blocks

[0205] Lipopeptide 10

[0206]

[0207] This lipopeptide 10 is based on IEKKIEA (SEQ ID NO: 12). The defgabc heptad repeat motif is fused to the universal T helper cell epitope SEQ-ID NO: 6 and an additional residue at the C-terminus (SEQ ID NO: 11) to improve solubility and stability and allow antigen conjugation, and is conjugated to S-[2,3-bis(palmitoyloxy)-(2)-propyl]-cysteine ​​via a GG linker. The synthesis of lipopeptide 10 was performed essentially as described in WO 2008 / 068017 and the product was purified by RP-HPLC (Zorbax C8 column, 30% to 100% MeCN in H2O (+0.1% CHOOH) in 10 min) purity: 95%, t R =4.87 min. MALDI-TOF: C 312 H 552 N 74 O 85 m / z calculated for S3: 6796.4; m / z found: 6797.4 [M+H] + .

[0208] Lipopeptides 11

[0209]

[0210] This lipopeptide 11 contains a coiled-coil domain with a serine at the "c" position of the heptad repeat sequence "defgabc" IEKKIES (SEQ ID NO: 13). Lipopeptide 11 was synthesized by RP-HPLC as described above for lipopeptide 10, purified, and analyzed by analytical RP-HPLC and MALDI-MS. HPLC (Zorbax C8 column, 30% to 100% MeCN in H2O (+0.1% CHOOH) in 10 minutes): purity: 90%, t R = 4.81 min; MALDI-MS: calculated C 312 H 552 N 74 O 89 MW of S3: 6860.46 Da; found: 6860.61 Da (±0.05%).

[0211] Lipopeptide LBB-1

[0212]

[0213] This lipopeptide building block LBB-1 corresponds to lipopeptide 10, except that the lipid Pam2-Cys has an R configuration at the chiral 2-propyl carbon atom and an R configuration at the chiral carbon of the cysteinyl moiety. Building block LBB-1 was analyzed by RP-HPLC (Agilent VariTide RPC, 0% to 95% MeCN in H2O (+0.1% TFA) in 63 minutes: purity 97.0%, t R =45.58; C 312 H 552 N 74 O 85 ESI-MS MW calculated value of S3: 6796.46 Da; found value: 6793.1.

[0214] Lipopeptide LBB- 2

[0215]

[0216] This lipopeptide building block LBB-2 corresponds to lipopeptide 11, except that the lipid Pam2-Cys has an R configuration at the chiral 2-propyl carbon atom and an R configuration at the chiral carbon of the cysteinyl moiety. Building block LBB-2 was analyzed by RP-HPLC (Agilent VariTide RPC, 0% to 95% MeCN in H2O (+0.1% TFA), in 63 minutes: purity 97.0%, t R = 45.58 min. MALDI-MS: calculated C 312 H 552 N 74 O 89 MW of S3: 6860.46 Da; found: 6861.1 Da (±0.05%).

[0217] Example 2

[0218] Synthesis of cyclic RSV peptides and conjugates comprising the peptides

[0219] The applicants have described cyclic peptides that can also be used, and preferably as antigens for the conjugates of the present invention, in their application PCT / EP2018 / 065714 filed on June 13, 2018. In particular, the design and synthesis of such cyclic peptides is described in Example 1 of PCT / EP2018 / 065714.

[0220] Furthermore, the preparation of conjugates comprising such peptides has been described in Example 2 of PCT / EP2018 / 065714, and their use for immunization against RSV infection has been described in detail in Examples 3-5 of PCT / EP2018 / 065714 and herein, in Example 3 of PCT / EP2018 / 065714, and their use for immunogenicity studies against RSV in mice has been described in Example 4 of PCT / EP2018 / 065714 for further intramuscular immunization experiments and in Example 5 for the generation of monoclonal antibodies.

[0221] The disclosure of PCT / EP2018 / 065714, as well as all disclosures of PCT / EP2018 / 065714, in particular those relating to specific cyclic peptides, conjugates and other component parts, are incorporated herein by reference in their entirety, and the generated biological data are specifically incorporated herein by reference in their entirety.

[0222] It is believed that the biological data and effects specifically described in Examples 3-5 of PCT / EP2018 / 065714 make it plausible to utilize the lipid building blocks, conjugates and SVLPs of the present invention and thus achieve said biological data and effects when adapted to the present invention and the features of the present invention, in view of the disclosed cyclic peptides, conjugates and component parts, and in particular in view of the described cyclic peptides, conjugates and compounds of Examples 1-2 of PCT / EP2018 / 065714. Therefore, in view of the adaptation of the present invention and the features of the present invention and the lipid building blocks, conjugates and SVLPs of the present invention herein, the scope of the present invention is believed to be further plausible by reference to said biological data and effects.

[0223] Regardless of whether it is incorporated by reference into PCT / EP2018 / 065714, some of its disclosures are repeated here in full or summarized form. For convenience, for the present invention and the description, the numbering of the cyclic peptide, conjugate and other component parts and the sequence of PCT / EP2018 / 065714 are maintained.

[0224] Example 1 of PCT / EP2018 / 065714 describes maleimido-peptide 1 (SEQ ID NO: 14) and (aminooxy)acetyl peptide 2 (SEQ ID NO: 15), particularly as reference examples because, as disclosed in Example 3 of PCT / EP2018 / 065714, conjugates comprising these peptides do not generate an antibody response and neutralizing antibodies against RSV A2 and A / long chains.

[0225] Typically, the synthesis of the peptides useful and preferred for the present invention, including cyclic peptides, is performed using the Fmoc solid phase peptide synthesis (SPPS) method using Tentagel R Ram resin on a 0.5 mmol scale. After assembly, the peptide is cleaved from the resin and the side chain protecting groups are removed, and the peptide is purified by RP-HPLC on a preparative column and typically lyophilized to obtain the peptide as a white powder. Analysis by RP-HPLC is then performed and the purity, t R , ESI-MS and MW.

[0226] Another preferred cyclic peptide of the present invention, the synthesis and characterization of which has been described in PCT / EP2018 / 065714, is as follows:

[0227] (Aminooxy)acetyl peptide 3

[0228]

[0229] In (aminooxy)acetyl peptide 3 (SEQ ID NO: 16), the (aminooxy)acetyl moiety (denoted as "AOAc") is coupled to the N-terminus of the peptide via a 21-amino-3,6,9,12,15,18-hexaoxaheneicosane-21-linker, and D-Ala (denoted as "a") is coupled to the C-terminus as an amide to confer exoprotease stability. (aminooxy)acetyl peptide 3 contains two disulfide bonds.

[0230] (Aminooxy)acetyl peptide 4

[0231]

[0232] (Aminooxy) acetyl peptide 4 (SEQ ID NO: 17) is derived from the F-protein of RSV virus when carrying out several sequence modifications. (Aminooxy) acetyl moiety (expressed as "AOAc") is coupled to the N-terminus of the peptide, and D-Ala (expressed as "a") is coupled to the C-terminus as an amide to confer exoproteinase stability. (Aminooxy) acetyl peptide 4 contains two disulfide bonds, as depicted in the above formula. (Aminooxy) acetyl peptide 4 is assembled by standard Fmoc SPPS. AOAc is coupled to bis-Boc-aminooxy-acetic acid, N-hydroxysuccinimide ester (Boc2-Aoa-OSu). After assembly is complete, the peptide is cleaved from the resin and the side chain protecting groups are removed by treating with 87.5% TFA, 5% TA, 5% H2O, 2.5% EDT for 2.5 hours. In order to cyclize by air oxidation, the reduced peptide is dissolved in 0.33M ammonium bicarbonate buffer at pH 7.8 and stirred overnight. The cyclic peptide was then purified by RP-HPLC on a preparative C18 column and lyophilized to give 4 as a white powder. Analytical RP-HPLC (Vydac 218TP54, 5 μm, 4.6 mm × 250 mm column, 0%–60% MeCN in H 2 O (+0.1% TFA), over 40 min): Purity: 90.4%; t R = 25.07 min. MALDI-MS: C 134 H 226 N 44 O 49 Calculated MW of S4: 3365.78 Da; found MW: 3365.80 Da (±0.01%).

[0233] (Aminooxy)acetyl peptide 5

[0234]

[0235] In (aminooxy)acetyl peptide 5 (SEQ ID NO: 18), an (aminooxy)acetyl moiety (denoted as "AOAc") is coupled to a lysine side chain in the peptide, the N-terminus is acetylated, and D-Ala (denoted as "a") is coupled to the C-terminus. (aminooxy)acetyl peptide 5 contains two disulfide bonds.

[0236] (Aminooxy)acetyl peptide 6

[0237]

[0238] In (aminooxy)acetyl peptide 6 (SEQ ID NO: 19), an (aminooxy)acetyl moiety (denoted as "AOAc") is coupled to the N-terminus of the peptide, and D-Ala (denoted as "a") is coupled to the C-terminus. (aminooxy)acetyl peptide 6 contains two disulfide bonds.

[0239] (Aminooxy)acetyl peptide 7

[0240]

[0241] In (aminooxy)acetyl peptide 7 (SEQ ID NO: 20), the (aminooxy)acetyl moiety (denoted as "AOAc") is coupled to the N-terminus, and D-Ala (denoted as "a") is coupled to the C-terminus. (aminooxy)acetyl peptide 7 contains two disulfide bonds.

[0242] Preparation of conjugates for immunization

[0243] Example 2 of PCT / EP2018 / 065714 describes further preparation of conjugates comprising cyclic RSV peptides by the general scheme ABC, wherein “A” represents the antigen, “B” represents the linker, and “C” represents the lipopeptide building block, and “X” represents the attachment to the lipopeptide building block C.

[0244] Antigen A1 comprises SEQ ID NO:37, Antigen A2 comprises SEQ ID NO:38, Antigen A3 comprises SEQ ID NO:2, and Antigen A4 comprises SEQ ID NO:3.

[0245] Antigen A:

[0246]

[0247] Connector B:

[0248]

[0249] Lipopeptide building block C:

[0250]

[0251] Furthermore, further specific linkers, lipopeptides and conjugates are described in Example 2 of PCT / EP2018 / 065714, the specific disclosure of which is incorporated herein by reference in its entirety and is as follows:

[0252] Maleimide PEG6 aldehyde linker compound 8

[0253]

[0254] Linker compound 8 was synthesized by reacting SM-PEG6 (Thermo Fisher Scientific) with aminoacetaldehyde dimethyl acetal in H2O. SM-PEG6 (7.6 mg, 12.6 μmol) was suspended in 0.3 ml of H2O, and 17 μl of a 1:10 (v / v) solution of aminoacetaldehyde dimethyl acetal in H2O was added. The mixture was stirred at room temperature for 90 minutes. The cross-linker was purified by RP-HPLC on a C8 column and lyophilized. ESI-MS: C 26 H 45 N3O 12 MW calculated: 591.66; MW found: 591.32 (±0.05%).

[0255] For hydrolysis of dimethyl acetal, linker 8 (20 mg) was treated with 95% TFA, 5% H2O for 5 minutes. TFA was removed in vacuo. ESI-MS C 24 H 39 N3O 11 : 545.59 Da; MW found: 545.28 Da (±0.05%).

[0256] Maleimide C3 aldehyde linker compound 9

[0257]

[0258] Described in Example 1 above Lipopeptide 10 and Lipopeptide 11 .

[0259] Conjugate 12 (Antigen A3 + Linker B3 + Lipopeptide Building Block C1)

[0260]

[0261] Conjugate 13 (antigen A3 + linker B3 + lipopeptide C2)

[0262]

[0263] Conjugate 14 (Antigen A3+Linker B4+Lipopeptide C1)

[0264]

[0265] Conjugate 15 (antigen A3 + linker B2 + lipopeptide C1)

[0266]

[0267] Conjugate 16 (Antigen A4+Linker B3+Lipopeptide C1)

[0268]

[0269] Conjugate 17 (antigen A1 + linker B1 + lipopeptide C1)

[0270]

[0271] Conjugate 18 (antigen A2 + linker B2 + lipopeptide C1)

[0272]

[0273] Example 3 of PCT / EP2018 / 065714 describes immunogenicity studies against RSV in mice, the specific disclosure of which is incorporated herein by reference in its entirety.

[0274] The results of Example 3 of PCT / EP2018 / 065714 show that immunization with conjugates 12, 13 and 15 can induce high neutralization titers in serum without the use of a common adjuvant. Lower but detectable titers were found in the serum of animals immunized with conjugates 14 and 16, but no titers were found in the serum of animals immunized with the corresponding linear or monocyclic peptide antigens (conjugates 17 and 18). Immunization with conjugate 12 resulted in complete protection of the lungs and reduced lung tissue pathology after the attack, but after immunization with FI-RSV, only a partial reduction in the virus in the lungs and a strong tissue pathology were observed when attacked. Immunization with 13 also significantly reduced viral replication and lung tissue pathology in the lungs.

[0275] Example 4 of PCT / EP2018 / 065714 describes an intramuscular immunization experiment, the specific disclosure of which is incorporated herein by reference in its entirety.

[0276] The results of Example 4 of PCT / EP2018 / 065714 show that animals immunized with 150 μg or 300 μg of conjugate 12 showed a strong reduction in viral titers (approximately 3×Log10) compared to the PBS control group. Most animals had no detectable virus in their lungs (Log10 titer ≤ 2.6 PFU / g). Vaccination with FI-RSV, 15 μg or 300 μg of conjugate 12 showed a strong reduction in viral titers (approximately 3×Log10). Animals immunized with PBS showed less and less protection (decreased by about 2×Log10). Adjuvants did not seem to improve protection. Animals immunized with PBS portrayed the pathology typically associated with primary RSV infection. Animals vaccinated with FI-RSV showed strong pathology, which suggests vaccine-related disease enhancement. All animals immunized with conjugate 12 showed no signs of vaccine-related disease enhancement, and pathology scores were equal to or lower than those of PBS control animals. The results show that, in the absence of co-administration of an adjuvant, two intramuscular immunizations with conjugate 12 were sufficient to achieve high levels of protective neutralizing antibodies, and immunization with conjugate 12 did not cause respiratory disease enhancement over a large dose range.

[0277] The generation of monoclonal antibodies is described in Example 5, the specific disclosure of which is incorporated herein by reference in its entirety.

[0278] The results of Example 5 of PCT / EP2018 / 065714 show that after immunization with conjugate 12, antibodies with KD in the sub-nanomolar range and neutralization titers similar to or better than palivizumab can be generated in mice.

[0279] Example 6 describes the generation of sequence variants of cyclic peptides and their synthesis, the specific disclosure of which is incorporated herein by reference in its entirety.

[0280] (Aminooxy)acetyl peptide 19 (having SEQ ID NO: 39)

[0281]

[0282] (Aminooxy)acetyl peptide 20 (having SEQ ID NO: 40)

[0283]

[0284] (Aminooxy)acetyl peptide 21 (having SEQ ID NO:41)

[0285]

[0286] (Aminooxy)acetyl peptide 22 (having SEQ ID NO: 42)

[0287]

[0288] (Aminooxy)acetyl peptide 23 (having SEQ ID NO: 43)

[0289]

[0290] (Aminooxy)acetyl peptide 24 (having SEQ ID NO: 44)

[0291]

[0292] (Aminooxy)acetyl peptide 25 (having SEQ ID NO: 45)

[0293]

[0294] (Aminooxy)acetyl peptide 26 (having SEQ ID NO: 46)

[0295]

[0296] (Aminooxy)acetyl peptide 27 (having SEQ ID NO: 47)

[0297]

[0298] (Aminooxy)acetyl peptide 28 (having SEQ ID NO: 48)

[0299]

[0300] (Aminooxy)acetyl peptide 29 (having SEQ ID NO: 49)

[0301]

[0302] (Aminooxy)acetyl peptide 30 (having SEQ ID NO: 50)

[0303]

[0304] (Aminooxy)acetyl peptide 31 (having SEQ ID NO: 51)

[0305]

[0306] (Aminooxy)acetyl peptide 32 (having SEQ ID NO: 52)

[0307]

[0308] (Aminooxy)acetyl peptide 33 (having SEQ ID NO: 53)

[0309]

[0310] (Aminooxy)acetyl peptide 34 (having SEQ ID NO: 54)

[0311]

[0312] (Aminooxy)acetyl peptide 35 (having SEQ ID NO: 55)

[0313]

[0314] (Aminooxy)acetyl peptide 36 (having SEQ ID NO: 56)

[0315]

[0316] (Aminooxy)acetyl peptide 37 (having SEQ ID NO: 57)

[0317]

[0318] Conjugate 38

[0319]

[0320] Conjugate 38 was prepared and purified as described below for conjugate 39, except that the lipopeptide building block LBB-2 was used instead of LBB-1. Analytical UPLC (ACQUITY UPLC BEH C8, 1.7 μm, 2.1×150 mm, 10% to 90% MeCN in H 2 O (+0.1% TFA), over 45 minutes, 40° C.): purity 79%, t R =32.43 min. MALDI-MS: C 470 H 815 N 121 O 144 MW calculated for S7: 10689.8 Da; found: 10746.9

[0321] Conjugate 39

[0322]

[0323] To prepare conjugate 39, a solution of (aminooxy)acetyl peptide 4 (3 mg, 0.9 μmol) in 0.25 ml of 0.1 M sodium acetate buffer at pH 3.5 containing linker 8 (1.9 mg, 3.6 μmol) was added to 0.25 ml of 0.1 M sodium acetate buffer at pH 3.5. The mixture was stirred for 2.5 hours and the oxime was purified by RP-HPLC on a preparative C8 column. The intermediate was analyzed by analytical UPLC (ACQUITY UPLC BEH C8, 1.7 μm, 2.1×150 mm, 10% to 70% MeCN in H 2 O (+0.1% TFA), over 60 minutes, 70° C.): purity 95%, t R =16.59 min. ESI-MS: C 158 H 263 N 47 O 59The calculated MW of S4 is 3893.35 Da; the found MW is 3892.35 (± 0.01%). The oxime (2.0 mg, 0.5 μmol) was dissolved in 0.5 ml of H2O and added to a solution of the lipopeptide building block LBB-1 (3.1 mg, 0.5 μmol) in 2 ml of 50% MeCN. The pH was adjusted to pH = 6.5 with 0.1 N NaOH / 0.1 N HCl, and the mixture was stirred at room temperature for 2.5 hours. Conjugate 47 was purified by RP-HPLC on a C8 column. TFA was removed using AG-X2 anion exchange resin (acetate form). The conjugate was analyzed by analytical UPLC and MS. UPLC (ACQUITY UPLC BEH C8, 1.7 μm, 2.1×150 mm, 40% to 80% MeCN in H2O (+0.1% TFA), over 50 min, 40°C): purity 94%, t R = 20.75 min. ESI-MS: C 470 H 815 N 121 O 148 MW calculated for S7: 10753.81 Da; found: 10751.1 Da (±0.05%).

[0324] Example 3

[0325] Synthesis of malarial peptide antigens and conjugates comprising said peptides

[0326] Antigen 43

[0327]

[0328] The peptide sequence is taken from the NPNA repeat region of the circumsporozoite protein (CS) of the malarial parasite Plasmodium falciparum. An additional D-alanine-amide (denoted as "a-NH2") is added at the C-terminus to improve stability. A γ-maleimidobutyryl (GMB) group has been added to allow conjugation to cysteine ​​residues. Synthesis of antigen 43 was performed in substance and the product was purified by RP-HPLC as described in WO 2008 / 068017. Analytical reverse phase RP-HPLC (Zorbax C18 column, 5% to 30% MeCN in H2O (+0.1% TFA), within 25 min: purity >95%, tR = 13.02 min. LC-MS (Zorbax C18 column, 5% to 100% MeCN in H2O (+0.1% CHOOH), within 10 min: tR = 3.15 min; ESI-MS m / z = 746.5 [M+3H]3+

[0329] Conjugate 44

[0330]

[0331] To couple antigen 43 to lipopeptide 10, a stirred solution of 43 (2.7 mg, 1.2 μmol, 1.2 equiv) in H2O / MeCN 1:1 (1 ml, 2.7 mg, 1.2 μmol, 1.2 equiv) was added dropwise to a solution of lipopeptide 10 (6.8 mg, 1.0 μmol) in H2O / MeCN 1:1 (0.5 ml). The pH was carefully adjusted to pH 6.5 using 0.1 N NaOH, and the mixture was stirred for 3 hours. After completion of the coupling reaction, the mixture was diluted with H2O containing 0.1% TFA (2 ml), and the conjugate was purified by reverse phase RP-HPLC using a gradient of 50% to 100% MeCN / H2O (+0.1% TFA) on a C4 semi-preparative column (Interchrom) over 17 minutes. Analytical reverse phase RP-HPLC (Interchrom C4 column, 25% to 100% MeCN in H2O (+0.1% TFA) in 25 min): purity >97%, tR = 19.64 min; MALDI-TOF: C 403 H 686 N 106 O 120 Measured m / z value of S3: 9032.6: Found m / z value: 9032.2 [M+H]+.

[0332] Conjugate 45

[0333]

[0334] Synthesis and purification of conjugate 45 were performed essentially as described above for conjugate 44, except that LBB-1 was used instead of lipopeptide 10. Analytical reverse phase RP-HPLC (Interchrom C4 column, 25% to 100% MeCN in H2O (+0.1% TFA) over 25 min): purity >97%, tR = 19.64 min. MALDI-TOF: C 403 H 686 N 106 O 120 S3 m / z measured value: 9032.6: m / z found value: 9032.4 [M+H]+

[0335] Conjugate 46

[0336]

[0337] Synthesis and purification of conjugate 46 were performed essentially as described above for conjugate 44, except that LBB-2 was used instead of lipopeptide 10. UPLC (ACQUITY UPLC BEH C8, 1.7 μm, 2.1×150 mm, 10% to 90% MeCN in H 2 O (+0.1% TFA) over 10 min, 25° C.): >79% purity, t R = 35.76 min); MALDI-TOF: m / z calculated for C 4 O 3 H 6 B 7 N 1 O 7 O 12 B 3 S 3 : 9095.74; m / z found: 9096.6

[0338] Example 4

[0339] Avidity index of conjugates including malarial peptide antigens

[0340] For mouse immunogenicity studies, Balb / c mice (5 per group) were immunized sc with the formulations described in Table 2.

[0341] Table 2: Formulation

[0342] serial number describe dose adjuvant way 1 0.1 ml PBS containing 44 40 μg none SC 2 0.1 ml PBS containing 45 40 μg none SC 3 0.1 ml PBS containing 46 40 μg none SC

[0343] Immunizations were performed on days 0, 28, and 56. Blood was collected on day 66. Relative avidity indices were determined by KSCN elution ELISA using 2M KSCN for elution essentially as described (Perciani et al., J Clin Lab Anal 2007; 21(3):201-6).

[0344] IgG endpoint dilution ELISA was performed essentially as in WO 2008 / 068017. For KSCN elution ELISA (Nunc immunoplate Polysorb F96), the plates were coated overnight with 50 μl of 5 μg / ml antigen solution in PBS and then washed with 0.05% Wash 3 times with 150 μl PBS at 20°C and wash with two-fold serum dilutions in a solution containing 0.5% skim milk powder and 0.05% Replicates of PBS (50 μl per well) were incubated for 2.5 hours. The plates were washed three times with PBS at 20°C. Half of the wells were incubated with PBS (pH 7.2) containing 2M KSCN for 15 minutes, and the other half of the wells were incubated with plain PBS for the same amount of time. The plates were then washed and incubated with 50 μl of alkaline phosphatase-conjugated rabbit anti-mouse IgG antibody (Sigma, gamma chain specific) at room temperature in a 5% PBS solution containing 0.5% skim milk powder and 0.05% 20% PBS at a dilution of 1:20'000 for 1 hour, with PBS + 0.05% The plates were washed three times again and incubated in the dark at room temperature with 50 μl of a 1 mg / ml solution of p-nitrophenyl phosphate (Sigma) in 50 mM sodium carbonate, 1 mM MgCl2, pH 9.6. After the appropriate time, the absorbance at 405 nm was read on a SpectraMax M5 microplate reader. The avidity index of each serum was then calculated as the ratio of the area under the curve obtained for the KSCN-treated wells to the area under the curve obtained for the corresponding control (Perciani et al., J. Clin. Lab. Anal. 2007; 21(3): 201-6).

[0345] Table 3 and Figure 1 Shown are the mean log10 IgG ELISA endpoint dilution titers and mean avidity index ± one standard error of the mean.

[0346] Table 3: Average affinity index.

[0347]

[0348] The results showed that, without co-administration of an adjuvant, immunization with conjugate 45 comprising the heptad repeat motif IEKKIEA and the lipid Pam2Cys having an (R) configuration at the 2-propyl carbon resulted in higher affinity antibodies than immunization with 44 comprising a mixture of (R) and (S) diastereomers at the 2-propyl carbon, and further showed that immunization with conjugate 46 comprising IEKKIES instead of IEKKIEA resulted in a higher mean avidity index than 44 (P=0.0291) but a significantly lower endpoint dilution titer (P=0.0011).

[0349] Example 5

[0350] Avidity index of conjugates including RSV antigens

[0351] For mouse immunogenicity studies, Balb / c mice (5 per group) were immunized with the formulations in Table 4.

[0352] Table 4: Formulation

[0353] serial number describe dose adjuvant way 1 0.1 ml PBS containing 47 50 μg none SC 2 0.1 ml PBS containing 38 50 μg none SC

[0354] Immunizations were performed on days 0, 28, and 56. Blood was collected on day 66. Relative avidity indices were determined by KSCN elution ELISA using 2M KSCN for elution essentially as described (Perciani et al., J. Clin. Lab. Anal. 2007; 21(3):201-6).

[0355] Table 5 shows the mean affinity index ± one standard error of the mean.

[0356] Table 5: Average affinity index.

[0357]

[0358] Example 6

[0359] Immunogenicity and efficacy of conjugate 38 after two subcutaneous administrations in mice

[0360] For this experiment, 6-8 week old female BALB / c mice (10 animals per group) were immunized twice subcutaneously with 150 μg of conjugate 38 in 0.1 ml PBS (Table 6, No. 1). Two control groups (10 animals per group) were immunized twice with FI-RSV and PBS, respectively (Table 6, Nos. 2 and 3).

[0361] Table 6: Formulation

[0362] serial number describe dose adjuvant way 1 0.1 ml PBS containing 38 150 μg none SC 2 FI-RSV 1:100 none SC 3 PBS 0 none SC

[0363] Animals were immunized on day 0 and day 21. On day 42 (D42), all animals were bled and further treated with 10 6 pfu RSV A2 live virus intranasal challenge. Five days later, on day 47 (D47), animals were sacrificed, completely bled, and lungs were harvested and bisected for virus titration and histopathological analysis.

[0364] Serum from D0 and D42 of the group immunized with conjugate 38, FI-RSV and PBS (previously bled to attack animals) was analyzed by plaque reduction neutralization test (PRNT) for antibodies against the RSV A2 chain. The test serum was heat inactivated at 56°C for 30 minutes, diluted with EMEM at 1:10, and further serially diluted at 1:4. The diluted serum samples were incubated with an equal volume of RSV A2 (25-50 PFU) for 1 hour at room temperature and inoculated into the confluent HEp-2 monolayer in 24-well plates in duplicate. After incubation for one hour at 37°C in a 5% CO2 incubator, the wells were covered with 0.75% methylcellulose culture medium. After incubation for 4 days, the covering was removed, and the cells were fixed and stained with 0.1% crystal violet for one hour, and then rinsed and air-dried. Neutralization titers were calculated as the reciprocal serum dilution required to achieve a 60% reduction in plaques compared to the virus control wells. The Log2 viral PRNT titers of RSV A2 are summarized in Table 7.

[0365] Table 7: Introduction of neutralizing antibodies.

[0366]

[0367] Two immunizations with conjugate 38 induced high titers of neutralizing antibodies at a dose of 150 μg. FI-RSV and PBS resulted in neutralizing antibody titers below the detection limit (<4.32).

[0368] For virus titration, lung homogenate from D47 was clarified by centrifugation and diluted in EMEM. Converging HEp-2 monolayers were infected in duplicate in 24-well plates with diluted homogenate. After incubation at 37°C for one hour in a 5% CO2 incubator, the wells were covered with 0.75% methylcellulose culture medium. After incubation for 4 days, the covering was removed, and the cells were fixed and stained with 0.1% crystal violet for one hour, and then rinsed and air-dried. Plaques were counted, and the plaque forming units per gram of tissue were calculated. The geometric mean viral titer of the D47 (5 days after the attack) sample is summarized in Table 8 below.

[0369] In order to carry out lung histopathological analysis, as mentioned above, lungs are dissected, formalin fixed and embedded in paraffin. With hematoxylin and eosin (H&E) lung sections are dyed, to be used for analyzing four parameters of lung inflammation: peribronchitis (peribronchiolitis), perivasculitis (perivasculitis), interstitial pneumonia (interstitialpneumonia) and alveolitis (alveolitis). With 0-4 severity scale, slides are blindly scored. Subsequently, scoring is converted into 0-100% histopathological scale. The average pathological score of D47 sample is summarized in the following table 8.

[0370] Table 8: Lung virus titers and pathological scores on day 47.

[0371]

[0372] All animals immunized with PBS showed the maximum viral titer in the lungs five days after the attack (average titer of about 4.76Log10PFU / g). Animals immunized with 150 μg of conjugate 38 showed a strong reduction in viral titer (average titer of about 2.58Log10PFU / g), and the lungs of most animals had undetectable virus (Log10 titer≤2.6PFU / g). Animals immunized with FI-RSV showed less protection (average titer of about 3.95Log10PFU / g). On the histopathological level, animals immunized with PBS depicted the pathology typically associated with primary RSV infection. Animals vaccinated with FI-RSV showed strong pathology, which suggests that the vaccine-related disease enhances. All animals immunized with conjugate 38 showed no signs of vaccine-related disease enhancing, and pathological scores were equal to or lower than those of PBS control animals (Table 8).

[0373] The results showed that two subcutaneous immunizations with conjugate 38 without co-administration of an adjuvant were sufficient to elicit high titers of protective neutralizing antibodies to protect against RSV A2 viral replication in the lungs, and that immunization with conjugate 38 did not cause enhancement of respiratory disease.

Claims

1. A lipopeptide building block consisting of: (i) a peptide portion comprising a coiled-coil peptide segment, wherein the coiled-coil peptide segment consists of the sequence (IEKKIES)4 (SEQ ID NO: 62); (ii) a lipid portion comprising formula LM-I where R 1 and R 2 Independently C 11-15 alkyl; and wherein R 3 is hydrogen or -C(O)C 11-15 alkyl; And wherein the lipid portion is linked to the peptide portion, wherein the wavy line in formula LM-I indicates the site of connection to the peptide portion.

2. The lipopeptide building block according to claim 1, wherein said R 1 and R 2 Independently -C 11 H 23 、-C 13 H 27 or -C 15 H 31 .

3. The lipopeptide building block of claim 1, wherein said R 1 and R 2 -C 15 H 31 .

4. The lipopeptide building block of claim 1, wherein said R 3 H or -C(O)C 15 H 31 .

5. The lipopeptide building block of claim 1, wherein the lipid portion is linked to the N-terminus of the peptide portion.

6. The lipopeptide building block of claim 1 , wherein the lipid portion consists of the formula LM-I* where R 3 is hydrogen or -C(O)C 11-15 alkyl.

7. The lipopeptide building block according to claim 6, wherein said R 3 H or -C(O)C 15 H 31 .

8. A lipopeptide building block according to claim 6 or claim 7, wherein the lipid portion is linked to the N-terminus of the peptide portion.

9. The lipopeptide building block of claim 1 , wherein the lipid portion is composed of the formula LM-I*1 10. The lipopeptide building block of claim 1, wherein the lipid portion and the peptide portion are linked directly or through a coupling moiety.

11. The lipopeptide building block of claim 10, wherein the coupling moiety is an amino acid linker.

12. The lipopeptide building block according to claim 11, wherein the amino acid linker consists of 2 to 15 amino acids.

13. The lipopeptide building block according to claim 11, wherein the amino acid linker consists of 2 to 10 amino acids.

14. The lipopeptide building block according to claim 11, wherein the amino acid linker consists of 2 to 5 amino acids.

15. The lipopeptide building block of claim 1, wherein the peptide portion further comprises a T helper cell epitope.

16. The lipopeptide building block of claim 15, wherein the T helper cell epitope comprises a sequence selected from the group consisting of: (i) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87; and (ii) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87, wherein one, two or three amino acids are exchanged for other amino acids or are deleted.

17. The lipopeptide building block of claim 15, wherein the T helper cell epitope consists of a sequence selected from the group consisting of: (i) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87; and (ii) SEQ ID NO: 6, SEQ ID NO: 63 to SEQ ID NO: 87, wherein one, two or three amino acids are exchanged with other amino acids or are deleted.

18. The lipopeptide building block of claim 1, wherein the lipopeptide building block has the formula LBB-2 19. A conjugate comprising: (a) a lipopeptide building block according to any one of claims 1 to 8; as well as (b) antigens, The antigen is linked to the lipopeptide building block directly or through a linker.

20. The conjugate of claim 19, wherein the antigen is a tumor antigen, an autoantigen, a polypeptide of a pathogen, an allergen, or a hapten.

21. The conjugate according to claim 19, wherein the conjugate is selected from any one of formula (38), (40), (41) or (42) 22. The conjugate of claim 19, wherein the conjugate has formula (38) 23. A bundle of conjugates comprising 2, 3, 4, 5, 6 or 7 conjugates according to any one of claims 20 to 22.

24. A bundle of conjugates comprising 2, 3, 4 or 5 conjugates according to any one of claims 20 to 22.

25. A bundle of conjugates, comprising three conjugates according to any one of claims 20 to 22.

26. The bundle of any one of claims 23 to 25, wherein in the bundle the coiled-coil peptide segments of the peptide moieties form a left-handed α-helical coiled-coil, wherein the coiled-coil peptide segments have a parallel orientation in the coiled-coil.

27. A synthetic virus-like particle comprising at least one bundle of the conjugate according to any one of claims 23 to 26.

28. A pharmaceutical composition comprising an immunologically effective amount of the conjugate according to any one of claims 20 to 22 or the synthetic virus-like particle according to claim 27 together with a pharmaceutically acceptable diluent, carrier or excipient.

29. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is a vaccine.

30. Use of the conjugate according to any one of claims 20 to 22, the synthetic virus-like particle according to claim 27, or the pharmaceutical composition according to claim 28 or claim 29 in the preparation of a vaccine.

31. Use of the conjugate according to any one of claims 20 to 22, the synthetic virus-like particle according to claim 27, or the pharmaceutical composition according to claim 28 or claim 29 in the preparation of a vaccine for preventing a disease or for reducing the risk of a disease.

32. The use according to claim 31, wherein the disease is an infectious disease, cancer or allergy.

33. The use according to claim 31, wherein the disease is respiratory syncytial virus (RSV) infection.

34. A conjugate of formula (39) 35. A bundle of conjugates comprising 2, 3, 4, 5, 6 or 7 conjugates according to claim 34.

36. A bundle of conjugates comprising 2, 3, 4 or 5 conjugates according to claim 34.

37. A bundle of conjugates, comprising three conjugates according to claim 34.

38. The bundle of any one of claims 35 to 37, wherein in the bundle the coiled-coil peptide segments of the peptide moieties form a left-handed alpha-helical coiled-coil, wherein the coiled-coil peptide segments have a parallel orientation in the coiled-coil.

39. A synthetic virus-like particle comprising at least one bundle of the conjugate according to any one of claims 35 to 38.

40. A pharmaceutical composition comprising an immunologically effective amount of the conjugate of claim 34 or the synthetic virus-like particle of claim 39 together with a pharmaceutically acceptable diluent, carrier or excipient.

41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is a vaccine.

42. Use of the conjugate according to claim 34, the synthetic virus-like particle according to claim 39, or the pharmaceutical composition according to claim 40 or claim 41 in the preparation of a vaccine.

43. Use of the conjugate of claim 34, the synthetic virus-like particle of claim 39, or the pharmaceutical composition of claim 40 or claim 41 in the preparation of a vaccine for preventing a disease or for reducing the risk of a disease.

44. The use according to claim 43, wherein the disease is an infectious disease, cancer or allergy.

45. The use according to claim 43, wherein the disease is respiratory syncytial virus (RSV) infection.

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