Lipopeptide building blocks and aggregates

Abstract

Provided herein are a lipopeptide building block comprising a lipid moiety, an amphipathic domain, a hydrophilic polymer block, a protease cleavage site, and lipopeptide constructs, lipopeptide aggregates and immunogenic compositions comprising the same.

Description

LIPOPEPTIDE BUILDING BLOCKS AND AGGREGATES 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 650,122 filed May 21, 2024; and 63 / 802,423 filed May 8, 2025, the disclosure of each of which is incorporated by reference herein in its entirety. 2. SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14671-003-228_SEQLISTING.xml”, was created on May 16, 2025, and is 136,552 bytes in size. 3. FIELD

[0003] The present disclosure relates generally to lipopeptides, aggregates, nanoparticles and immunogenic compositions comprising the same for inducing an immune response in a subject to treat a disease, disorder, or condition. 4. BACKGROUND

[0004] Subunit vaccines and active immunotherapies hold significant potential for preventing and treating infectious and non-communicable diseases. However, despite great potential, only relatively few subunit vaccines have been licensed for use in humans to date. A significant challenge is that subunit vaccines often lack sufficient immunogenicity. Various strategies have been explored to increase the immunogenicity of subunit vaccines including co-administration of exogenous adjuvants (see, e.g., Perrie,Y., et al., Int. J. Pharm.364, 272- 280 (2008); Leroux-Roels, Geert, Vaccine 28S C25-C36 (2010) Facciolà, A. et al, Vaccines.10, 819 (2022)). However, exogenous adjuvants are associated with side-effects safety concerns due to an unspecific immune activation leading to induction of autoimmune diseases.

[0005] Other approaches focus on improving the delivery and presentation of the antigens in subunit vaccines. These approaches may promote trapping and retention of the antigens in local lymph nodes, as well as antigen presentation by antigen presenting cells. Such approaches include association with carrier proteins well known in the art, such as KLH, DT,TT, CRM197, or Protein D (see, e.g., EP 0594610) or particles (microparticles, nanoparticles), virus-like particles (VLPs) including phage particles, HBV core particles, HPV particles, and other VLPs, as well as liposomes, virosomes, ISCOMS (see, e.g., WO 00 / 3227, WO02 / 056905, WO 00 / 198333, WO 00 / 177158, WO 00 / 214478, WO 00 / 32227, WO 01 / 85208, WO 02 / 056905, WO 03 / 024480, WO 03 / 024481, WO 2016 / 112921, WO 92 / 19267, WO 2009 / 000433, WO 96 / 11711, WO 96 / 33739, EP 0109942), and delivery via attenuated viral or bacterial carriers acting as vectors or in nucleic acid vaccines such as adenoviruses, adeno-associated viruses (AAVs), measles, alphaviruses, and other vectors (see, e.g., WO 2001 / 02607, WO 2002 / 22080, WO 2019 / 086461, WO 2005 / 033321, WO 97 / 06270, and WO 2003 / 023026). Synthetic Virus-like Particles (SVLPs) have also been utilized for vaccine development. Specifically, SVLP-based vaccine candidates using synthetic lipopeptide building blocks to which antigens are conjugated have been developed (see, e.g., WO2015 / 082501, WO2018 / 229159, WO2020 / 127728, WO2022063990A, WO2021260176A1). Additionally, certain lipopeptides have been developed that include T- helper epitopes and coiled-coil domains consisting of 3-8 heptad repeats and self-assemble into SVLPs, where the stability is thought to increase with the number of heptad repeats (see, e.g., WO2008 / 068017 and WO2020 / 127728). Lipid nanoparticles (LNPs) including particles containing mixtures of lipids and short amphipathic peptides have also been developed for delivery of antigens (see, e.g., WO 2010 / 009277) and mRNA vaccines (see, e.g., WO 2020 / 219941, WO 2021 / 123332, and WO 2022 / 099003). All these approaches have certain limitations. Coupling antigens to LNPs, carrier proteins, and other carriers typically often results in variable products that elicit variable antibody responses. The production and purification of LNPs, liposomes, virosomes, and lipopeptides is highly complex due to the hydrophobic nature of the components, which may lead to unwanted particle aggregation during formulation, filling, and shipping, and may complicate purification processes and affect stability. Many proteins, vectors VLPs, and other carriers are immunogenic themselves, and resulting carrier-specific immune responses may impact the immunogenicity of other conjugate vaccines using the same carrier that are administered subsequently (see, e.g., McCluskie MJ, et al., Immunopharmacol Immunotoxicol.2016 Jun;38(3):184-96). There is, therefore, still a need for further, more polar, more accessible, and even more effective antigen delivery systems.5. SUMMARY

[0006] In one aspect, provided herein is a lipopeptide comprising: i) a lipid moiety, ii) an amphipathic domain; iii) a hydrophilic polymer block; and iv) a protease cleavage site, optionally wherein the amphipathic domain is an amphipathic peptide.

[0007] In one embodiment, the lipopeptide comprises the following moieties in the stated order, wherein the moieties are covalently connected to each other: (a) a lipid moiety; (b) an aminohexanoic acid linker; (c) an amphipathic peptide; (d) a hydrophilic polymer block; (e) a protease cleavage site; and (f) a T-helper epitope.

[0008] In one embodiment, the lipopeptide comprises the following moieties in the stated order, wherein the moieties are covalently connected to each other: (a) a lipid moiety; (b) a linker; (c) an amphipathic peptide of less than 20 amino acids; (d) a hydrophilic polymer block; (e) a protease cleavage site; and (f) a T-helper epitope.

[0009] In one embodiment, the lipopeptide comprises the following moieties in the stated order, wherein the moieties are covalently connected to each other: (a) a lipid moiety; (b) a linker; (c) an amphipathic peptide; (d) a hydrophilic polymer block consisting of between 10 to 19 repeat units; (e) a protease cleavage site; and (f) a T-helper epitope.

[0010] In one embodiment, the lipopeptide comprises the following moieties in the stated order, wherein the moieties are covalently connected to each other: (a) a lipid moiety; (b) a linker; (c) an amphipathic peptide;(d) a hydrophilic polymer block; (e) a protease cleavage site cleavable in the endolysosomal system of an antigen presenting cell; and (f) a T-helper epitope.

[0011] In certain embodiments, the order of the components a. to f. are in the amino- to carboxy-terminus direction of the peptide components.

[0012] In certain embodiments, the lipid moiety is a Toll-like receptor (TLR) agonist. In certain embodiments, the TLR agonist is a TLR-2 agonist.

[0013] In certain embodiments, the lipid moiety has a structure selected from the group consisting of LM 1, LM 2, LM 3, LM 4, LM 5, LM 6, LM 7, and LM 8. In certain embodiments, the lipid moiety is selected from the group consisting of Cys((R)-2,3- di(palmitoyloxy)-2-propyl); Cys((S)-2,3-di(palmitoyloxy)-2-propyl); Cys((R)-2,3- di(palmitoyloxy)-2-propyl-N-palmitoyl); Cys((R)-2,3-di(dilauroyloxy)-2-propyl-N- palmitoyl); unsaturated Pam3Cys; unsaturated Pam2Cys; N,N’-dipalmitoyl-2,3-diamino- propionamide; phosphatidylethanolamine; diphosphoryl hexaacyl lipid A; 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine; and 1,3-dipalmitoyl-glycero-2-phosphoethanolamine. In certain embodiments, the lipid moiety is Pam2Cys.

[0014] In certain embodiments, the linker is a peptide linker. In certain embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-163. In certain embodiments, the linker comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-163. In certain embodiments, the linker consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-163.

[0015] In certain embodiments, the amphipathic peptide is a coiled-coil domain. In certain embodiments, the coiled-coil domain directs formation of helical bundles. In certain embodiments, the coiled-coil domain comprises a heptad motif represented by (abcdefg)n, wherein each a and d independently represent a hydrophobic residue, each b, c, e, f, and g independently represent a polar residue, and n is 1-10.

[0016] In certain embodiments, the amphipathic domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-69. In certain embodiments, the amphipathic peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-69. In certain embodiments, the amphipathic peptide:(a) comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; or (b) comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; or (c) consists of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0017] In certain embodiments, the amino acid sequence (e.g., of the amphipathic peptide or linker) is chemically modified.

[0018] In certain embodiments, the hydrophilic polymer block comprises poly(ethylene glycol), poly(vinyl alcohol), poly(N-vinylpyrrolidone), polysarcosine, polyglutamic acid, poly(hydroxyethyl-L-asparagine), poly(hydroxyethyl-L-glutamine). In certain embodiments, the hydrophilic polymer block comprises poly(ethylene glycol).

[0019] In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by intracellular proteases. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by an asparaginyl endopeptidase. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a cathepsin protease. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a protease selected from the group consisting of legumain, cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin P, cathepsin S, and cathepsin W. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by cathepsin S. In certain embodiments, the protease cleavage site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86. In certain embodiments, the protease cleavage site comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86. In certain embodiments, the protease cleavage site consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86.

[0020] In certain embodiments, the T-helper epitope is selected from the group consisting of a flu, hepatitis B, tetanus toxin, diphtheria toxin, measles and covid T-helper epitope. Incertain embodiments, the T-helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-118. In certain embodiments, the T-helper cell epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-118.

[0021] In certain embodiments, the lipopeptide as described above further comprises one or more antigen epitopes. In certain embodiments, the one or more antigen epitopes comprises at least one of a B-cell epitope, CD4+ T-cell epitope, or CD8+ T-cell epitope. In certain embodiments, the one or more antigen epitopes comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124. In certain embodiments, the one or more antigen epitopes comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124. In certain embodiments, the one or more antigen epitopes consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124.

[0022] In another aspect, the present disclosure provides a lipopeptide aggregate comprising at least two lipopeptides as described above. In certain embodiments, the lipopeptide aggregate comprises an antigen, as described above. In certain embodiments, the lipopeptide aggregate is a micelle.

[0023] In another aspect, the present disclosure provides a nanoparticle comprising a lipopeptide as described above. In certain embodiments, the nanoparticle is self-assembled from the lipopeptide. In certain embodiments, a plurality of the lipopeptide aggregates or nanoparticles: (a) has a solubility in PBS buffer of at least 25 mg / mL as determined by laser nephelometry; and / or (b) has a particle size distribution in the range of about 15 nm to about 75 nm, preferably about 20 nm to about 40 nm as determined by dynamic light scattering (DLS) or electron microscopy; and / or (c) has a homogeneity of about 0.95 to about 1.15 as indicated by polydispersity index (Mw / Mn) as assessed by multi-angle light scattering; and / or (d) has a melting point (Tm) of at least about 66.72 °C as assessed by differential scanning calorimetry analysis (DSC); and / or (e) has an average spacing of the one or more antigen epitopes of about 5 nm to about 10 nm, as determined or predicted by one or more of crystallography, NMR spectroscopy, molecular modeling, and mutagenesis analysis; and / or(f) has a spacing of the one or more antigen epitopes that allows B-cell receptor (BCR) cross-linking, as predicted by cross-linking model (CLM).

[0024] In another aspect, the present disclosure provides a method of inducing generation of antibodies in a subject, wherein the method comprises administering the lipopeptide aggregate or nanoparticle as described above to the subject such that the subject generates antibodies against the antigen. In certain embodiments, the antigen is a self-antigen, and optionally wherein the self-antigen is PCSK9. In certain embodiments, the method reduces B- cell self-tolerance in the subject.

[0025] In another aspect, the present disclosure provides an immunogenic composition comprising a plurality of the lipopeptide aggregates or nanoparticles described above. In certain embodiments, the immunogenic composition further comprises an adjuvant. In certain embodiments, the adjuvant is selected from the group consisting of aluminum, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), monophosphoryl lipid A (MPL), squalene, cytosine phosphoguanine, QS-21, microcrystalline tyrosine, MF59, mannide monooleate, GLA, SLA, E6020, resiquimod (R848), imidazoquinolines, imiquimod, 3M-052, 2',3'-cGAMP, 3',3'- cGAMP, cGMP, cAMP, AMP, muramyl dipeptide, poly I:C, CpG and combinations thereof.

[0026] In certain embodiments, the lipopeptide aggregate, nanoparticle, or immunogenic composition induces an immune response. In certain embodiments, the lipopeptide aggregate, nanoparticle, or immunogenic composition induces antibodies against at least a portion of the lipopeptide or nanoparticle. In certain embodiments, the lipopeptide aggregate, nanoparticle, or immunogenic composition induces CD4+ T-cells against at least a portion of the lipopeptide or nanoparticle. In certain embodiments, the lipopeptide aggregate, nanoparticle, or immunogenic composition induces CD8+ T-cells against at least a portion of the lipopeptide or nanoparticle. 6. BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG.1 shows a schematic representation of a lipopeptide construct and lipopeptide aggregate comprising i) a lipid moiety (LM), ii) an amphipathic domain (AD), iii) a hydrophilic polymer block (HP), iv) a protease cleavage site (P), a T-helper cell epitope (TH) and an antigen epitope (A).

[0028] FIG.2A and 2B depict Lipopeptide Construct 1 and Lipopeptide Construct 2, respectively.

[0029] FIG.3 shows the results of the solubility assessment as described in the Examples.

[0030] FIG.4 depicts a transmission electron microscopy (TEM) image demonstrating that the lipopeptide aggregate particles are highly homogenous.

[0031] FIG.5 shows IgG Enzyme-Linked Immunosorbent Assay (ELISA) curves for sera of C57BL / 6 mice injected one (1°) or two (2°) times with Lipopeptide Construct 2 or vehicle (control). Shown are the mean signals ± error.

[0032] FIG.6 shows IgG endpoint titers (EPT) in sera from mice injected one or two times with Lipopeptide Construct 2 in PBS or vehicle (control). Shown are the individual endpoint dilution titers (circles) and the geometric mean values (horizontal bars).

[0033] FIG.7 shows (A) low density lipoprotein receptor (LDLR) expression in liver, (B) serum PCSK9 levels, and (C) total cholesterol (TC) levels from mice injected on Day 0, 21, and 42 with Lipopeptide Construct 2 (LPN) in PBS or Vehicle (control); (D) a Comparison of TC levels from day 0 versus Day 56 in LPC-2 group; and (E) Comparison of TC levels from day 0 vs 56 in vehicle group; ** P < 0.005; * P < 0.05; ns = not significant. 7. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure relates to a lipopeptide construct comprising 1) a lipopeptide building block (BB) (see Section 7.1 herein) comprising a lipid moiety (see Section 7.1.1 herein), an amphipathic domain (AD) (see Section 7.1.2 herein), a hydrophilic polymer block (HP) (see Section 7.1.3 herein), and a protease cleavage site (P) (see Section 7.1.4 herein), and optionally one or more antigens (A) (see Section 7.3 herein). In certain embodiments, the lipopeptide has the structure: BB-A, where BB represents the lipopeptide building block and A represents the one or more antigens. In certain embodiments, the lipopeptide has the structure: LM-AD-HP-P-A, wherein LM represents the lipid moiety, AD represents the amphipathic domain, HP represents the hydrophilic polymer block, P represents the protease cleavage site, and A represents the one or more antigens. In certain embodiments, the lipid moiety and the amphipathic domain are connected, either directly or indirectly through a linker (L) (see Section 7.4 herein). In certain embodiments, the lipopeptide building block and the antigen are connected, either directly or indirectly through a linker (L) (see Section 7.4 herein). Accordingly, in certain embodiments, the lipopeptide construct has the structure BB-L-A. In certain embodiments, the lipopeptide has the structure: LM-L-AD-HP-P-L-A, where each instance of linker (L) is optional. In certain embodiments, the lipopeptide building block further comprises a T-helper epitope (TH) (see Section 7.2herein), which can be connected, either directly or indirectly through a linker (L), to the one or more antigens. Accordingly, in certain embodiments, the lipopeptide construct has the structure LM-L-AD-HP-P-TH-L-A, where each instance of linker (L) is optional. In certain embodiments, the lipopeptide construct has the structure LM-L-AD-HP-P-TH-L-A, wherein the order of components is in the amino- to carboxy-terminus direction of the peptide components. FIG.1 shows a schematic representation of a lipopeptide construct and lipopeptide aggregate comprising i) a lipid moiety (LM), ii) an amphipathic domain (AD), iii) a hydrophilic polymer block (HP), iv) a protease cleavage site (P), a T-helper cell epitope (TH) and an antigen (A). As will be described in Section 7.3 herein, the lipopeptide construct may comprise one or more antigens, which may be the same or different (e.g., dual or multi- target). In certain embodiments, the lipopeptide construct can comprise the sequences set forth in Table 8 herein (see Section 7.6 herein).

[0035] In certain embodiments, the linker is a C3-10alkylamino acid, e.g., an e-amino caproic acid linker. In certain embodiments, the amphipathic domain is an amphipathic peptide. In certain embodiments, the amphipathic peptide has less than 20 amino acids. In certain embodiments, the hydrophilic polymer block consists of between 10-19 repeat units. In certain embodiments, the protease cleavage site is cleavable by proteolytic enzymes present in the endolysosomal system of cell, e.g., an antigen presenting cell.

[0036] In certain embodiments, a plurality of lipopeptide constructs provided herein are combined to form a lipopeptide aggregate. In certain embodiments, the plurality of lipopeptide constructs self-assemble to form lipopeptide aggregates. In certain embodiments, the lipopeptide aggregate is a micelle. In certain embodiments, the lipopeptide aggregate is a nanoparticle, or nanomicelle.

[0037] The present disclosure additionally provides an immunogenic composition comprising the lipopeptide aggregates (see Section 7.7 herein) and methods of using the lipopeptide aggregates (see Section 7.8 herein). 7.1. Lipopeptide Building Block

[0038] In certain embodiments, the lipopeptide construct comprises a lipopeptide building block. In certain embodiments, the lipopeptide building block comprises a lipid moiety as provided in Section 7.1.1 herein, an amphipathic domain as provided in Section 7.1.2 herein, a hydrophilic polymer block as provided in Section 7.1.3 herein, and a protease cleavage site as provided in Section 7.1.4 herein.7.1.1. Lipid Moiety

[0039] In certain embodiments, the lipid moiety comprises or consists of a Toll-like receptor (TLR) agonist, for example, a TLR-1, TLR-2, TLR-4, TLR-6, or TLR-10 agonist. In certain embodiments, the lipid moiety comprises or consists of a TLR-2 agonist.

[0040] In certain embodiments, the lipid moiety comprises hydrocarbyl chains. In certain embodiments, the lipid moiety comprises one or more hydrocarbyl chains. In certain embodiments, the lipid moiety comprises two hydrocarbyl chains. In certain embodiments, the lipid moiety comprises three hydrocarbyl chains. In certain embodiments, the lipid moiety is a phospholipid. The phospholipid can comprise one or more of ester- or ether-linked extended alkyl or alkenyl side chains, including a combination thereof. In certain embodiments, the phospholipid comprises enantiomers of 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine. In certain embodiments, the phospholipid comprises achiral analogues, including, but not limited to, 1,3-dipalmitoyl-glycero-2-phosphoethanolamine.

[0041] In certain embodiments, the lipid moiety is selected from one of Formula LM 1 to LM 8:, wherein R1and R2in formulas LM 1 and LM 2 are, independently of each other, hydrocarbyl or hydrocarbyl-C=O, and Y is H or COOH;,wherein R1, R2and R in formula LM 3 are independently of each other hydrocarbyl or hydrocarbyl-C=O; or R1and R2are independently of each other hydrocarbyl or hydrocarbyl- C=O, and R is H or acetyl or lower alkyl-C=O;, wherein R1and R2in formulas LM 4 and LM 5 are independently of each other hydrocarbyl or hydrocarbyl-C=O , and n is 1, 2, 3, or 4;, wherein R1and R2in formula LM 6 are independently of each other a hydrocarbyl, X is O or NH, and n is 1, 2, 3, or 4, or, wherein R1and R2in formulas LM 7 and LM 8 are independently of each other hydrocarbyl.

[0042] In certain embodiments, the lipid moiety is a di-palmitoyl-S-glycerylcysteinyl of formula LM 3, wherein R1and R2are each palmitoyl, and R3is H or acetyl.

[0043] In certain embodiments, each hydrocarbyl or hydrocarbyl chain, which are used synonymously herein, are independently a straight or branched alkyl chain consisting of at least 7 carbon atoms, between 8 and 50 carbon atoms, or between 8 and 25 carbon atoms, or a straight or branched alkenyl chain consisting of at least 7 carbon atoms, between 8 and 50 carbon atoms, or between 8 and 25 carbon atoms with one, two, or three double bonds. Alkenyl chains have one, two or three double bonds in the chain, each with E or Z geometry, as is customarily found in natural fatty acids and fatty alcohols. Also included in the definition of "hydrocarbyl" or "hydrocarbyl chain" are branched alkyl or alkenyl chains, for example an alkyl bearing a methyl or ethyl substituent at the second or third carbon atom counted from the end of the chain, as e.g., as in 2-ethyl-hexyl. The term, "lower alkyl" means alkyl chain with 1-7 carbon atoms, including, for example, 1-4 carbon atoms, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, or tert-butyl.

[0044] In certain embodiments, the lipid moiety iswherein represents attachment to the rest of the lipopeptide building block.

[0045] In certain embodiments, the lipid moiety is:wherein represents attachment to the rest of the lipopeptide building block.

[0046] In certain embodiments, the lipid moiety is:, wherein represents attachment to the rest of the lipopeptide building block and R3is -C(O)C11-15alkyl or –C(O)C15H31.wherein represents attachment to the rest of the lipopeptide building block and R3is -C(O)C11-15alkyl or –C(O)C15H31.wherein represents attachment to the rest of the lipopeptide building block.

[0049] In certain embodiments, the lipid moiety is:wherein represents attachment to the rest of the lipopeptide building block.wherein represents attachment to the rest of the lipopeptide building block.

[0051] In certain embodiments, the lipid moiety is selected from the group consisting of Cys((R)-2,3-di(palmitoyloxy)-2-propyl) (also referred to as Pam2Cys or dipalmitoyl-S- glyceryl cysteine); Cys((S)-2,3-di(palmitoyloxy)-2-propyl); Cys((R)-2,3-di(palmitoyloxy)-2- propyl-N-palmitoyl) (also referred to as Pam3Cys or tripalmitoyl-S-glyceryl cysteine); Cys((R)-2,3-di(dilauroyloxy)-2-propyl-N-palmitoyl); unsaturated Pam3Cys; unsaturated Pam2Cys; N,N’-dipalmitoyl-2,3-diamino-propionamide (also referred to as Pam2Dap), phosphatidylethanolamine; diphosphoryl hexaacyl lipid A; 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine; and 1,3-dipalmitoyl-glycero-2-phosphoethanolamine.

[0052] In certain embodiments, the lipid moiety comprises Pam2Cys. In certain embodiments, the lipid moiety consists of Pam2Cys. In certain embodiments, the lipid moiety comprises Pam3Cys. In certain embodiments, the lipid moiety consists of Pam3Cys. 7.1.2. Amphipathic Domain

[0053] The amphipathic domain can be a peptide comprising one or more repeat units of alternating hydrophobic residues (X) and hydrophilic residues (Y) with a general pattern of XmYnXoYp, wherein m is 1-5, n is 1-5, o is 1-5, and p is 0-5. In certain embodiments, the amphipathic domain comprises 20 or fewer amino acids, e.g., 3-20, 5-20, 10-20, or 15-20 amino acids. In certain embodiments, the amphipathic domain comprises less than 20 amino acids (e.g., the sum of m, n, o, and p is less than 20), such as 3-19, 5-19, 10-19, 15-19, or 17- 19 amino acids. In certain embodiments, the amphipathic domain has 19 amino acids.

[0054] In certain embodiments, the amphipathic domain has an amphipathic alpha helical structure with opposing polar and nonpolar faces oriented along the long axis of the helix. In certain embodiments, the amphipathic domain has amphipathic beta strand structure in which hydrophobic side chains point in one direction and polar side chains in the other. In certain embodiments, the amphipathic domain directs formation of helical bundles. In certain embodiments, the amphipathic domain directs formation of a coiled-coil, and therefore is or comprises a coiled-coil. In certain embodiments, the amphipathic domain directs formation of an amphipathic beta sheet with at least two parallel beta strands in which hydrophobic side chains point in one direction and polar side chains in the other.

[0055] In certain embodiments, the amphipathic domain is a peptide structure comprising one chain. In certain embodiments, the amphipathic domain is a peptide structure comprising at least two chains. In certain embodiments, the amphipathic domain comprises parallel beta strands. In certain embodiments, the amphipathic domain comprises at least two peptidechains are associated into a bundle. In certain embodiments, the at least two peptide chains each have an alpha helical secondary structure and are associated into a bundle. Accordingly, in certain embodiments, the amphipathic domain comprises, or consists of, a coiled-coil domain. In certain embodiments, the coiled-coil domain directs formation of helical bundles. Various amphipathic domains, including coiled-coil domains, that are suitable in certain embodiments provided herein are provided in WO 2008 / 068017, WO 2015 / 082501, WO 2018 / 229156, WO 2020 / 127728, WO 2009 / 109428, WO 2011 / 112999, US 9,044,514, US 9,994,646, US 7,811,577, and US 8,728,785, each of which are incorporated herein by reference in their entirety.

[0056] In certain embodiments, the amphipathic domain comprises multiple repeat units consecutively connected to each other. The repeat units of the amphipathic domain segment may be identical or may be different, e.g., may contain at least one discontinuity, such as an insertion, deletion, or exchange of at least one amino acid within the repeat unit. In certain embodiments, 1, 2, 3, or 4 amino acids within the repeat unit may be inserted, deleted, or exchanged.

[0057] In certain embodiments, the amphipathic domain comprises or consists of 2-10 repeat units including 2, 3, 4, 5, 6, 7, 8, 9, and 10 repeat units, 3-8 repeat units including 3, 4, 5, 6, 7, and 8 repeat units, or 4 repeat units.

[0058] The amphipathic domain can be based on canonical repeat units and non-canonical repeat units. In certain embodiments, the amphipathic domain comprises canonical tandem heptad repeats that may form right-handed amphipathic alpha-helices, which then assemble to form helical bundles with left-handed coiled-coils. In certain embodiments, the coiled-coil domain comprises non-canonical, non-heptad-based repeats that form coiled-coils that are not necessarily left-handed or even regular supercoils.

[0059] Repeat units of amphipathic domains have a sequence with a certain number of amino acids, wherein the positions of the amino acids are labelled as lowercase letters. In certain embodiments, the repeat unit of the coiled-coil domains consists of 7-15 amino acids or 7-11 amino acids. In certain embodiments, the coiled-coil domain comprises or consists of 7 amino acids, wherein the seven amino acid positions are designated with letters a, b, c, d, e, f, and g. In certain embodiments, the heptad motif includes amino acids having hydrophobic residues at positions a and d, and polar, helix-favoring residues at the other residues. In certain embodiments, each of a and d can independently be selected from Ala, Ile, Leu, Met, Val, Phe, Trp, and Tyr. In certain embodiments, residues a and d can each independently beGln or Asn. In certain embodiments, residues a and d each do not comprise Asn or Gln. In certain embodiments, residues b, c, e, f, and g are selected from Ala, Glu, Lys, and Gln.

[0060] In certain embodiments, the amphipathic domain comprises or consists of 2-10 repeat units or 3-8 repeat units. In certain embodiments, the amphipathic domain comprises or consists of 2 repeat units. In certain embodiments, the amphipathic domain comprises or consists of 20, 15, 11, or 7 amino acids. In certain embodiments, the amphipathic domain consists of 7 amino acids (heptad motif).

[0061] In certain embodiments, the amphipathic domain comprises 2-10 tandemly connected heptad motifs, wherein positions a and d in each heptad motif (abcdefg) contain alpha-amino acids belonging to Group 1 and / or to Group 2 as defined herein below. In certain embodiments, not more than two of all the a and d positions may be occupied by any amino acid residue belonging to Group 3, and not more than one of all the a and d positions may be occupied by any amino acid residue belonging to Group 4 or Group 5 or by glycine. In certain embodiments, positions b, c, e, f, and g are each independently selected from alpha- amino acids belonging to Groups 3, 4, and 5. In certain embodiments, at least one amino acid in positions b, c, e, f, and g are hydrophobic. In certain embodiments, at least two amino acids in positions b, c, e, f, and g are hydrophobic. In certain embodiments, at least three amino acids in positions b, c, e, f, and g are hydrophobic. In certain embodiments, positions b, c, e, f, and g are each independently selected from a hydrophobic amino acid. In certain embodiments, positions b, c, e, f, and g are each independently selected from amino acids belonging to Groups 1 and 2.

[0062] Group 1 comprises alpha-amino acid residues with small to medium sized hydrophobic side chains. A hydrophobic residue refers to an amino acid side chain that is uncharged at physiological pH and that is repelled by aqueous solution. These side chains generally do not contain hydrogen bond donor groups, such as primary and secondary amides, primary and secondary amines and the corresponding protonated salts thereof, thiols, alcohols, ureas or thioureas. However, they may contain hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, or tertiary amines. Genetically encoded amino acids in this group include alanine, isoleucine, leucine, methionine, and valine.

[0063] Group 2 comprises amino acid residues with aromatic or heteroaromatic side chains. An aromatic amino acid residue refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated aromatic pi-electron system. In addition, it may contain additional hydrophobic groups such as lower alkyl, aryl or halogen,hydrogen bond donor groups such as primary and secondary amines, and the corresponding protonated salts thereof, primary and secondary amides, alcohols, and hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, or tertiary amines. Genetically encoded aromatic amino acids include phenylalanine and tyrosine. A heteroaromatic amino acid residue refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated aromatic pi-system incorporating at least one heteroatom such as O, S, and N. In addition, such residues may contain hydrogen bond donor groups such as primary and secondary amides, primary and secondary amines and the corresponding protonated salts thereof, alcohols, and hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, or tertiary amines. Genetically encoded heteroaromatic amino acids include tryptophan and histidine.

[0064] Group 3 comprises amino acids containing side chains with polar non-charged residues. A polar non-charged residue refers to a hydrophilic side chain that is uncharged at physiological pH, but that is not repelled by aqueous solutions. Such side chains typically contain hydrogen bond donor groups such as primary and secondary amides, primary and secondary amines, thiols, and alcohols. These groups can form hydrogen bond networks with water molecules. In addition, they may also contain hydrogen bond acceptor groups such as ethers, thioethers, esters, tertiary amides, or tertiary amines. Genetically encoded polar non- charged amino acids include asparagine, cysteine, glutamine, serine, and threonine. Non- proteinogenic polar amino acids include citrulline and homocysteine.

[0065] Group 4 comprises amino acids containing side chains with polar cationic residues and acylated derivatives thereof, such as acylamino-derived residues and urea-derived residues. Polar cationic side chains refer to a basic side chain, which is protonated at physiological pH. Genetically encoded polar cationic amino acids include arginine, lysine, and histidine. Ornithine is an example for a urea-derived amino acid residue.

[0066] Group 5 comprises amino acids containing side chains with polar anionic residues. Polar anionic refers to an acidic side chain, which is deprotonated at physiological pH. Genetically encoded polar anionic amino acids include aspartic acid and glutamic acid, for example, a polar anionic residue is -(CH2)aCOOH, wherein “a” is 1-4.

[0067] In certain embodiments, the amphipathic domain includes 1-10 (e.g., between 2- 10) tandemly connected heptad motifs, represented by (abcdefg)n, wherein n is 1-10, and each (abcdefg) may have any one of the following sequences: • lxxlxxx (referring respectively to the positions abcdefg);• lxx2xxx (referring respectively to the positions abcdefg); • 2xxlxxx (referring respectively to the positions abcdefg); or • 2xx2xxx (referring respectively to the positions abcdefg), wherein 1 is a genetically encoded amino acid from Group 1, 2 is a genetically encoded amino acid from Group 2, and x is a genetically encoded amino acid from Groups 1, 2, 3, 4, or 5 or is glycine.

[0068] In certain embodiment, alpha-amino acids with a hydrophobic side chain (Group 1) are independently selected from alanine, isoleucine, leucine, methionine and valine; alpha- amino acids with aromatic or hetero-aromatic residue (Group 2) are independently selected from phenylalanine, tyrosine, tryptophan, and histidine; alpha-amino acids with polar non- charged residue (Group 3) are independently selected from asparagine, cysteine, glutamine, serine, and threonine; alpha-amino acids with polar cationic residue (Group 4) are independently selected from arginine, lysine, and histidine; and alpha-amino acids with polar anionic residue (Group 5) are independently selected from aspartic acid and glutamic acid.

[0069] In certain embodiments, the amphipathic domain comprises or consists of amino acid sequences identified in naturally occurring peptides and proteins, but excluding those of human origin, for example, coiled-coil domains identified in viral and bacterial proteins.

[0070] In certain embodiments, the amphipathic domain can be (X1X2X3X4X5X6X7)n, wherein X1and X2are each independently a hydrophobic amino acid residue, glutamine, or asparagine; X2, X3, X4, X6, and X7 are each independently a hydrophilic, charged, polar or neutral amino acid residue; and n is 1-10. In certain embodiments, the amphipathic domain can be (IEKKIEX1)n, wherein X1is selected from Ala, Gly, Thr, and Ser; and n is 2. In certain embodiments, the coiled-coil domain includes E / K salt bridges between b and f positions. In certain embodiments, the amphipathic domain includes a cysteine residue for conjugation.

[0071] Exemplary amino acid sequences that can be included in the amphipathic domain are listed in Table 1 below, wherein X1 is selected from Val, Ile, Leu, Trp, and Phe; X2 is selected from Glu, Lys, Arg, Tyr, His, Ser, Thr, Asn, Gln, and Ala; X3is selected from Glu, Asp, Lys, Arg, His, Ser, Thr, Asn, Gln, and Ala; X4is selected from Glu, Lys, Arg, His, Ser, Thr, Asn, Gln, and Ala; X5 is selected from Val, Ile, Leu, Met, Asn, Gln, Trp, and Phe; X6 is selected from Glu, Asp, Lys, Arg, Tyr, His, Ser, Thr, Asn, Gln, Ala, and Gly; X7 is selected from Glu, Lys, Ser, Thr, Asn, Gln, Tyr, Ala, and Gly; n is 1-10; and m is 1-10, and variantsthereof in which one, two, or three amino acids are replaced by other amino acids or are deleted. Table 1. Amphipathic Domain Sequences Sequence SEQ ID NO: (X1X2X3X4X5X6X7)n 1 (X5X6X7X1X2X3X4)n 2 (X1X2X1X3X5X6X7)n3 (X1EKKX5X6X7)n 4 (IEKKIEX6)n 5 IEKKIEAIEKKIEAIEKKIEAIEKKIEA 6 (IEEKIKX6)n7 (IKEEIKX6)n 8 (ISAKIEA)n 9 (IEAIEKK)n10 (ISAKIEA)n(IEKKIEX6)m 11 IEKKIEAIEKKIEAX5X6 12 IEKKIEAIEKKIEAI 13 IEKKIEAIEKKIEX6 14 IEKKIEAIEKKIE 15 IEKKIEAIEKKI 16 ISAKIEAIEKKIEAIEKKX5X617 ISAKIEAIEKKIEAIEKKI 18 IKKEIEAIKKEQEAIKKKIEAIEKEIE 19 IAALKQEIAALKKENAALKFEIAALKQ 20 IDQLSSDVQTLNAKVDQLSNDVNAMRSDVQAAKDDAAR 21 IEKKIEAIEKKIEAIEKKIEAIEKKIEAIEKKNEAIEKKIEAIEKKIEAIEKK IEAIEKKIES22LEARIRELEARIK 23 LEELRRRIEELERRIR 24 MKQIEDKIEEIESKQKKIENEIARIKKL 25 IKKIENEIARIKKL 26 IEEIESKQKKIENEIAEIKKL 27 LAKVSAKADAAQSTANEALAKANAAQSTATDALSKANAAQSTADQ AMSTANSANQKAEEANEKVERM28IDQLSSDVQTLNAKVDQLSNDVNAMRSDVQAAKDDAARANQRLDN MATKY29IDQLSSDVQTLNAKVDQLSNDVNAQRSDVQAAKDDAARANQRLDNI KKEIEAIKKEQEAIKKKIEAIEKEIEAINAS30IDQLSSDVQTLNAKVDQLSNDVNAQRSDVQAAKDDAARANQRLDNI KKEIEAIKKEQEAIKKKIEAIEKEIEAINASVVNIQKEIDRLNEVAKNLN 31 ES IDQLSSDVQTLNAKVDQLSNDVNAMRSDVQAAKDDAARANQFNSAI GKIQDSLSSTASALGKLQDVVNQNAQALNTLVQ32IWKLHEEFLKKFEELLKLHEERLKKM 33 VEALEKKVAALESKVQALEKKVEAL 34Sequence SEQ ID NO: IAEIAAIEYEQAAIKEEIAAIKDKIAAIKEYIAAI 35 IEKIAAIKEEQAAIEEEIQAIKEEIAAIKYLIAQI 36 IAEIAAIKYKQAAIKNEIAAIKQEIAAIEQMIAAI 37 MKEMATLLTSLGVIQSAQEFESSRDASYVFARRALKSANYAEMTFNV CGLILSAEKSSARKVDENKQLLKQIQESVESFRDIYKRFSEYQKEQNSL 38 LMSNLSTL LKQIQESVESFRDIYKRFSEYQKEQNSLLMSNLSTL 39 LFSDVQDIKTALAKIHEDNQKIISKLESLLLLKGEVESIKKQINRQNISIS TLEGHLSSIMIAIPGLL40IKTALAKIHEDNQKIISKLESLLLLKGEVESIKKQINRQNISISTLEGHLS SIMIAI41MKQLEDKVEELLSKNYHLENEVARLKKLVGER 42 MKQIEDKLEEILSKLYHIENELARIKKLLGER 43 MKVKQLVDKVEELLSKNYHLVNEVARLVKLVGER 44 MKVKQLEDVVEELLSVNYHLENVVARLKKLVGE 45 IENYNQLKEDYNTLKRELSDRDDEVKRLREDIAKENELRTKAEEEAD KLNKEVEDLTASLFDEANNLVADARMEKYAIEILNKRLTEQLREKDM 46 LLDTLTLQLKNL IEKKIEE 47 IKKEIEAIKKEQEAIKKKEAIEK 48 IEDKIEEIESKQKKIENEIARIKKL 49 KIKKIENEIARIKKL 50 LAKVSAKADAAQSTANEALAKANAAQSTATDALSKANAAQSTADQ AMSTANSANQKAEEANEKVERMFKKAMMK52AEMTFNVCGLILSAEKSSARKVDENKQLLKQIQESVESFRDIYKRFSE YQKEQNSLLMSNLSTL53VEALEKKVAALECKVQALEKKVEALEHGW 55 IWKLHEEFLCKFEELLKLHEERLKKM 56 AALKCGVQELEKGAEAGEGGW 57 ALEKKVAALESKVQALEKKVEALEHGW 58 AAKAAAAKAAAAKAA 59 IMRIGKFLEHLKAAA 60 LSRAIAKGKDNLKEYK 61 FTVSFKLR 62 DWLKAFYDKVAEKLKEAFLA 63 ELLEKWKEALAALAEKLK 64 FWLKAFYDKVAEKLKEAF 65 DWLKAFYDKVAEKLKEAFRLTRKRGLKLA 66 DWLKAFYDKVAEKLKEA 67 KLKLLLLLKLK 68 X1X2X1X3X4X5X6 69

[0072] In certain embodiments, the amphipathic domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-69.

[0073] In certain embodiments, the amphipathic domain comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1-69.

[0074] In certain embodiments, the amphipathic domain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-69.

[0075] In certain embodiments, the amphipathic domain comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0076] In certain embodiments, the amphipathic domain comprises an amino acid sequence consisting of an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0077] In certain embodiments, the amphipathic domain consists of an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0078] In certain embodiments, one or more of the amino acids of the amphipathic domain are chemically modified. 7.1.3. Hydrophilic Polymer Block

[0079] In certain embodiments, the hydrophilic polymer block comprises a poly(ethylene glycol) oligomer, a poly(vinyl alcohol) oligomer, a polyoxazolines oligomer, a poly(amino acid) oligomer, a polysarcosine oligomer, or a polysaccharide.

[0080] In certain embodiments, the hydrophilic polymer block comprises a poly(ethylene glycol) oligomer. In certain embodiments, the hydrophilic polymer block consists of a poly(ethylene glycol) oligomer. In certain embodiments, the poly(ethylene glycol) oligomer is monodisperse. In certain embodiments, the poly(ethylene glycol) oligomer comprises or consists of 2-45 repeat units, 4-24 repeat units, 10-19 repeat units, or 6-12 repeat units. In certain embodiments, the poly(ethylene glycol) oligomer comprises or consists of 12 repeat units. In certain embodiments, the poly(ethylene glycol) oligomer comprises or consists of 12 repeat units. Various hydrophilic polymer blocks that are suitable in certain embodiments provided herein are provided in WO 92 / 16221, the disclosure of which is incorporated herein by reference in its entirety.

[0081] In certain embodiments, the hydrophilic polymer block comprises a poly(vinyl alcohol) oligomer. In certain embodiments, the hydrophilic polymer block consists of a poly(vinyl alcohol) oligomer. In certain embodiments the poly(vinyl alcohol) oligomer is monodisperse. In certain embodiments the poly(vinyl alcohol) oligomer comprises or consists of 2-45 repeat units, 4-24 repeat units, 10-19 repeat units, or 6-12 repeat units. In certain embodiments, the hydrophilic polymer block comprises or consists of 12 repeat units.

[0082] In certain embodiments, the hydrophilic polymer block comprises a polyoxazolines oligomer. In certain embodiments, the hydrophilic polymer block consists of a polyoxazoline oligomer. In certain embodiments, the poly(N-vinylpyrrolidone) oligomer comprises or consists of 2-45 repeat units or 4-24 repeat units, 10-19 repeat units, or 6-12 repeat units. In certain embodiments, the poly(N-vinylpyrrolidone) oligomer comprises or consists of 12 repeat units.

[0083] In certain embodiments, the hydrophilic polymer block comprises a poly(amino acid) oligomer. In certain embodiments, the hydrophilic polymer block consists of a poly(amino acid) oligomer. In certain embodiments, the poly(amino acid) oligomer is monodisperse. In certain embodiments, the poly(amino acid) is polyglutamic acid (PGA), poly(hydroxyethyl-l-asparagine) (PHEA), or poly(hydroxyethyl-l-glutamine) (PHEG). In certain embodiments the poly(amino acid) oligomer comprises or consists of 2-45 repeat units, 4-24 repeat units, 10-19 repeat units, or 6-12 repeat units. In certain embodiments, the hydrophilic polymer block comprises or consists of 12 repeat units.

[0084] In certain embodiments, the hydrophilic polymer block comprises a polysarcosine oligomer. In certain embodiments, the hydrophilic polymer block consists of a polysarcosine oligomer. In certain embodiments the polysarcosine oligomer is monodisperse. In certain embodiments the polysarcosine oligomer comprises or consists of 2-45 repeat units, 4-24 repeat units, 10-19 repeat units, or 6-12 repeat units. In certain embodiments, the polysarcosine oligomer comprises or consists of 12 repeat units.

[0085] In certain embodiments, the hydrophilic polymer block comprises a polysaccharide. In certain embodiments, the hydrophilic polymer block consists of a polysaccharide oligomer. In certain embodiments the polysaccharide oligomer is monodisperse. In certain embodiments the polysaccharide oligomer comprises or consists of 2-45 repeat units, 4-24 repeat units, 10-19 repeat units, or 6-12 repeat units. In certain embodiments, the polysaccharide oligomer comprises or consists of 12 repeat units.

[0086] In certain embodiments, the hydrophilic polymer block comprises poly(ethylene glycol), poly(vinyl alcohol), poly(N-vinylpyrrolidone), polysarcosine, polyglutamic acid, poly(hydroxyethyl-L-asparagine), or poly(hydroxyethyl-L-glutamine). In certain embodiments, the hydrophilic polymer block consists of poly(ethylene glycol), poly(vinyl alcohol), poly(N-vinylpyrrolidone), polysarcosine, polyglutamic acid, poly(hydroxyethyl-L- asparagine), or poly(hydroxyethyl-L-glutamine). 7.1.4. Protease Cleavage Site

[0087] In certain embodiments, the lipopeptide building block comprises a protease cleavage site, e.g., an amino acid sequence susceptible to cleavage by intracellular proteases, to facilitate intracellular processing of the lipopeptide building block. In certain embodiments, the protease cleavage site is incorporated at or near the C-terminus of the lipopeptide building block. In certain embodiments, the protease cleavage site is incorporated at the C-terminus of the lipopeptide building block. In certain embodiments, the protease cleavage site is conjugated to the hydrophilic polymer block.

[0088] In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by intracellular proteases. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a protease selected from the group consisting of a serine protease, an aspartic protease, a cysteine protease. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a cysteine protease, such as Legumain. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a cathepsin. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a protease selected from the group consisting of asparaginyl endopeptidase (e.g., Legumain), cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin P, cathepsin S, and cathepsin W. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by asparaginyl endopeptidase. In certain embodiments, the protease cleavage site comprises an amino acid sequence susceptible to cleavage by cathepsin S.

[0089] In certain embodiments, the protease cleavage site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86. In certain embodiments, the protease cleavage site comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 82 and 83. In certain embodiments, the protease cleavage site comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86. In certain embodiments, the protease cleavage site comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 82 and 83. In certain embodiments, the protease cleavage site consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86. In certain embodiments, the protease cleavage site consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 82 and 83.

[0090] Exemplary protease cleavage sites that can be included in the lipopeptide building block are provided in Table 2 below, wherein Z1is succinyl, Z2is citrulline, Z3is homophenylalanine, Z4 is beta-alanine, and Z5 is norvaline. Table 2. Exemplary Protease Cleavage Sites Sequence SEQ ID NO: Z1TANL 70 AANL 71 PAN 72 FR 73 FK 74 VK 75 VZ2 76 VVR 77 AAD 78 RSZ2GZ3 79 Z1Z4LAL 80 TVGLR 81 PMGLP 82 PMGAP 83 KVSVR 84 RGFFP 85 RPKPVEZ5WRK 86 7.2. T-helper Epitope

[0091] The lipopeptide construct can comprise a T-helper cell epitope. In certain embodiments, the T-helper epitope is connected directly or indirectly through a linker to a component of the lipopeptide building block. In certain embodiments, the T-helper is connected, either directly or indirectly through a linker, through its N- or C-terminus, to the C-terminal of the protease cleavage site, or to an amino acid side chain of the protease cleavage site.

[0092] In certain embodiments, the T-helper epitope is selected from a flu, hepatitis B, tetanus toxin, diphtheria toxin, measles, covid and common cold T-helper epitope. In certain embodiments, the T-helper epitope is selected from a flu, hepatitis B, tetanus toxin, diphtheria toxin, measles, and covid T-helper epitope. In certain embodiments, the T-helper epitope is a flu T-helper epitope. In certain embodiments, the T-helper epitope is a tetanus toxin T-helper epitope. In certain embodiments, the T-helper epitope is a diphtheria toxin T- helper epitope. In certain embodiments, the T-helper epitope is a covid T-helper epitope. In certain embodiments, the T-helper epitope is a common cold T-helper epitope. The T-helper epitopes that can be included in the lipopeptide aggregate are provided in Table 3 below, and variants thereof in which one, two, or three amino acids are replaced by other amino acids or are deleted. Table 3. Exemplary T-helper Epitope Sequences Source: SequenceaSEQ ID NO: Tetanus toxin QYIKANSKFIGITE 87 Tetanus toxin IREDNNITLKLDRCNN 88 Tetanus toxin VSIDKFRIFCKANPK 89 Tetanus toxin FNNFTVSFWLRVPKVSASHLET 90 Tetanus toxin LKFIIKRYTPNNEIDS 91 Diphtheria toxin PVFAGANYAAWAVNVAQVID 92 Diphtheria toxin VHHNTEEIVAQSIALSSLMV 93 Diphtheria toxin QSIALSSLMVAQAIPLVGEL 94 Diphtheria toxin VDIGFAAYNFVESIINLFQV 95 Diphtheria toxin QGESGHDIKITAENTPLPIA 96 Diphtheria toxin GVLLPTIPGKLDVNKSKTHI 97 Diphtheria toxin TIDKISDVSTIVPYIGPALN 98 Chlamydia protein ALNIWDRFDVFCTLGATTGYLKGNS 99 E. coli protein GLQGKIADAVKAKG 100 E. coli protein GLAAGLVGMAADAMVEDVN 101 E. coli protein STETGNQHHYQTRVVSNANK 102 Hepatitis B core protein PHHTALRQAILCWGELMTLA 103 Hepatitis B Surface antigen FFLLTRILTIPQSLD 104 Influenza Matrix YSGPLKAEIAQRLEDV 105 Influenza HA SRGLFGAIAGFIEGGWQ 106 Influenza HA PKYVKQNTLKLAT 107 Influenza HA PKYVKQNTLKLAR 108 Influenza HA PKYVKQNTLKLRT 109 Measles F protein GILESRGIKARITHVDTESY 110 Measles F protein LSEIKGVIVHRLEGV 111 Measles N protein SAGKVSSTLASELG 112 P. falciparum CSP IEKKIAKMEKASSVFNVVNS 113 Artificial (PADRE) aKFVAAWTLKAAab114Source: SequenceaSEQ ID NO: Artificial (PADRE) aK-Chx-VAAWTLKAAab115 SARS-CoV2 N ASAFFGMSRIGMEVT 116 SARS-CoV2 M NRFLYIIKLIFLWLLWPVTL 117 Rhinovirus capsid DSTITSQDVANAVVGYGV 118 a References: SEQ ID NOs: 70-74 and 86-88: Eur. J. Immunol.2001, 31, 3816-3824; SEQ ID NOs: 55-60: JID 2000, 181, 1001-1009; SEQ ID NOs: 82-85 and 93-94: US 5,759,551; SEQ ID NO: 89-92 Nat. Comm. 2012, 665; SEQ ID NO: 95: J Virol.200442–51; SEQ ID NO: 96: Nature 1988, 336, 778-780; SEQ ID NOs: 97-98: Immunity 1994, 1, 751–761. SEQ ID NO: 99: Vaccines (Basel).2023,11, 1090; SEQ ID NO: 100: J Allergy Clin Immunol Glob.2022, 3,112-121. SEQ ID NO: 101: Cells 2021, 10, 2294. b “a” denotes D-Ala and “Chx” denotes cyclohexylalanine.

[0093] In certain embodiments, the T-helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-118. In certain embodiments, the T- helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-91. In certain embodiments, the T-helper epitope comprises an amino acid sequence of SEQ ID NO: 90. In certain embodiments, the T-helper epitope comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 87-118. In certain embodiments, the T-helper epitope comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 87-91. In certain embodiments, the T-helper epitope comprises an amino acid sequence consisting of SEQ ID NO: 90. In certain embodiments, the T-helper epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 87- 118. In certain embodiments, the T-helper epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-91. In certain embodiments, the T- helper epitope consists of an amino acid sequence of SEQ ID NO: 90. 7.3. Antigen

[0094] References to the term, “antigen” herein also includes antigen epitopes, e.g., a lipopeptide construct as provided herein may not include an entire antigen, but one or more fragments thereof, e.g., a fragment that binds to a corresponding antigen receptor on an immune cell and is recognized by an immune system, e.g., by antibodies, B cells, or T cells. The lipopeptide construct can comprise one or more epitopes of one or more antigens, each of which may be the same or different. In certain embodiments, the lipopeptide construct comprises epitopes of one antigen. In certain embodiments, the lipopeptide construct comprises two different epitopes of one antigen. In certain embodiments, the lipopeptideconstruct comprises two different epitopes, each to a different antigen (e.g., the lipopeptide construct is a dual target construct that can evoke an immune response to both antigens).

[0095] Suitable antigen(s) can be a molecule capable of being bound by an antibody. In certain embodiments, the one or more antigen(s) comprise a peptide or a protein. In certain embodiments, the peptide can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be >100 amino acids. In certain embodiments, the antigen comprises a mimotope. In certain embodiments, the mimotope can be a molecule mimicking a natural peptidic or carbohydrate epitope, including peptidic compounds containing one or more non-natural amino acids, e.g., D-amino acids, β-amino acids, γ-amino acids, δ-amino acids, or ε-amino acids, and other replacements known in the art. In certain embodiments, the mimotope is fixed in a protein-like conformation. In certain embodiments, the antigen comprises a hapten such as a drug, hormone, or toxins or a carbohydrate.

[0096] In certain embodiments, the antigen(s) is connected directly or indirectly through a linker to a component of the lipopeptide building block. In certain embodiments, the antigen(s) is connected, either directly or indirectly through a linker, through its N- or C- terminus, to the C-terminal of protease cleavage site, or to an amino acid side chain of the protease cleavage site. In certain embodiments, the antigen(s) is connected directly or indirectly through a linker to the T-helper epitope. In certain embodiments, the antigen(s) is connected, either directly or indirectly through a linker, through its N- or C-terminus, to the N- or to the C-terminal of the T-helper epitope, or to an amino acid side chain of the T-helper epitope. In certain embodiments, the antigen(s) is connected to a component of the lipopeptide building block or the T-helper epitope through a side chain residue of the antigen, for example, through a terminal or internal aspartic acid, glutamic acid, lysine, ornithine (Orn), 1,3-diaminopropionic acid (Dap), or 1,4-diaminobutyric acid (Dab), 4-amino-proline (Amp), or cysteine side chain.

[0097] In certain embodiments, the average spacing of an epitope of the antigen is in the range of about 5 nm to 10 nm. The structural arrangement of the epitope residues within the antigen's 3D structure may be analyzed using various techniques, including crystallography, NMR spectroscopy, molecular modeling, and mutagenesis analysis. In certain embodiments, this spacing may result in lipopeptide constructs and aggregates thereof that evoke productionof antibodies with improved inhibition and neutralization potency. In certain embodiments, the spacing of the epitope of the antigen allows B-cell receptor (BCR) cross-linking as predicted by cross-linking model (CLM), which leads to B-cell activation.

[0098] Without wishing to be bound by theory, it is thought that the provided antigens provide targeted responses to both linear and conformational epitopes and exclude disease- enhancing epitopes.

[0099] In certain embodiments, the lipopeptide construct comprises one or more antigens is independently selected from a B-cell epitope, a CD4+ T-cell epitope, and a CD8+ T-cell epitope. In certain embodiments, each of the one or more antigen(s) comprises at least one of a B-cell epitope, a CD4+ T-cell epitope, and a CD8+ T-cell epitope. In certain embodiments, the antigen comprises a B-cell epitope. In certain embodiments, the lipopeptide construct comprises a CD4+ T-cell epitope. In certain embodiments, the lipopeptide construct comprises a CD8+ T-cell epitope.

[0100] In certain embodiments, one or more of the antigen(s) independently comprises one or more non-genetically encoded amino acids, such as ornithine (Orn), 1,3- diaminopropionic acid (Dap), or 1,4-diaminobutyric acid (Dab), or 4-amino-proline (Amp). In certain embodiments, one or more of the antigen(s) independently comprises one or more D-amino acids.

[0101] In certain embodiments, the lipopeptide construct comprises a self-antigen. In certain embodiments, the lipopeptide construct comprises an antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124, shown in Table 4 below. In certain embodiments, lipopeptide construct comprises a Proprotein Convertase Subtilisin / Kexin type 9 (PCSK9) epitope. In certain embodiments, the lipopeptide construct comprises an amino acid sequence of SEQ ID NO: 119, which is one example of a PCSK9 epitope. In certain embodiments, the antigen comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124. In certain embodiments, the antigen comprises an amino acid sequence consisting of an amino acid sequence of SEQ ID NO: 119. In certain embodiments, the antigen consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124. In certain embodiments, the antigen consists of an amino acid sequence of SEQ ID NO: 119.Table 4. Exemplary Peptide Antigens Sequence SEQ ID NO: SIPWNLERGTPPRYRADEYQPK119PEEDGTRFHRQASK120GDIIGASSDCSTCFMSQSGT 121 SIPWNLER 122 SIINFEKL 123 NPNANPNANPNANPNANPNA 124 7.4. Linkers

[0102] The components of the lipopeptide aggregate can be connected via various linkers. The linkers can include linkers having the structure L1 to L16 below and / or short peptides of 2-20 amino acids (as discussed below). The amphipathic domain (AD) can be covalently connected to the lipid moiety (LM) at or near one terminus, i.e., the N-terminus or the C- terminus. In certain embodiments, the amphipathic domain is connected to the lipid moiety via the N-terminus of the amphipathic domain. In certain embodiments, the lipid moiety is directly connected to the amphipathic domain to form the structure: LM-AD. In certain embodiments, the lipid moiety is indirectly connected to the amphipathic domain via a linker to form the structure: LM-L-AD. Additionally, the antigen can be connected to the lipopeptide building block (BB), for example, to the amphipathic domain or to the T-helper epitope, via a linker, to form the structure BB-L-A.

[0103] If the components of the lipopeptide aggregate, for example, the amphipathic domain and the lipid moiety, or the antigen and the BB, are directly connected, this can be accomplished, for example, through an amide bond between a lipid moiety carbonyl function and an amino function, e.g., the N-terminal amino function, of the amphipathic domain. In certain embodiments, certain lipid moieties, e.g., LM 1, LM 2, and LM 8 (as defined below), are connected through an amide bond between their amine function and a carboxy function, e.g., the C-terminal carboxy function, of the amphipathic domain.

[0104] A person of ordinary skill in the art would know that a wide variety of suitable linkers and coupling strategies exist, including, but not limited to, linkers based on dicarboxylic acid derivatives, linkers containing one or multiple ethylene glycol units, amino acid residues (including α-, β-, γ-, δ-amino acids), or sugar (carbohydrate) units, or containing heterocyclic rings. In certain embodiments, the linker can have the structure of L1 to L16, shown in Table 5 below, wherein n is 1-45, e.g., between 1 and 45 and m is between 1 and 45, for example, wherein n is 1-20, e.g., between 1 and 20, and m is 1-20, e.g., between 1 and20, shown with the connecting functional group C=O and / or X, wherein X is O or NH, and wherein represents attachment to adjacent lipopeptide building block components. Table 5. Linkers L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14L15 L16

[0105] In certain embodiments, linkers L1 to L16 may be connected to the lipid moiety and amphipathic domain as follows.

[0106] In certain embodiments, a carbonyl function shown for L1 to L16 may be connected to an amino function of a suitable lipid moiety and / or an amino function, e.g., the N-terminal amino function, of the amphipathic domain through an amide bond. In certain embodiments, a carbonyl function shown for L1 to L16 may be connected to a lipid moiety by replacement of the corresponding carbonyl function in particular lipid moieties LM 3 to LM 7.

[0107] In certain embodiments, a functional group X shown for L1 to L16 (with the meaning -NH or -O) may be connected to a carbonyl function of a suitable lipid moiety and / or a carboxy function, e.g., the C-terminal carboxy function, of the amphipathic domain through an amide bond (for X = NH) or through an ester bond (for X = O).

[0108] In certain embodiments, the terminal -CH2 group of L8 may be connected to an amino function of a suitable lipid moiety, an amino function, e.g., the N-terminal amino function, of the amphipathic domain, or a carbonyl function of a suitable lipid moiety. One of ordinary skill in the art would appreciate that the linker can be connected via a terminus of the amphipathic domain, or through a side chain residue of the amphipathic domain, for example, through a terminal or internal aspartic acid, glutamic acid, lysine, ornithine, or cysteine side chain.

[0109] In certain embodiments, the lipid moiety can be connected to the amphipathic domain through a single amino acid (including α-, β-, γ-, δ-amino acid). In certain embodiments, the lipid moiety can be connected to the amphipathic domain through an ε- amino acid. In certain embodiments, the lipid moiety can be connected to the amphipathic domain through an alkylamino acid linker, such as a C3-10 alkylamino acid linker. For example, in certain embodiments, the lipid moiety can be connected to the amphipathicdomain through a 5-aminopentanoic acid, 6-aminohexanoic acid, or 7-aminoheptanoic acid linker. In certain embodiments, the lipid moiety is connected to the amphipathic domain through an ε-amino caproic acid (εAhx, also known as 6-aminohexanoic acid) linker, shown below:, wherein represents attachment to adjacent components, e.g., the lipid moiety and the amphipathic domain. In certain embodiments, the amino moiety is connected to the lipid moiety and the carboxyl moiety is connected to the amphipathic domain.

[0110] In certain embodiments, the lipid moiety can be connected to the amphipathic domain through a peptide linker of 2-20 amino acids. In certain embodiments, the peptide linker can have the sequence according to one of SEQ ID NOs: 125-146 herein, wherein n is 1-5 and m is 1-5. Table 6. Exemplary Peptide Linkers for Linking the Lipid Moiety Sequence SEQ ID NO: GS 125 SG 126 GGn127 SnKm 128 GSAGSAAGSGEF 129 GFLGG 130 AGNRVRRSVG 131 ANTRHSESDK 132 GTGGKQSSDK 133 GNGNKSGSDD 134 SNIEIFNAKG 135 TTDKKEIKAY 136 SFGGNHKLSS 137 GSQNLAPLEE 138 GQDSDQQKDG 139 GNDDGKDKDG 140 GNNSSKDKEA 141 SLPGLGSKST 142 STSEVIGEKI 143 SSNAK 144 GSQNLAPLEEK 145 GSQN 146

[0111] The antigen can be conjugated directly or through a linker, either at the N- or C- terminus of the antigen and is connected either to the N- or to the C-terminal of the lipopeptide building block, or optionally to an amino acid side chain. Alternatively, the antigen can be conjugated to the lipopeptide building block through a side chain residue of the antigen, such as a terminal or internal aspartic acid, glutamic acid, lysine, ornithine (Orn), 1,3-diaminopropionic acid (Dap), or 1,4-diaminobutyric acid (Dab), 4-amino-proline (Amp), or cysteine side chain.

[0112] Suitable linkers for linking the antigen to the lipopeptide building block include short peptides of 2-20 amino acids, hydroxyalkyl- or aminoalkyl-carboxylic acids, substituted or unsubstituted polyalkenyloxy glycols, e.g., containing 1-12 C2and / or C3alkenyloxy units, polyalkenyloxy glycol block co-polymers (e.g., PLURONICS®), mono-, di-, tri- and oligosaccharides, which may comprise acetyl, glycerol-phosphate or other substituents at one or more positions, polysaccharides such as poly(salicylic acid) and derivatives (e.g., peptide conjugates) thereof, proteinogenic or non-proteinogenic amino acids, and C1-C8saturated or unsaturated hydrocarbons, and may comprise one or more of the following functional groups: a disulphide bond, amine, amide, acetal, ester, ether, thioether, hydrazone, hydrazide, imine, oxime, urea, thiourea, carbonate, iminocarbonate, amidine, amide, imide, an alkyl succinimide, which may also be hydrolyzed to an amide, sulphonamide, sulfone, or a heterocyclic ring comprising one or more atoms selected from nitrogen and oxygen, for example a triazole, and combinations thereof. Any method used for conjugating peptides or other antigens to an antigen delivery system such as carrier protein, polymer, dendrimer, aggregate or virus-like particle, can be used to conjugate the antigen to the lipopeptide building block component. Table 7 sets forth exemplary peptide linker sequences. In certain embodiments, the peptide linker can have the sequence according to one of SEQ ID NOs: 147 to 163 herein, wherein z is any amino acid, n is 1-4 (e.g., between 1 and 4), m is 1-4 (e.g., between 1 and 4), and o is 1-4 (e.g., between 1 and 4), x is 1-6 (e.g., between 1 and 6), and PEG is NH(CH2CH2O)xCH2CH2CO, wherein x is 1-8.Table 7. Exemplary Linkers for Linking the Antigen Sequence SEQ ID NO: (Gnzm)o 147 Gn148 Kn 149 Sn150 A(EAAAK)nA 151 (AP)n152 KKKGGGGS 153 KKKGGS 154 GGGGSGGGG 155 KKKGGGGSGGGGS 156 SLPGLGSKSGNN 157 {PEG}Sn158 {PEG}Gn 159 KKK{PEG}Sn161 KKK{PEG}GnSm 162 KKKCA 163

[0113] In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 147 to 163.

[0114] In certain embodiments, the peptide linker comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 147 to 163.

[0115] In certain embodiments, the peptide linker consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 147 to 163. 7.5. Exemplary Lipopeptide Constructs

[0116] In certain embodiments, the lipopeptide construct can have the structure set forth and described in Table 8, wherein and amino-PEG12-propionyl is NH(CH2CH2O)xCH2CH2CO, wherein x is 12.Table 8. Exemplary Lipopeptide Aggregate Constructs Construct No.Sequence1{Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl-PMGLPFNNFTVSFWLRVPKVSASHLEQKKKCA-NH2{Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- 2 PMGLPFNNFTVSFWLRVPKVSASHLEQKKKC{H- SIPWNLERGTPPRYRADEYQPK{3-succinimido-propionyl-}-NH2}A-NH2 {Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- 3 PMGLPFNNFTVSFWLRVPKVSASHLEQKKKC{3-succinimido- propionyl-PEEDGTRFHRQASK}A-NH2} {Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- 4 PMGLPFNNFTVSFWLRVPKVSASHLEQKKKC{3-succinimido- propionyl-GDIIGASSDCSTCFMSQSGT-NH2}A-NH2{Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- 5 PMGLPFNNFTVSFWLRVPKVSASHLEQKKKC{3-succinimido- propionyl-SIINFEKL-OH}A-NH2{Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- 6 PMGLPFNNFTVSFWLRVPKVSASHLEQKKKC{3-succinimido- propionyl-NPNANPNANPNANPNANPNA-NH2}A-NH2 7.6. Lipopeptide Aggregates

[0117] In certain embodiments, the lipopeptide constructs having attributes as described in any of Sections 7.1-7.5 may aggregate to form a plurality of lipopeptide aggregates. In certain embodiments, the lipopeptide constructs self-assemble to form the plurality of lipopeptide aggregates. In certain embodiments, the lipopeptide aggregates are micelles. In certain embodiments, the plurality of lipopeptide aggregates are nanoparticles, e.g., nanomicelles. In certain embodiments, the plurality of lipopeptide aggregates have a particle size distribution, e.g., as measured by dynamic light scattering, of about 10 nm to about 100 nm. In certain embodiments, the plurality of lipopeptide aggregates have a size distribution, e.g., as measured by dynamic light scattering, of about 30 nm to about 70 nm. In certain embodiments, the plurality of lipopeptide aggregates have a size distribution, e.g., as measured by dynamic light scattering, of about 30 nm to about 50 nm. In certain embodiments, the plurality of lipopeptide aggregates have a size distribution, e.g., as measured by dynamic light scattering, of about 15 nm to about 75 nm, preferably about 20 nm to about 40 nm. As used herein, and unless otherwise defined, the term “about” is used to encompass values within (e.g., ±) 10% of the named value. Particle size may be measured, e.g., by dynamic light scattering (DLS), by transmission electron microscopy (TEM), or scanning electron microscopy (SEM).

[0118] In certain embodiments, the plurality of lipopeptide aggregates has a homogeneity, or polydispersity index (PD, Mw / Mn) of about 1 to about 1.2, about 1 to about 1.15, about 1 to about 1.1 or about 1 to about 1.05.

[0119] In certain embodiments, the lipopeptide aggregate comprises a lipopeptide construct selected from the group consisting of Constructs 1-6 as set forth in Table 8. In certain embodiments, the lipopeptide aggregate comprises a lipopeptide construct selected from the group consisting of Constructs 1-4 as set forth in Table 8.

[0120] In certain embodiments, a lipopeptide aggregate is formed from and comprises two or more different lipopeptide constructs. For example, in one embodiment, a lipopeptide aggregate may comprise lipopeptide constructs displaying one or more epitopes of a first antigen and a lipopeptide constructs displaying one or more epitopes of a second antigen. In such embodiments, an aggregate would be able to evoke an immune response to both antigens. In another example, a lipopeptide aggregate may comprise lipopeptide constructs that vary in domains comprising chemical moieties that may naturally vary in bulk compositions, e.g., linkers with PEG, or lipid moieties with a distribution of hydrocarbyl chain lengths. 7.7. Immunogenic Compositions

[0121] The present disclosure additionally provides an immunogenic composition comprising the lipopeptide aggregates, e.g., as described in Section 7.6. In certain embodiments, the immunogenic composition comprises a single type of lipopeptide aggregate, e.g., all or substantially all of the lipopeptide constructs and aggregates display the same antigen epitopes. In certain embodiments, an immunogenic composition may comprise a first plurality of lipopeptide aggregates that display one or more epitopes of a first antigen and a second plurality of lipopeptide aggregates that display one or more epitopes of a second antigen. In such embodiments, the immunogenic composition is a dual target composition that can evoke an immune response to both antigens. In any embodiment, an immunogenic composition may comprise lipopeptide aggregates assembled from lipopeptide constructs varying in domains comprising chemical moieties that may naturally vary in bulk compositions, e.g., linkers with PEG, or lipid moieties with a distribution of hydrocarbyl chain lengths.

[0122] In certain embodiments, the immunogenic composition is a vaccine, for example, a malaria vaccine. In certain embodiments, the immunogenic composition is a vaccine. Incertain embodiments, the immunogenic composition is a pharmaceutical composition comprising the lipopeptide aggregates, e.g., as described in Section 7.6, either alone or in combination with one or more pharmaceutically acceptable carriers or excipients. In certain embodiments, the immunogenic pharmaceutical composition comprises the lipopeptide aggregates, e.g., as described in Section 7.6, either alone or in combination with one or more pharmaceutically acceptable carriers and one or more adjuvants.

[0123] In certain embodiments, the immunogenic pharmaceutical composition comprises one or more adjuvants selected from, for example, a mineral salt (e.g., aluminium hydroxide, aluminium phosphate, aluminium sulfate, calcium phosphate), monophosphoryl lipid A (MPL), plant extracts containing saponins (e.g., QS-21), imidazo-quinolines (e.g., Imiquimod), muramyl dipeptides and tripeptides, lipopeptides, oil-in- water emulsions (e.g., Montanide ISA 720), cytokines (e.g., IL-2 or GM-CSF), mycobacterial and bacterial derivatives (e.g., Freund's complete adjuvant), BCG, nucleic acid derivatives (e.g., polyIC), or any other adjuvant known to those skilled in the art. In certain embodiments, the immunogenic pharmaceutical composition comprises an adjuvant selected from the group consisting of aluminum, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), monophosphoryl lipid A (MPL), squalene, cytosine phosphoguanine, QS-21, microcrystalline tyrosine, MF59, mannide monooleate, GLA, SLA, E6020, resiquimod (R848), imidazoquinolines, imiquimod, 3M-052, 2',3'-cGAMP, 3',3'-cGAMP, cGMP, cAMP, AMP, muramyl dipeptide, poly I:C, CpG and combinations thereof.

[0124] Non-limiting examples of suitable excipients include preservatives, stabilizers, wetting agents, tonicity adjusting agents and / or emulsifiers, solubilizers, salts for regulating osmotic pressure, and buffering substances to stabilize the pH, including, but not limited to, sodium phosphate, potassium phosphate, sodium chloride, polysorbate 80, mannitol, sucrose, trehalose, amino acids such as glycine, histidine or the monosodium salt of glutamic acid, and proteins such as human serum albumin.

[0125] In certain embodiments, the immunogenic pharmaceutical composition may be a controlled release composition, e.g., by encapsulating the lipopeptide aggregates in or attaching the lipopeptide aggregates to bio-degradable polymers, for example, polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphipathic block copolymers of hydrogels. The lipopeptide aggregates may be formulated in liposomes.

[0126] The immunogenic pharmaceutical composition can be prepared according to conventional means known to a person of ordinary skill in the art, for example, using dissolution and lyophilization processes.

[0127] As would be appreciated by a person of ordinary skill in the art, the dosage of the active ingredient, e.g., the lipopeptide aggregates, e.g., as described in Section 7.6, depends upon the intended recipient (e.g., species), its age, weight, and individual condition, and the administration route. An optimal dosage for a particular active ingredient and a particular target population can be determined by standard studies involving observation of appropriate immune responses in subjects. In certain embodiments, the immunogenic composition comprises from about 0.05% to about 99%, from about 0.05% to about 75%, from about 0.05% to about 50%, from about 0.05% to about 25%, from about 0.05% to about 10%, from about 0.1% to about 50%, from about 0.1% to about 25%, from about 0.1% to about 10%, from about 1% to about 90%, from about 1% to about 75%, from about 1% to about 50%, from about 1% to about 10%, from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, or from about 10% to about 25% of the active ingredient, e.g., the lipopeptide aggregates, e.g., as described in Section 7.6.

[0128] Advantageously, in certain embodiments, the lipopeptide aggregates provided herein have a solubility in PBS buffer of at least 10 mg / mL, such as about 10 mg / mL to about 40 mg / mL, or about 15 mg / mL to about 35 mg / mL, about 20 mg / mL to about 30 mg / mL, or about 25 mg / mL, as measured, e.g., by laser nephelometry. In certain embodiments, the lipopeptide aggregates provided herein are more soluble as measured, e.g., by laser nephelometry, than certain virus-like particles described, e.g., in WO 2008 / 068017 A1, WO 2015 / 082501 A1, WO 2018 / 229156 A1, WO 2020 / 127728 A1, WO 2021 / 260176 A1, WO 2022 / 063990 A1, WO 2022 / 103927 A1, and US 10,806,702 B2, each of which are incorporated herein by reference in its entirety.

[0129] Advantageously, in certain embodiments, the lipopeptide aggregates provided herein have a higher stability as evidenced by melting point (Tm), as measured by DSC, than certain virus-like particles described, e.g., in WO 2008 / 068017 A1, WO 2015 / 082501 A1, WO 2018 / 229156 A1, WO 2020 / 127728 A1, WO 2021 / 260176 A1, WO 2022 / 063990 A1, WO 2022 / 103927 A1, and US 10,806,702 B2. In certain embodiments, the lipopeptide aggregates provided herein and displaying at least one antigen epitope, e.g., a PCSK9 epitope, have a melting point of at least about 65 °C.7.8. Methods

[0130] The one or more antigens together with the rest of the lipopeptide construct, as provided herein, stimulates an immune response in the subject and causes the subject to generate antibodies against the lipopeptide construct, e.g., one or more of the antigens. Accordingly, provided herein is a method of inducing generation of antibodies in a subject, wherein the method comprises administering a lipopeptide aggregate, e.g., as described in Section 7.6 or an immunogenic composition, e.g., as described in Section 7.7, such that the subject generates antibodies against the one or more antigens. In certain embodiments, the lipopeptide aggregate induces a response by one or both of CD4+ or CD8+ T-cells.

[0131] In certain embodiments, the antigen is a self-antigen. In certain embodiments, administering the lipopeptide aggregate, e.g., as described in Section 7.6 or an immunogenic composition, e.g., as described in Section 7.7, reduces B-cell self-tolerance in the subject, e.g., reduces the subject’s immune system’s ability to distinguish between self-antigens (molecules belonging to the body) and foreign antigens (molecules from outside the body), preventing B cells from attacking the body's own tissues.

[0132] In certain embodiments, the antigen is a self-antigen. In certain embodiments, the antigen comprises a PCSK9 epitope. Accordingly, provided herein is a method of inducing generation of PCSK9 antibodies in a subject, wherein the method comprises administering a lipopeptide aggregate e.g., as described in Section 7.6, displaying one or more PCSK9 epitopes, such that the subject generates antibodies against PCSK9. In certain embodiments, the PCSK9 epitope comprises or consists of the amino acid sequence of SEQ ID NO: 119.

[0133] PCSK9 binds to LDL receptor on the liver, reducing its ability to remove LDL from the bloodstream, leading to higher LDL levels. PCSK9 inhibitors prevent this binding, allowing the liver to effectively clear LDL cholesterol. As such, a lipopeptide construct provided herein, displaying a PCSK9 epitope, may be used to treat conditions characterized by high levels of LDL cholesterol, such as hyperlipidemia. 8. EXAMPLES Example 1: Synthesis of Lipopeptide Building Blocks

[0134] The lipopeptide building blocks provided herein were produced by chemical synthesis using Fmoc solid phase peptide synthesis (SPPS). The peptide chain was assembled stepwise on a solid support using Fmoc-protected amino acids and standard SPPS methods. The hydrophilic polymer block was incorporated at the appropriate position using during theassembly of the peptide using standard SPPS methods. Following assembly of the peptide and removal of the N-terminal protecting groups, the lipid was coupled to the N-terminus using standard SPPS methods. Following assembly, the lipopeptide aggregate was cleaved and the sidechain protecting groups were removed by treatment with acid (e.g., TFA). The crude lipopeptide was then purified by high performance liquid chromatography (HPLC) using a reversed phase column. For storage, the lipopeptide was lyophilized or dialyzed in a suitable storage buffer. The purity and identity were determined by HPLC and mass spectrometry (MS). Antigens were conjugated to lipopeptide building blocks and constructs provided herein using common bioconjugation reactions. The resulting lipopeptide constructs were purified by HPLC using a reversed phase column or by or size exclusion chromatography (SEC), ion exchange chromatography (IEC), or tangential flow filtration (TFF). For storage, the lipopeptide construct was lyophilized or dialyzed in a suitable storage buffer. The purity and identity of the lipopeptide construct was determined by HPLC and mass spectrometry (MS). Details of the synthesis of representative Lipopeptide Constructs are described in further detail below. Synthesis of Exemplary Lipopeptide Constructs and Control Peptides Lipopeptide Construct 1 {Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- PMGLPFNNFTVSFWLRVPKVSASHLEQKKKCA-NH2 (1)

[0135] This lipopeptide construct comprises the lipid H-Cys((R)-2,3-di(palmitoyloxy)- propyl) (Pam2Cys), a ε-aminocaproic acid linker (εAhx), the artificially designed amphiphilic peptide sequence ISAKIEAIEKKIEAIEKKI (SEQ ID NO: 18), a hydrophilic polymer block comprising dodecaethylene glycol (PEG12), a cathepsin S cleavage site with sequence PMGLP (SEQ ID NO: 82), the T-helper epitope FNNFTVSFWLRVPKVSASHLEQ (SEQ ID NO: 90), a KKKCA linker (SEQ ID NO: 163) for conjugation of antigens, and a C-terminal carboxamide. Lipopeptide Construct 1 is shown in FIG.2A.

[0136] Lipopeptide 1 was synthesized by SPPS on ChemMatrix Rink amide resin (loading 0.4 mmol / g), using dicyclohexylcarbodiimide (DIC) and 6-chloro-1- hydroxybenzotriazole (Cl-HOBt) for activation and 20% piperidine in dimethyl fumarate (DMF) for Fmoc removal. After each coupling step, any remaining free amino groups were capped with acetic anhydride. Amino-PEG12-propionic acid was coupled using O-(N-Fmoc- 2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol and DIC / Cl-HOBt for activation.{Pam2Cys} was coupled to the N-terminus using Fmoc-Cys((R)-2,3-di(palmitoyloxy)propyl- OH. For cleavage, the resin was treated with TFA / triisopropylsilane / water at a ratio of 95:2.5:2.5 for 3 h. The resin was filtered, the filtrate was precipitated and washed with cold ethyl ether, dried in vacuo, and the Lipopeptide 1 was purified by HPLC on a C18 column. Analytical HPLC (4.6x250mm Vydac 218TP54 column, 0-95% acetonitrile in water + 0.1% TFA in 63.3 min.): tR= 52.08 min. Purity: 95.1%. MALDI-MS: MW calculated for C338H573N73O88S3: 7163.9; MW found = 7164.6 Da (± 0.01%). For analysis of micelle formation, the lipopeptide was dissolved at 1 mg / mL in Dulbecco’s PBS (D-PBS) containing 0.1 mM Tris(2-carboxyethyl)phosphine (TCEP) and equilibrated for 30 min at room temperature. Dynamic light scattering (DLS) was measured on a Wyatt DynaPro Titan DLS instrument. Measurements were evaluated using the DYNAMICS software version 6.9 (Wyatt). The size distribution was monomodal with a Rh = 14.6 and a PDI of 0.02 at 25 °C. This indicates that the lipopeptide self-assembles into highly homogenous aggregates in the 30-nanometer size range. Lipopeptide Construct 2 {Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-amino-PEG12-propionyl- PMGLPFNNFTVSFWLRVPKVSASHLEQKKKC{H- SIPWNLERGTPPRYRADEYQPK{3-succinimido-propionyl-}-NH2}A-NH2 (2)

[0137] Lipopeptide Construct 2 comprises Lipopeptide Construct 1 and peptide H- SIPWNLERGTPPRYRADEYQPK-NH2(SEQ ID NO: 119), which is linked to the Cys residue in Lipopeptide Construct 1 via its lysine side chain using a 3-maleimido-propionyl linker. Lipopeptide Construct 2 is shown in FIG.2B.

[0138] The maleimidopeptide H-SIPWNLERGTPPRYRADEYQPK{3-maleimido propionyl-}-NH2(119-MALC3), was synthesized using standard Fmoc SPPS methods, using DIC / OxymaPure for activation and piperazine for Fmoc removal, and using the orthogonally protected amino acids Fmoc-Lys(ivdDe)-OH and Boc-Ser(tBu)-OH for the coupling of Lys and Ser, respectively. Following assembly of the peptide, the ivdDe protecting group was selectively removed using 2% hydrazine in DMF. After ivdDe removal, 3-maleimido propionic acid was coupled to the free side chain amine using DIC / OxymaPure for activation. The peptide was cleaved and the side chain protecting groups were removed as described above for Lipopeptide Construct 1. The 119-MALC3 was purified by HPLC on a C18 column and lyophilized. Analytical HPLC (4.6x250 mm Accucore XL C18 column, 5-95% acetonitrile in water (+ 0.1% TFA) in 12 min.): tR = 7.98 min. Purity: 96.5%. ESI-MS: MWcalculated for C127H187N37O37: 2824.12; MW found = 2824.76 Da (± 0.01%). For conjugation, Lipopeptide Construct 1 (6 mg) was dissolved in 0.5 mL 50% acetonitrile / water. A solution of 119-MalC3 (4 mg) in 0.5 mL 50% acetonitrile / water was added and the pH was increased to 6.5. The mixture was gently shaken for 2 h, and the conjugate was purified by HPLC on a C18 column. The TFA salt form was converted into the chloride form using AGX8 resin (Biorad) and the conjugate was sterile-filtered and lyophilized. Analytical UPLC (Acquity UPLC BEH C18 column, 5-95% acetonitrile in water + 0.2% formic acid in 9 min): tR = 5.21 min. Purity: 96.5%. ESI-MS: MW calc. for C465H758N110O125S3: 9986.00; MW found = 9989.89 Da (± 0.01%). DLS: Rh = 16.3 nm, PDI = 0.05.

[0139] The following peptides in Table 9 were synthesized for comparison with Lipopeptide Construct 1 using standard Fmoc-SPPS methods and HPLC purified as previously described. Table 9: Control peptides ID Sequence Peptide 3 Ac-ISAKIEAIEKKIEAIEKKI-NH2 Lipopeptide 4{Pam2Cys}-GG-IEKKIEAIEKKIEAIEKKIEAIEKKIEA-IEKKIAKMEKASSVFNVVNS-KKKCA-NH2Lipopeptide 5 {Pam2Cys}-εAhx-ISAKIEAIEKKIEAIEKKI-PMGLP-FNNFTVSFWLRVPKVSASHLEQ-KKKCA-NH2Analytical Characterization

[0140] Peptide 3 comprises an N-terminal acetyl group, the artificially designed amphiphilic peptide sequence ISAKIEAIEKKIEAIEKKI (SEQ ID NO: 18), and a C-terminal carboxamide. Purity (HPLC): 94.1%. ESI-MS: MW calc: 2195.7 Da; MW found = 2195.8 Da (± 0.01%).

[0141] Lipopeptide 4 comprises the lipid H-Cys((R)-2,3-di(palmitoyloxy)-propyl) (Pam2Cys), GG (GGn, SEQ ID NO:127, wherein n is 1) as a linker, the artificially designed amphiphilic peptide sequence IEKKIEAIEKKIEAIEKKIEAIEKKIEA (SEQ ID NO:6) and the T-helper epitope IEKKIAKMEKASSVFNVVNS (SEQ ID NO: 113), a KKKCA linker (SEQ ID NO: 163), and a C-terminal carboxamide. Purity (UPLC): 91.7%. ESI-MS: MW calc: 6796.3 Da; MW found = 6796.3 Da (± 0.01%).

[0142] Lipopeptide 5 comprises the lipid H-Cys((R)-2,3-di(palmitoyloxy)-propyl) (Pam2Cys), a ε-aminocaproic acid linker (εAhx), the artificially designed amphiphilic peptide sequence ISAKIEAIEKKIEAIEKKI (SEQ ID NO: 18), a cathepsin S cleavage sitewith sequence PMGLP (SEQ ID NO: 82), the T-helper epitope FNNFTVSFWLRVPKVSASHLEQ (SEQ ID NO: 90), a KKKCA linker (SEQ ID NO: 163), and a C-terminal carboxamide. Purity (UPLC): 99.0%. ESI-MS: MW calc: 6168.8 Da; MW found = 6796.9 Da (± 0.01%). Example 3: Solubility Assessment

[0143] The solubility of control Lipopeptides 4 and 5 and Lipopeptide Construct 1 was assessed by laser nephelometry. This method detects insoluble particles and formation of aggregates in liquid samples by measuring forward scattered light. Sample preparation: 5 mg aliquots of each compound in lyophilized form were suspended with 180 µL dd H2O, equilibrated at room temperature for 20 min, diluted with 20 µL 10X PBS buffer, pH 7.2, and 3-fold dilution series were prepared in triplicates in a 96-well plate in PBS buffer. Measurements: The microplate was read on NEPHELOstar Plus microplate nephelometer (BMG Labtech) in forward scatter mode (620 nm). The counts were recorded as Relative Nephelometric Units (RNU) and plotted versus the concentration after subtracting the average signal from PBS buffer (blank subtraction), as shown in FIG.3. A standard curve was measured using Formazin standards. The solubility threshold was defined as lowest concentration at which measured RNU exceed 80'000 RNU (10 NTU). Results: An increase in RNU was observed for all samples. For control Lipopeptide 4 the counts increased from ~ 3,000 to 30,000 RNU at the highest concentration, indicating that the solubility of this lipopeptide is ≥ 25 mg / mL. In contrast, Lipopeptide 5 produced a sharp increase in RNU from ~75’000 RNU to ~ 1’700’000 RNU indicating aggregation at concentrations ≥ 0.3 mg / mL. Surprisingly, the solubility of Lipopeptide Construct 1, which contains an additional internal hydrophilic polymer block between the amphipathic domain and the protease cleavage site, remained < 23’000 RNU over the measured concentration range, indicating that the solubility threshold of this lipopeptide is well below 25 mg / mL. Interpretation: Lipopeptide 5 exhibits aggregation and turbidity beginning at ~ 0.3 mg / mL, consistent with limited aqueous solubility. In contrast, Lipopeptide Construct 1 displays minimal forward scattering up to at least 25 mg / mL, indicative of a highly soluble, non- aggregating formulation. The assay demonstrates that Lipopeptide Construct 1 has a solubility at least 80-fold greater than Lipopeptide 5 under identical buffered aqueous conditions.

[0144] The solubility of Lipopeptide Construct 1 and certain virus-like particles as described previously (e.g., in WO 2008 / 068017 A1, WO 2015 / 082501 A1, WO 2018 / 229156A1, WO 2020 / 127728 A1, WO 2021 / 260176 A1, WO 2022 / 063990 A1, WO 2022 / 103927 A1, and US 10,806,702 B2) are assessed by laser nephelometry as described above for control Lipopeptides 4 and 5 and Lipopeptide Construct 1. The assay demonstrates that Lipopeptide Construct 1 has a higher solubility than the virus-like particles previously provided under identical buffered aqueous conditions. Example 2: Secondary Structure Content

[0145] The secondary structure content of Peptide 3, Lipopeptides 4 and 5 and Lipopeptide Construct 1 was assessed by Far UV CD-spectroscopy. Sample preparation: 5 mg aliquots of each compound in lyophilized form were suspended with 900 µL dd H2O, equilibrated at room temperature for 20 min, the concentration was assessed using absorbance at 205 nm, the concentration was adjusted to a final concentration of 0.1 mg / mL using dd H2O, 10X PBS and PBS buffer. Measurements: Spectra were measured in a 1 mm cuvette using a J-810 spectropolarimeter (Jasco Corp), wavelength range of 180–260 nm, scan speed of 50 nm / min, response time of 4 seconds, and width of 1 nm. Baseline measurement: 300 μL of PBS was loaded and scanned to record a blank spectrum, which was subtracted from sample spectra. Sample measurements: 300 μL of each diluted sample was loaded and measured. All Spectra were collected over three scans and processed. Results: All samples showed one maximum near 190 nm and two minima at 208 and 222 nm, consistent with a high helical content. Deconvolution using SELCON3 (see, e.g., N. Sreerama and Woody 1993) yielded a helix content of ≥ 80% for Peptide 3, Lipopeptide 4 and Lipopeptide 5, and a helix content of ~50% for Lipopeptide Construct 1.

[0146] The ratios of the minima at 222 nm and 208 nm were 1.04–1.05 for Lipopeptides 4 and 5, indicating stable coiled-coil structures and 0.97 for Peptide 3 indicating weaker helix-helix interactions. Interpretation: This result indicates that Lipopeptide Construct 1 adopts a well-defined stable structure with significant helical content in physiological buffer at room temperature. Example 3: Thermal Stability

[0147] The stability of Peptide 3, Lipopeptides 4 and 5 and Lipopeptide Construct 1 in PBS was assessed by differential scanning calorimetry analysis (DSC) to determine the denaturation temperature (Tm). Sample preparation: Samples were prepared at final concentrations of 2-3 mg / mL in PBS buffer as described above for the CD analysis. Measurements: DSC thermograms were measured on a MicroCal VP-DSC instrument(Malvern) in 400 µL of buffer, in the range from 23 °C to 94 °C. For determination of Tm, the DSC thermograms were normalized for the sample concentrations, corrected for the instrument baseline by subtraction of the corresponding buffer-buffer scan and fitted by non- linear fitting to determine Tm. Lipopeptide 5 could not be assayed due to precipitation. Results: All measured DSC thermograms exhibited symmetrical well-defined peaks, indicative of two state cooperative unfolding. Tm= 58.01 °C, 57.24 °C, and 66.72 °C were determined for Peptide 3, Lipopeptide 4, Lipopeptide Construct 1, respectively. Interpretation: the peptides exhibit high conformational homogeneity and high cooperativity of unfolding. Peptide 3 and Lipopeptide 4 exhibit comparable thermal stability in PBS despite the lipid moiety in Lipopeptide 4. Lipopeptide Construct 1 exhibits greater thermal stability than Peptide 3 and Lipopeptide 4. The higher Tm indicates that Lipopeptide Construct 1 has superior thermal stability compared to previously provided lipopeptides.

[0148] The thermal stability of Lipopeptide Construct 1 and certain virus-like particles as described previously (e.g., in WO 2008 / 068017 A1, WO 2015 / 082501 A1, WO 2018 / 229156 A1, WO 2020 / 127728 A1, WO 2021 / 260176 A1, WO 2022 / 063990 A1, WO 2022 / 103927 A1, and US 10,806,702 B2) are assessed by DSC as described above for control Lipopeptides 4 and 5 and Lipopeptide Construct 1. The DSC analysis demonstrates that Lipopeptide Construct 1 has a higher thermal stability than the virus-like particles previously provided. Example 4: Molecular Mass

[0149] To characterize solution properties and determine the absolute molecular mass of Lipopeptide Construct 1 particles in aqueous buffer, SEC-MALS was used. Sample preparation: A sample was prepared at final concentrations of 2-3 mg / mL in PBS buffer as described above for CD analysis. Measurements: SEC-MALS was performed using UPLC (Waters), a PL aquagel-OH mixed (8 μm particle size) GPC column, 0.1 M NaNO3 as mobile phase, a flow rate of 1 mL / min column temperature of 30 °C, and a MALS Detector (Wyatt) (angles 15° and 90°). Data collection and molecular weight calculations were performed by ASTRA software (Wyatt). Two runs where performed. The refractive index increment (dn / dc) used for calculations was 0.15. Results: Mp = 618.5 ± 7.5 Da; Mn = 612.5 ± 8.5 Da; Mw= 640 ± 17 Da; Mz= 673 ± 30 Da; PD (Mw / Mn) = 1.04 ± 0.1. Interpretation: The determined Mw corresponds to ~ 87-90 copies of Lipopeptide Construct 1 per particle. The PD close to 1 is indicative of highly uniform samples with a very narrow molecular weightdispersity. The result indicates that Lipopeptide Construct 1 forms molecularly defined highly homogenous particles in aqueous buffers. Example 5: Lipopeptide Aggregates

[0150] For the formation of lipopeptide aggregates, the lipopeptide building blocks and lipopeptide constructs provided herein are reconstituted, diluted, or dialyzed into a pharmacologically acceptable buffer and equilibrated for five or more minutes. As shown in FIG.4, the particles are well-dispersed and highly homogenous. Example 6: In Vivo Studies with Lipopeptide Construct 2

[0151] The immunogenicity of Lipopeptide Construct 2 was determined by injecting five C56BL / 6 mice subcutaneously with 75 µg of Lipopeptide Construct 2 in 0.1 mL D- PBS without adjuvant. Five additional mice were injected with D-PBS (vehicle control). Mice were injected on Days 0 and 21 and bled on Days 0, 21, and 42. The sera were analyzed by ELISA. Briefly, 96-well plates (Maxisorp, Nunc) were coated overnight with antigen, washed with PBS containing 0.05% TWEEN 20 (PBST), blocked with PBS containing 5% skim milk powder (MPBS) for 1 h, washed three times with PBST and incubated with serial dilutions of sera for 2 h. The plates were then washed again with PBST, incubated with goat anti-mouse IgG-HRP conjugate (Invitrogen) for 1 h, washed and TMB solution (Merck Millipore) was added. The color reaction was stopped with TMB stop reagent solution (Merck Millipore). The optical density (OD) at 450 nm was read on a plate reader (Biotek Synergy H1M) (FIG.5). ELISA endpoint titers were calculated by non-linear regression using the GraphPad 10 software (FIG.6). Endpoint titers correspond to the dilution yielding two times the mean absorbance of PBS-treated mice. Example 7: Effect of Lipopeptide Construct 2 Immunization on LDL Receptor and Cholesterol Levels In Vivo

[0152] Mouse study: Two groups of C56BL / 6 mice (n = 5 per group) were injected subcutaneously with either 75 µg Lipopeptide Construct 2 (LPC-2) in PBS or vehicle control on days 0, 21 and 46 as described previously in Example 6. Blood samples were collected in days 0, 21, 46, and 56 (final bleeds). The mice were sacrificed, and livers were taken, homogenized using a mechanical homogenizer and RIPA lysis buffer with 1x PMSF, sodium orthovanadate 1x and protease inhibitors (1 tablet / 50 mL) (0.5 mL buffer / 100 mg) on ice. The sera were analyzed for IgG by ELISA as described above and total cholesterol was measured using the cholesterol oxidase-DAOS method using a LabAssay total cholesterol kit(Fujifilm Wako) according to the manufacturer’s protocol. Day 56 serum PCSK9 and LDL receptor (LDLR) levels in liver homogenates were measured using quantitative mouse PCSK9 and LDLR sandwich ELISA kits (Quantikine, R&D Systems), according to the manufacturer’s protocols. Curve fitting and statistical analyses were performed GraphPad Prism 10 software using unpaired t-test for comparisons of groups and different timepoints. Results: Geometric IgG Titers are summarized in Table 10. Table 10. IgG Titers Day 0 Day 21 Day 42 Day 56Vehicle < 1 < 1 < 1 < 1 Lipopeptide Construct 2 (LPC-2) < 1 3.2 ± 0.4 4.8 ± 0.3 5.7 ± 0.2

[0153] Day 21, 46, and 56 sera from Lipopeptide Construct 2 (LPC-2) injected mice contained high titers of antigen-specific IgG antibodies (IgG titer > 1:1,000). No antigen- specific IgG could be detected sera from the vehicle group and in Day 0 sera (IgG titer < 1:10). No IgG could be detected in any sera against Peptide 3 (IgG titers < 1:10), indicating that there was no response against the Lipopeptide Construct 2 core structure.

[0154] Lipopeptide Construct 2-immunized mice exhibited an increase in LDLR levels versus vehicle immunized mice (as shown in FIG.7A). Mice immunized with Lipopeptide Construct 2 exhibited significantly higher LDLR levels (p = 0.0077) and higher serum PCSK9 levels (p = 0.0381) (as shown in FIG.7B). All mice immunized with Lipopeptide Construct 2 exhibited a significant reduction in cholesterol (e.g., p = 0.0021, for day 0 vs day 56), whereas the mice from the vehicle group exhibited no significant change (p ≥ 0.05) (as shown in FIGs.6C-E). Interpretation: Immunization with Lipopeptide Construct 2 induced high titers of antigen-specific IgG antibodies that blocked the PCSK9-LDLR interaction and significantly increased LDLR expression in vivo, leading to more efficient cholesterol uptake in the liver and a significant reduction of serum cholesterol levels. Similar as observed upon administration of monoclonal PCSK9-blocking antibodies, a moderate increase in serum PCSK9 levels was observed. This increase may be explained by higher steady-state levels and slower clearance of Ab-bound PCSK9 versus free PCSK9 (Zhang L, et al., Int J Biol Sci.2012;8(3):310-27). The results indicate that immunization with Lipopeptide Construct 2 elicits high titers of antibodies with PCSK9-blocking, cholesterol lowering potential in vivo.

[0155] Immunogenicity of Lipopeptide Construct 2 and certain virus-like particles as described previously (e.g., in WO 2008 / 068017 A1, WO 2015 / 082501 A1, WO 2018 / 229156A1, WO 2020 / 127728 A1, WO 2021 / 260176 A1, WO 2022 / 063990 A1, WO 2022 / 103927 A1, and US 10,806,702 B2) are assessed as described above. Analyses demonstrates that immunization with Lipopeptide Construct 2 elicits higher titers of antibodies with a greater potential for PCSK9-blocking, cholesterol lowering potential in vivo than the virus-like particles previously provided.

Claims

CLAIMS 1. A lipopeptide comprising: i) a lipid moiety, ii) an amphipathic domain; iii) a hydrophilic polymer block; and iv) a protease cleavage site, optionally wherein the amphipathic domain is an amphipathic peptide.

2. A lipopeptide comprising the following moieties in the stated order and wherein the moieties are covalently connected to each other: a. a lipid moiety; b. an aminohexanoic acid linker; c. an amphipathic peptide; d. a hydrophilic polymer block; e. a protease cleavage site; and f. a T-helper epitope.

3. A lipopeptide comprising the following moieties in the stated order and wherein the moieties are covalently connected to each other: a. a lipid moiety; b. a linker; c. an amphipathic peptide of less than 20 amino acids; d. a hydrophilic polymer block; e. a protease cleavage site; and f. a T-helper epitope.

4. A lipopeptide comprising the following moieties in the stated order and wherein the moieties are covalently connected to each other: a. a lipid moiety; b. a linker; c. an amphipathic peptide; d. a hydrophilic polymer block consisting of between 10 to 19 repeat units; e. a protease cleavage site; and f. a T-helper epitope.

5. A lipopeptide comprising the following moieties in the stated order and wherein the moieties are covalently connected to each other:a. a lipid moiety; b. a linker; c. an amphipathic peptide; d. a hydrophilic polymer block; e. a protease cleavage site cleavable in the endolysosomal system of an antigen presenting cell; and f. a T-helper epitope.

6. The lipopeptide of any one of the preceding claims wherein the order of the components a. to f. ae in the amino- to carboxy-terminus direction of the peptide components.

7. The lipopeptide of any one of claims 1-6, wherein the lipid moiety is a Toll-like receptor (TLR) agonist.

8. The lipopeptide of claim 7, wherein the TLR agonist is a TLR-2 agonist.

9. The lipopeptide of any one of claims 1-8, wherein the lipid moiety has a structure selected from the group consisting of LM 1, LM 2, LM 3, LM 4, LM 5, LM 6, LM 7, and LM 8.

10. The lipopeptide of any one of claims 1-9, wherein the lipid moiety is selected from the group consisting of Cys((R)-2,3-di(palmitoyloxy)-2-propyl); Cys((S)-2,3- di(palmitoyloxy)-2-propyl); Cys((R)-2,3-di(palmitoyloxy)-2-propyl-N-palmitoyl); Cys((R)- 2,3-di(dilauroyloxy)-2-propyl-N-palmitoyl); unsaturated Pam3Cys; unsaturated Pam2Cys; N,N’-dipalmitoyl-2,3-diamino-propionamide; phosphatidylethanolamine; diphosphoryl hexaacyl lipid A; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; and 1,3-dipalmitoyl- glycero-2-phosphoethanolamine.

11. The lipopeptide of any one of claims 1-10 wherein the lipid moiety is Pam2Cys.

12. The lipopeptide of any one of claims 1-11, wherein the linker is a peptide linker.

13. The lipopeptide of any one of claims 1-11, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-163.

14. The lipopeptide of any one of claims 1-13, wherein the linker comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-163.

15. The lipopeptide of claim 14, wherein the linker consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 125-163.

16. The lipopeptide of any one of claims 1-15, wherein the amphipathic peptide is a coiled-coil domain.

17. The lipopeptide of claim 16, wherein the coiled-coil domain directs formation of helical bundles.

18. The lipopeptide of claim 16 or claim 17, wherein the coiled-coil domain comprises a heptad motif represented by (abcdefg)n, wherein each a and d independently represent a hydrophobic residue, each b, c, e, f, and g independently represent a polar residue, and n is 1- 10.

19. The lipopeptide of any one of claims 1-18, wherein the amphipathic peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-69.

20. The lipopeptide of claim 19, wherein the amphipathic peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-69.

21. The lipopeptide of any one of claims 1-20, wherein the amphipathic peptide: a. comprises the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; or b. comprises an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18; or c. consists of the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO:

18.

22. The lipopeptide of claim 21, wherein the amino acid sequence is chemically modified.

23. The lipopeptide of any one of claims 1-22, wherein the hydrophilic polymer block comprises poly(ethylene glycol), poly(vinyl alcohol), poly(N-vinylpyrrolidone), polysarcosine, polyglutamic acid, poly(hydroxyethyl-L-asparagine), poly(hydroxyethyl-L- glutamine).

24. The lipopeptide of claim 23, wherein the hydrophilic polymer block comprises poly(ethylene glycol).

25. The lipopeptide of any one of claims 1-24, wherein the protease cleavage site comprises an amino acid sequence susceptible to cleavage by intracellular proteases.

26. The lipopeptide of any one of claims 1-25, wherein the protease cleavage site comprises an amino acid sequence susceptible to cleavage by an asparagine endopeptidase.

27. The lipopeptide of any one of claims 1-26, wherein the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a cathepsin protease.

28. The lipopeptide of any one of claims 1-27, wherein the protease cleavage site comprises an amino acid sequence susceptible to cleavage by a protease selected from the group consisting of legumain, cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L, cathepsin O, cathepsin P, cathepsin S, and cathepsin W.

29. The lipopeptide of any one of claims 1-28, wherein the protease cleavage site comprises an amino acid sequence susceptible to cleavage by cathepsin S.

30. The lipopeptide of any one of claims 1-29, wherein the protease cleavage site comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86.

31. The lipopeptide of any one of claims 1-30, wherein the protease cleavage site comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86.

32. The lipopeptide of any one of claims 1-31, wherein the protease cleavage site consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70-86.

33. The lipopeptide of any one of claims 1-32, wherein the T-helper epitope is selected from the group consisting of a flu, hepatitis B, tetanus toxin, diphtheria toxin, measles and covid T-helper epitope.

34. The lipopeptide of claim 33, wherein the T-helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-118.

35. The lipopeptide of claim 34, wherein the T-helper cell epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-118.

36. The lipopeptide of any one of claims 1-35, further comprising one or more antigen epitopes.

37. The lipopeptide of claim 36, wherein the one or more antigen epitopes comprises at least one of a B-cell epitope, CD4+ T-cell epitope, or CD8+ T-cell epitope.

38. The lipopeptide of claim 35 or 36, wherein the one or more antigen epitopes comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119- 124.

39. The lipopeptide of claim 35 or 36, wherein the one or more antigen epitopes comprises an amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124.

40. The lipopeptide of claim 35 or 36, wherein the one or more antigen epitopes consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-124.

41. A lipopeptide aggregate comprising at least two lipopeptides of any one of claims 1- 35.

42. A lipopeptide aggregate comprising at least two lipopeptides of any one of claims 36- 41.

43. The lipopeptide aggregate of claim 41 or 42, wherein the lipopeptide aggregate is a micelle.

44. A nanoparticle comprising the lipopeptide of any one of the claims 1-35, optionally wherein the lipopeptide further comprises an antigen.

45. A nanoparticle comprising the lipopeptide of any one of the claims 36-40.

46. The nanoparticle of claim 44 or 45, wherein the nanoparticle is self-assembled from a plurality of the lipopeptides.

47. The nanoparticle of any one of claims 41-46, wherein a plurality of the lipopeptide aggregates or nanoparticles: a. has a solubility in PBS buffer of at least 25 mg / mL as determined by laser nephelometry; and / or b. has a particle size distribution in the range of about 15 nm to about 75 nm, preferably about 20 nm to about 40 nm, as determined by dynamic light scattering (DLS) or electron microscopy; and / or c. has a homogeneity of about 0.95 to about 1.15 as indicated by polydispersity index (Mw / Mn) as assessed by multi-angle light scattering; and / or d. has a melting point (Tm) of at least about 66.72 °C as assessed by differential scanning calorimetry analysis (DSC); and / or e. has an average spacing of the one or more antigen epitopes of about 5 nm to about 10 nm, as determined or predicted by one or more of crystallography, NMR spectroscopy, molecular modeling, and mutagenesis analysis; and / or f. has a spacing of the one or more antigen epitopes that allows B-cell receptor (BCR) cross-linking, as predicted by cross-linking model (CLM).

48. A method of inducing generation of antibodies in a subject, wherein the method comprises administering the lipopeptide aggregate or nanoparticle of any one of claims 42-47 to the subject such that the subject generates antibodies against the antigen.

49. The method of claim 48, wherein the antigen is a self-antigen, and optionally wherein the self-antigen is PCSK9.

50. The method of claim 49, wherein the method reduces B-cell self-tolerance in the subject.

51. An immunogenic composition comprising the lipopeptide aggregate of any one of claims 41-43 or the nanoparticle of any one of claims 44-47.

52. The immunogenic composition of claim 51, further comprising an adjuvant.

53. The immunogenic composition of claim 52, wherein the adjuvant is selected from the group consisting of aluminum, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), monophosphoryl lipid A (MPL), squalene, cytosine phosphoguanine, QS-21, microcrystalline tyrosine, MF59, mannide monooleate, GLA, SLA, E6020, resiquimod (R848), imidazoquinolines, imiquimod, 3M-052, 2',3'-cGAMP, 3',3'-cGAMP, cGMP, cAMP, AMP, muramyl dipeptide, poly I:C, CpG and combinations thereof.

54. The lipopeptide aggregate, nanoparticle, or immunogenic composition of any one of claims 41-53, wherein the lipopeptide aggregate, nanoparticle, or immunogenic composition induces an immune response.

55. The lipopeptide aggregate, nanoparticle, or immunogenic composition of claim 54, wherein the lipopeptide aggregate, nanoparticle, or immunogenic composition induces generation of antibodies against at least a portion of the lipopeptide or nanoparticle.

56. The lipopeptide aggregate, nanoparticle, or immunogenic composition of claim 54 or 55, wherein the lipopeptide aggregate, nanoparticle, or immunogenic composition induces CD4+ T-cells against at least a portion of the lipopeptide or nanoparticle.

57. The lipopeptide aggregate, nanoparticle, or immunogenic composition of claim 54 or 55, wherein the lipopeptide aggregate, nanoparticle, or immunogenic composition induces CD8+ T-cells against at least a portion of the lipopeptide or nanoparticle.

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