Supramolecular peptide vaccines and compositions and methods thereof

Abstract

Aspects of the application relate to a protein comprising a charge-complementary peptide and an antigen. In some embodiments, an antigen comprises a peptide or protein, or a fragment thereof, located on a pathogen, such as a virus, a bacterium, a fungus, or a parasite. In some embodiments, the pathogen is pathogenic to mammals, such as humans. In some embodiments, an antigen comprises a tumor antigen. Aspects of the application also relate to methods including, but not limited to, a method comprising contacting the proteins with a cell and a method comprising administering the proteins to a subject. In some embodiments, the proteins are administered to a subject for the purposes of vaccinating the subject and / or for the purposes of cancer immunotherapy. Further aspects of the application relate to compositions, such as pharmaceutical compositions, comprising the proteins or a supramolecular structure thereof, such as a particle, a granule, or a hydrogel.

Description

[0001] SUPRAMOLECULAR PEPTIDE VACCINES AND COMPOSITIONS AND METHODS THEREOF RELATED APPLICATIONS

[0002] The application claims the benefit under 35 U. S. C. § 119(e) of U. S. Provisional Application number 63 / 711,088, filed October 23, 2024, which is incorporated by reference herein in its entirety.

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0004] The contents of the electronic sequence listing (U119770257WO00-SEQ-MFM.xml; Size: 16,756 bytes; and Date of Creation: October 23, 2025) is herein incorporated by reference in their entirety.

[0005] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under Grant No. EB032922 awarded by the National Institutes of Health and Grant No. CBET 1743432 awarded by the National Science Foundation. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Self-assembly is the spontaneous organization of molecules into a precise supramolecular architecture without any external guidance. Throughout nature, biomolecule self-assembly gives rise to various functional biomaterials that can perform complex tasks, such as molecular sensing and recognition, chemical synthesis, motility, and compartmentalization, as well as multi-scale hierarchical organization. There is increasing interest in biomolecule self-assembly for bottom-up fabrication of biomaterials for various technological applications.

[0009] SUMMARY

[0010] Aspects of the application relate to a protein comprising a co-assembling peptide and an antigen. In some embodiments, the co-assembling peptide comprises a positively charged peptide (e.g., wherein the antigen is fused to the N-terminus and / or the C-terminus of the positively charged peptide). In some embodiments, the co-assembling peptide comprises a negatively

[0011] #14496674v1 charged peptide (e.g., wherein the antigen is fused to the N-terminus and / or the C -terminus of the negatively charged peptide). In some embodiments, the antigen is a peptide or protein located on a pathogen (e.g., a virus, a bacterium, a fungus, or a parasite). In some embodiments, the pathogen is a mammalian pathogen (e.g., a human pathogen). In some embodiments, the antigen is a tumor antigen.

[0012] Aspects of the application also relate to a composition (e.g., a pharmaceutical composition, such as a vaccine) comprising a protein that comprises a co-assembling peptide and an antigen. In some embodiments, the composition comprises an assembly formed by coassembling peptides, such as a supramolecular structure (e.g., a particle, a granule, or a hydrogel), that comprises the protein. For example, in some aspects, the application relates to a vaccine.

[0013] In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises: (a) a protein and a co-assembling peptide, wherein the protein comprises a positively charged co-assembling peptide and an antigen (e.g., wherein the antigen comprises a peptide); and the co-assembling peptide comprises a negatively charged co-assembling peptide; and (b) an adjuvant.

[0014] In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises: (a) a protein and a co-assembling peptide, wherein the protein comprises a negatively charged co-assembling peptide and an antigen (e.g., wherein the antigen comprises a protein) and the co-assembling peptide comprises a positively charged co-assembling peptide; and (b) an adjuvant.

[0015] In some embodiments, the antigen is a peptide or protein located on a pathogen. In some embodiments, the pathogen is a mammalian pathogen. In some embodiments, the mammalian pathogen is a human pathogen.

[0016] In some embodiments, the antigen is a tumor antigen.

[0017] In some embodiments, the composition (e.g., wherein the composition is a pharmaceutical composition, such as a vaccine) comprises a crowding agent. In some embodiments, the crowding agent comprises polyethylene glycol (PEG).

[0018] In some embodiments, the adjuvant comprises a nucleic acid. In some embodiments, the nucleic acid comprises cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a single-stranded, unmethylated DNA, or a double-stranded RNA.

[0019] #14496674v1 In some embodiments, the composition (e.g., wherein the composition is a pharmaceutical composition, such as a vaccine) comprises a supramolecular structure that comprises the protein.

[0020] Aspects of the application also relate to methods. In some aspects, a method comprises contacting a protein that comprises a co-assembling peptide (e.g., wherein the protein is provided in a composition described herein) and an antigen with a cell (e.g., a mammalian cell, such as a human cell, including, but not limited to, an ex vivo cell or an in vitro cell). In some aspects, a method comprises administering a protein that comprises a co-assembling peptide and an antigen (e.g., wherein the protein is provided in a composition described herein) to a subject (e.g., a mammalian subject, such as a human subject). For example, in some aspects, the application relates to a method of vaccination.

[0021] In some embodiments, a method comprises administering a composition (e.g., a pharmaceutical composition, such as a vaccine) to a subject in need thereof, wherein the composition comprises a protein and a co-assembling peptide, wherein: (i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or (ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide.

[0022] In some embodiments, the protein comprises the negatively charged co-assembling peptide and the antigen, and the co-assembling peptide comprises the positively charged peptide. In some embodiments, the antigen comprises a protein.

[0023] In some embodiments, the protein comprises the positively charged co-assembling peptide and the antigen, and the co-assembling peptide comprises the negatively charged peptide. In some embodiments, the antigen comprises a peptide.

[0024] In some embodiments, the subject has or is at risk of having a disease or a disorder associated with reduced immunity or lack of immunity to the antigen.

[0025] In some embodiments, the antigen is a peptide or protein located on a pathogen. In some embodiments, the pathogen is a mammalian pathogen. In some embodiments, the mammalian pathogen is a human pathogen. In some embodiments, the subject is at risk of infection with the pathogen. In some embodiments, the subject has been exposed to the pathogen or is at risk of exposure to the pathogen.

[0026] #14496674v1 In some embodiments, the antigen is a tumor antigen. In some embodiments, the subject has or is at risk of having cancer. In some embodiments, the cancer is associated with expression of the tumor antigen.

[0027] In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject.

[0028] In some embodiments, the composition (e.g., wherein the composition is a pharmaceutical composition, such as a vaccine) further comprises a crowding agent. In some embodiments, the crowding agent comprises polyethylene glycol (PEG).

[0029] In some embodiments, the composition (e.g., wherein the composition is a pharmaceutical composition, such as a vaccine) further comprises an adjuvant. In some embodiments, the adjuvant comprises a nucleic acid. In some embodiments, the nucleic acid comprises cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a singlestranded, unmethylated DNA, or a double-stranded RNA.

[0030] In some aspects, a method comprises contacting a positively charged peptide with a negatively charged peptide, thereby producing a supramolecular structure, wherein the positively charged peptide and / or the negatively charged peptide comprises an antigen. In some embodiments, the positively charged peptide and the negatively charged peptide are contacted in the presence of a crowding agent (e.g., a crowding agent described herein, such as polyethylene glycol (PEG)). For example, in some aspects, the application relates to a method of producing a composition (e.g., a pharmaceutical composition, such as a vaccine) described herein.

[0031] In some embodiments, a method of producing a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises contacting a positively charged co-assembling peptide with a negatively co-assembling charged peptide, thereby producing a supramolecular structure, wherein the positively charged peptide and / or the negatively charged peptide comprises an antigen (e.g., an antigen described herein). In some embodiments, the positively charged peptide and the negatively charged peptide are contacted in the presence of a crowding agent (e.g., a crowding agent described herein).

[0032] Accordingly, further aspects of the application relate to uses of a protein (e.g., a protein comprising a co-assembling peptide described herein) or a composition comprising the protein (e.g., wherein the composition is a pharmaceutical composition described herein). For example, in some aspects, the application relates to a composition for use in a vaccine, wherein the

[0033] #14496674v1 composition comprises a protein and a co-assembling peptide, wherein: (i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or (ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide. As a further example, in some aspects, the application relates to a composition for use in a method of vaccination, wherein the method comprises administering to a subject a composition comprising a protein and a co-assembling peptide, wherein: (i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or (ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the coassembling peptide comprises a positively charged co-assembling peptide. As a further example, in some aspects, the application relates to use of a composition in the manufacture of a vaccine, the composition comprising a protein and a co-assembling peptide, wherein: (i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or (ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide.

[0034] BRIEF DESCRIPTION OF DRAWINGS FIG. 1A-1C show non-limiting embodiments of co-assembly tags based on charge (CATCH) complementarity peptides and supramolecular compositions (e.g., pharmaceutical compositions, such as vaccines) thereof.

[0035] FIG. 2 shows CATCH can form nanofibrils or nano-sized granules depending on formation conditions.

[0036] FIGs. 3A-3B shows CATCH granules undergo endosomal escape following entry into cells. Brightfield (FIG. 3A) and YFP channel (FIG. 3B) of an MDA (fibroblast) cell line expressing Gal8-YFP (Yellow Fluorescent Protein) following treatment with CATCH Granules. Gal8 is a cytosolic protein that recruits to endosomes following their disruption.

[0037] FIG. 4 show's CATCH can be linked to peptides to serve as an antigen delivery vehicle. FIGs. 5A-5D shows CATCH granules transfect a dendritic cell line (DC2.4s). FIG. 5A show's fluorescence microscopy overlay of GFP and DAPI signal from DC2.4s transfected with

[0038] #14436674' / : CATCH granules. FIG. 5B shows flow cytometry of GFP intensity of DC2.4s following treatment with CATCH granules. FIG. 5C shows fluorescence microscopy of DC2.4s treated with CATCH Granules containing CATCH(6K+). FIG. 5D shows flow cytometry data of GFP positive cells for DC2.4s treated with CATCH(6K+) granules or their individual components.

[0039] FIG. 6 shows CATCH granules induce significant cross presentation in DC2.4s. Flow cytometry of MHCI-SIINFEKL intensity of DC2.4s that are untreated, treated with CATCH granules, or treated with SIINFEKL (SEQ ID NO: 13) peptide (Positive Control).

[0040] FIG. 7 shows the presence of a population of GFP+ and MHCI-SIINFEKL+ cells indicates entry of granules into DC2.4s and cross-presentation; Figures: Flow cytometry of GFP vs MHCI-SIINFEKL intensity of DC2.4s that are untreated, treated with CATCH Granules, or treated with SIINFEKL (SEQ ID NO: 13) peptide (Positive Control).

[0041] FIGs. 8A-8C shows CATCH peptides conjugated to an antigen retain transfection ability. FIG. 8A shows non-limiting examples of a CATCH peptide fused to an antigen (e.g., a peptide or protein, such as MHC-I restricted ovalbumin epitope, MHC-II restricted ovalbumin, restricted GP100, full-length ovalbumin). FIGs. 8B-8C show fluorescence microscopy and flow cytometry of DC2.4s treated with CATCH granules containing varying percentages of CATCH(6K+)-SIINFEKL indicate that transfection of dendritic cells is maintained with antigen incorporation.

[0042] FIG. 9 shows contour plots of GFP intensity vs. MHCI-SIINFEKL intensity for DC2.4s that were treated with CATCH granules containing 0% (Left) or

[0043] 100% (Right) of CATCH(6K+)-SIINFEKL.

[0044] FIG. 10 shows dynamic light scattering measurements of CATCH(6E-)-Nanoluciferase (left panel) and CATCH(6E-)-GFP assemblies (right panel) prepared with 0%, 10%, or 100% CATCH(+)-SIINFEKL.

[0045] FIG. 11 shows MHCI-SIINFEKL presentation of DC2.4s treated with CATCH assemblies with 0, 10, 50, or 100% modification with SIINFEKL (SEQ ID NO: 13) antigen compared to untreated, CATCH(+)-antigen alone, and a SIINFEKL (SEQ ID NO: 13) positive control.

[0046] FIGs. 12A-12B show non-limiting examples of adjuvants that can be used to promote entry of CATCH assemblies into cells.

[0047] FIGs. 13A-13B show data from analyzing samples containing CpG alone and CATCH assemblies formed at 1:1, 1:2, 1:10, 1:100 ratios of CATCH(6K+) to CpG (FIG. 13A) and concentrations of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) within

[0048] #14496674v1 each sample for different starting concentrations or ratios of CATCH(6K+) to cGAMP (FIG. 13B).

[0049] FIG. 14 shows CD86 (left panel) and MHCI-SIINFEKL (right panel) detected in bone marrow-derived dendritic cells treated with CpG incorporated CATCH assemblies at a 1:10 ratio of CpG to CATCH(6K+).

[0050] FIG. 15 shows microscopy (left panel) and DLS (right panel) analyses of splenic tissue following intravenous injection of CATCH assemblies comprising CATCH(+)-SIINFEKL.

[0051] DETAILED DESCRIPTION

[0052] Aspects of the application relate to a protein comprising a charge-complementary peptide (e.g., CATCH peptides) and an antigen (e.g., wherein the protein is a fusion protein), which can be used in compositions and methods for vaccinating a subject (e.g., vaccinating a subject against a pathogen) and / or administering an immunotherapy to a subject (e.g., administering a cancer immunotherapy to a subject), such as a mammalian subject (e.g., a human subject). See, e.g., FIGs. 1 A-1C. The application is based, at least in part, on the surprising and unexpected discovery that compositions comprising co-assembling peptides can be used to deliver an antigen to immune cells (e.g., dendritic cells), which results in presentation of the antigen on the surface of the immune cells. Accordingly, the co-assembling peptides can be useful as vaccines (e.g., to elicit an immune response against an antigen in a subject). The application is also based, at least in part, on the surprising and unexpected discovery that compositions comprising co-assembling peptides can be formulated with one or more additional components that promote the formation of assemblies, such as supramolecular structures (e.g., granules, a particle, and / or a hydrogel), and that can be used to deliver an antigen to immune cells (e.g., dendritic cells), which results in presentation of the antigen on the surface of the immune cells. In some embodiments, the one or more additional components comprise one or more crowding agents (e.g., an intermolecular crowding agent, such as polyethylene glycol (PEG)). In some embodiments, the one or more additional components comprise one or more adjuvants, such as one or more nucleic acids (e.g., a nucleic acid for a single-stranded, unmethylated DNA, a synthetic double-stranded RNA and / or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP)). In some embodiments, the one or more adjuvants comprise an adjuvant that targets and / or affects an endosomal process (e.g., a TLR-dependent process, such as a TLR3-dependent process or a TLR9-dependent

[0053] #14496674v1 process). In some embodiments, the one or more adjuvants comprise an adjuvant that targets and / or affects a cytosomal process (e g.. STING pathway-associated process). Accordingly, compositions and methods described herein can be used for the induction of antigen-specific anti-tumor therapy or for the immunization of a subject (e.g., immunization against a pathogen).

[0054] As used herein, “co-assembling peptides”, “co-assembly peptides”, or “charge-complementary peptides” refers to positive peptides and negative peptides that each comprise one or more charge-complementary segments which are capable of binding to each other under certain conditions to form larger order structures (e.g., assemblies, such as supramolecular structures including, but not limited to, granules, particles, hydrogels, etc.). Accordingly, coassembling peptides comprise self-assembly properties under conditions which stimulate granule (e.g., particle) formation (e.g., in the presence of at least one crowding agent and when present in a neutral aqueous solution, such as a buffer).

[0055] Non-limiting embodiments of charge-complementary peptides are depicted in FIGs. 1, 2, 4, and 8 A. Further descriptions of charge-complementary peptides can also be found in, for example, U. S. Patent No. 10,906,939, International Patent Application No. PCT / US2022 / 025426 (published as WO 2022 / 225,987), and International Patent Application No. PCT / US2023 / 065823 (published as WO 2023 / 201,370), which are each incorporated by reference herein in their entirety.

[0056] In some embodiments, the one or more charge-complementary segments comprise arrangements of charged amino acids (e.g., cationic amino acids and anionic amino acids) and hydrophobic amino acids. In some embodiments, the one or more charge-complementary segments may further comprise polar amino acids. Accordingly, as used herein “cationic amino acids” refer to residues with positively charged side (or “R” groups) groups and can include, but are not limited to, lysine, arginine, and histidine. As used herein, “anionic amino acids” refers to residues that have negatively charged side groups and can include, but are not limited to, aspartate and glutamate. As used herein “polar amino acids” refers to residue that have polar, uncharged side groups, and can include, but are not limited to, serine, threonine, cysteine, proline, asparagine, and glutamine. As used herein “hydrophobic amino acids” refers to residues that have nonpolar, aliphatic or aromatic side groups and can include, but are not limited to, glycine, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tyrosine, and tryptophan. Amino acids can each be selected from natural and non-natural amino acids. Natural and non-

[0057] #14496674v1 natural amino acids can also be selected from a cationic amino acid, an anionic amino acid, a hydrophobic amino acid, or a polar amino acid. In some embodiments, the amino acids (e g., hydrophobic, anionic, cationic, polar, etc.) can be modified, such as iodinated, fluorinated, or otherwise labeled.

[0058] In some embodiments, co-assembling peptides are at least 3 amino acids in length. In some embodiments, co-assembling peptides comprise 3-50 amino acids in length. In some embodiments, co-assembling peptides comprise 3-5, 5-8, 8-11, 11-15, 15-20, 20-25, 25-30, 30-35, 40-45, 45-50, or more amino acids in length. In some embodiments, co-assembling peptides, comprise or consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26, 27, 28, 29, 30, 31, 32, 33. 34. 35, 36, 37, 38, 39, 40, 41, 42. 43, 44, 45, 46, 47, 48, 49, 50, or more amino acids in length. In some embodiments, co-assembling peptides are the same length. However, in other embodiments, the lengths of the co-assembling peptides may differ and still form interactions that drive granule formation.

[0059] While, in some aspects, co-assembling peptides structures / sequences may be described herein by referring to the order of amino acids found therein relative to the N-terminus or the C-terminus of the peptide, other aspects of the present disclosure may refer to amino acids in coassembling peptides by their position relative to other structures / sequences within the peptide. Accordingly, referring to amino acids in co-assembling peptides by describing their relative positions (e.g., as being N-terminal to, C-terminal to, flanking, etc. or as “first”, “second”, “third”, etc.) should not be considered to limit the sequence or structure of the given peptide embodiment unless explicitly stated. The following embodiments relate to co-assembling peptide structures which includes, but is not limited to, the one or more charge-complementary segments that the peptides comprise.

[0060] In some embodiments, the co-assembling functional properties of the charged, coassembling peptides of the present disclosure stem from the structure of said peptides encoded by their primary sequences which includes a core sequence of alternating hydrophobic and hydrophilic residues. The “core sequence” refers to a charged (e.g., either positive or negative) segment of amino acids comprising either at least two hydrophobic residues which flank a cationic residue or anionic residue, or a hydrophobic residue flanked by at least one cationic residue or anionic residue and a hydrophilic residue (e.g., either polar or charged) (see Table 1). As such, both a hydrophobic core component and a charged core component, provided by the

[0061] #14496674v1 appropriate residues comprising said side chain chemistries, promote granule (e.g., particle) formation (see Table 1). As such, the core structure must be sufficiently hydrophobic and charged, while other residues found in the N-terminal and / or C-terminal direction of the core structure may be cationic or anionic to further promote self-assembly.

[0062] In some embodiments, one core sequence is found in a positive peptide and / or negative peptide. In some embodiments, a positive peptide and / or a negative peptide may comprise two, three, four, or more core sequences. In some embodiments, more than one core sequence is found in a positive peptide and / or negative peptide located adjacent to each other in the primary sequence. In some embodiments, more than one core sequence is found in a positive peptide and / or negative peptide and separated by one or more (e.g., two, three, four, or more) residues. In some embodiments, a positive peptide and / or a negative peptide comprise more than one core sequence comprising the same amino acid arrangements (e.g., a positive peptide with a core sequence of KFK and KFK or, as a further example, a negative peptide with a core sequence of DFD and DFD). In some embodiments, a positive peptide and / or a negative peptide comprise more than one core sequence comprising different amino acid arrangements (e.g., a positive peptide with a core sequence of KFK and QFK or, as a further example, a negative peptide with a core sequence of DFD and QFD ).

[0063] In some embodiments, a core sequence comprises one or more amino acids which are located N-terminal to the core sequence and / or C-terminal to the core sequence. In some embodiments, a core sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids which are located N-terminal to the core sequence and / or C-terminal to the core sequence. In some embodiments, a core sequence is flanked by sequences in the N-terminal direction and C-terminal direction (e.g., a core sequence with an equal or non-equal number of amino acids flanking it in the N-terminal and C-terminal direction). In some embodiments, the flanking sequences may be the same sequence or different sequences. In some embodiments, however, a core sequence is found at the N-terminus, the C -terminus, or both termini.

[0064] In some embodiments, a positive peptide may comprise 1-50 core sequences of at least 3 amino acids in length (see, e.g., the positively-charged core sequences as described in Table 1 herein). In some embodiments, a positive peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (e.g., up to 15, up to 20, up to 25, etc.) core sequences of at least 3 amino acids in length, optionally wherein any or all of the core sequences are separated by at least one amino acid. In

[0065] #14496674v1 some embodiments, a positive peptide may comprise 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-7, 7-12, 12-17, 17-22, 22-27, 27-32, 32-37, 37-42, 42-47, 47-50, 3-8, 8-13. 13-18, 18-23, 23-28, 28-33, 33-38, 38-43, 43-48, 48-50, 4-9, 9-14, 14-19, 19-24, 24-29, 29-34, 34-39, 39-44. 44-49, 49-50, 5-10, or 45-50 or more core sequences of at least 3 amino acids in length, optionally wherein any or all of the core sequences are separated by at least one amino acid. In some embodiments, wherein more than one core sequence is found in a positive peptide, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct core sequences of at least 3 amino acids in length which differ in amino acid sequence by at least one amino acid.

[0066] In some embodiments, a negative peptide may comprise 1-50 core sequences of at least 3 amino acids in length (see, e.g., the negatively-charged core sequences as described in Table 1 herein). In some embodiments, a negative peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (e.g., up to 15, up to 20, up to 25, etc.) core sequences of at least 3 amino acids in length, optionally wherein any or all of the core sequences are separated by at least one amino acid. In some embodiments, a negative peptide may comprise 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-7, 7-12, 12-17, 17-22, 22-27, 27-32, 32-37, 37-42, 42-47, 47-50, 3-8, 8-13, 13-18, 18-23, 23-28. 28-33, 33-38, 38-43, 43-48, 48-50, 4-9, 9-14, 14-19, 19-24, 24-29, 29-34, 34-39, 39-44, 44-49, 49-50, 5-10, or 45-50 or more core sequences of at least 3 amino acids in length, optionally wherein any or all of the core sequences are separated by at least one amino acid. In some embodiments, wherein more than one core sequence is found in a negative peptide, there may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct core sequences of at least 3 amino acids in length which differ in amino acid sequence by at least one amino acid.

[0067] Apart from the co-assembling peptide species provided herein, other species within the genus of co-assembling peptides may be designed using the teachings provided herein. For example, charge-complementarity between the positive and negative peptides is necessary for granule (e.g., particle) formation. Accordingly, a minimum of two charged residues (e.g., at least one cationic residue in the positive peptide and at least one anionic residue in the negative peptide) may be necessary to electrostatically repress similarly charged peptides and prevent their association.

[0068] Additionally, the present disclosure describes types of amino acid substitutions that can be made to engineer co-assembling peptides. For example, for the hydrophobic component of the core sequence, phenylalanine could be switched to tryptophan but could not be switched to a

[0069] #14496674v1 polar or charged residue (see Table 1). In a separate example, the charged residues of the core component could comprise arginine instead of lysine and / or aspartate instead of glutamate (see Table 1). For other residues found either in the core structure or other positions of the charged peptides, polar residues could be swapped as appropriate (e.g., asparagine substituted for glutamine; see Table 1).

[0070] Table 1 shown below provides primary amino acid sequences that are found in coassembling peptides. For example, Table 1 describes specific examples of co-assembling peptide sequences and core sequences that may be used to design other co-assembling peptides capable of forming granules.

[0071] Table 1, Non -Limiting Embodiments of Co-assembling Peptide Core Sequences

[0072]

[0073]

[0074] #14496674V1

[0075]

[0076] In some embodiments, a positive peptide can include at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids which can each be independently selected from a cationic amino acid and a hydrophobic amino acid, and wherein at least one amino acid of said amino acids can be a cationic amino acid and at least one

[0077] #14496674v1 said amino acids can be a hydrophobic amino acid. In some embodiments, the positive peptide may comprise the at least 3 amino acids, the at least 4 amino acids, the at least 5 amino acids, or the at least 7 amino acids which are arranged in alternating order by side chain chemistry (e.g., cationic amino acid-hydrophobic amino acid-cationic amino acid, such as to be part of a cationic core sequence that binds to an anionic core sequence).

[0078] In some embodiments, a positive peptide comprises a cationic core sequence comprising at least 3 amino acids, wherein a first hydrophobic amino acid is flanked by at least one cationic amino acid and a first hydrophilic amino acid which is either cationic or polar. In some embodiments, the positive peptide further comprises a second hydrophobic amino acid N-terminal to the cationic core sequence, a third hydrophobic amino acid C-terminal to the cationic core sequence, or the second hydrophobic amino acid and the third hydrophobic amino acid. In some embodiments, the positive peptide further comprises a second hydrophilic amino acid N-terminal to the second hydrophobic amino acid and a third hydrophilic amino acid C-terminal to the third hydrophobic amino acid, wherein the second hydrophilic amino acid and the third hydrophilic amino acid are either cationic or polar. In some embodiments, the positive peptide further comprises one or more (e.g., 1, 2, 3, 4, or more) cationic or polar amino acids that are found N-terminal to and / or C-terminal to the second hydrophilic amino acid and the third hydrophilic amino acid.

[0079] In some embodiments, a positive peptide comprises a cationic core sequence comprising at least 3 amino acids, wherein a first hydrophobic amino acid and a second hydrophobic amino acid flank a cationic amino acid. In some embodiments, the positive peptide further comprises a first hydrophilic amino acid N-terminal to the cationic core sequence, a second hydrophilic amino acid C-terminal to the cationic core sequence, or both the first hydrophilic amino acid and the second hydrophilic amino acid, wherein the first hydrophilic amino and the second hydrophilic amino acid are either cationic or polar. In some embodiments, the positive peptide further comprises a third hydrophobic amino acid N-terminal to the first hydrophilic amino and a fourth hydrophobic amino acid C-terminal to the second hydrophilic amino acid. In some embodiments, the positive peptide further comprises one or more (e.g., 1, 2, 3, 4, or more) hydrophilic amino acids that are found N-terminal to third hydrophobic amino acid and / or C-terminal to the fourth hydrophobic amino acid.

[0080] #14496674v1 In some embodiments, the positive peptide segment comprises a sequence of QQKFKFKFKQQ (SEQ ID NO: 1) (also referred to herein as CATCH (4+)), KQKFKFKFKQK (SEQ ID NO: 3) (also referred to herein as CATCH (6+)), KQQFKFKFKQQ (SEQ ID NO: 5), QQKFQFQFKQQ (SEQ ID NO: 6) (also referred to herein as CATCH (2+)), or KQQFKFKFQQK (SEQ ID NO: 8) (also referred to herein as CATCH (4+), SQQKFKFKFKQQWENLYFQ (SEQ ID NO: 10), or Ac-QQKPKPKPKQQ-Am [mCATCH(+)] (SEQ ID NO: 12).

[0081] In some embodiments, a negative peptide can include at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids which can each be independently selected from an anionic amino acid and a hydrophobic amino acid, and wherein at least one amino acid of said amino acids can be an anionic amino acid and at least one said amino acids can be a hydrophobic amino acid. In some embodiments, the negative peptide may comprise the at least 3 amino acids, the at least 4 amino acids, the at least 5 amino acids, or the at least 7 amino acids which are arranged in alternating order based on side chain chemistry (e.g., anionic amino acid-hydrophobic amino acid-anionic amino acid, such as to be part of an anionic core sequence).

[0082] In some embodiments, a negative peptide comprises an anionic core sequence comprising at least 3 amino acids, wherein a first hydrophobic amino acid is flanked by at least one anionic amino acid and a first hydrophilic amino acid which is either anionic or polar. In some embodiments, the negative peptide further comprises a second hydrophobic amino acid N-terminal to the anionic core sequence, a third hydrophobic amino acid C-terminal to the anionic core sequence, or the second hydrophobic amino acid and the third hydrophobic amino acid. In some embodiments, the negative peptide further comprises a second hydrophilic amino acid N-terminal to the second hydrophobic amino acid and a third hydrophilic amino acid C-terminal to the third hydrophobic amino acid, wherein the second hydrophilic amino acid and the third hydrophilic amino acid are either anionic or polar. In some embodiments, the negative peptide further comprises one or more (e.g., 1, 2, 3, 4, or more) anionic or polar amino acids that are found N-terminal to and / or C-terminal to the second hydrophilic amino acid and the third hydrophilic amino acid.

[0083] In some embodiments, a negative peptide comprises an anionic core sequence comprising at least 3 amino acids, wherein a first hydrophobic amino acid and a second

[0084] #14496674v1 hydrophobic amino acid flank an anionic amino acid. In some embodiments, the negative peptide further comprises a first hydrophilic amino acid N-terminal to the anionic core sequence, a second hydrophilic amino acid C-terminal to the anionic core sequence, or both the first hydrophilic amino acid and the second hydrophilic amino acid, wherein the first hydrophilic amino and the second hydrophilic amino acid are either anionic or polar. In some embodiments, the negative peptide further comprises a third hydrophobic amino acid N-terminal to the first hydrophilic amino and a fourth hydrophobic amino acid C-terminal to the second hydrophilic amino acid. In some embodiments, the negative peptide further comprises one or more (e.g., 1, 2, 3, 4, or more) hydrophilic amino acids that are found N-terminal to third hydrophobic amino acid and / or C-terminal to the fourth hydrophobic amino acid.

[0085] In some embodiments, a charge-complementary peptide is a positively charged peptide. In some embodiments, a positively peptide comprises an amino acid sequence of QQKFKFKFKQQ (SEQ ID NO: 1), KQKFKFKFKQK (SEQ ID NO: 3). KQQFKFKFKQQ (SEQ ID NO: 5), QQKFQFQFKQQ (SEQ ID NO: 6), KQQFKFKFQQK (SEQ ID NO: 8), SQQKFKFKFKQQWENLYFQ (SEQ ID NO: 10), or Ac-QQKPKPKPKQQ-Am (SEQ ID NO: 12). In some embodiments, a charge-complementary peptide is a negatively charged peptide. In some embodiments, a negatively charged peptide comprises an amino acid sequence of In some embodiments, the negative peptide segment comprises a sequence of EQEFEFEFEQE (SEQ ID NO: 2), QQEFEFEFEQQ (SEQ ID NO: 4), QQEFQFQFEQQ (SEQ ID NO: 7), SEQEFEFEFEQEWENLYFQ (SEQ ID NO: 9), or Ac-EQEPEPEPEQE-Am (SEQ ID NO: 11).

[0086] In some embodiments, an antigen is fused to the N-terminus and / or the C-terminus of a charge-complementary peptide. In some embodiments, an antigen is fused to the N-terminus and / or the C-terminus of a positively charged peptide. In some embodiments, an antigen is fused to the N-terminus and / or the C-terminus of a negatively charged peptide. In some embodiments, an antigen is fused to a charge-complementary peptide directly (e.g., wherein the antigen is linked by a covalent bond to the N-terminus or the C-terminus of the charge-complementary peptide). In some embodiments, an antigen is fused to a charge-complementary peptide via a linker (e.g., wherein the antigen is connected to a linker and the linker is connected to the N-terminus or the C-terminus of the charge-complementary peptide). In some embodiments, a linker is an amino acid linker, such as a flexible linker (e.g., a hydrophilic linker, such as a glycine-serine-rich linker).

[0087] #14496674v1 In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises a protein comprising a charge-complementary peptide and an antigen (e.g., wherein the charge-complementary peptide comprises a positively charged peptide or a negatively charged peptide). In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises a plurality of proteins (e.g., two, three, four, five, or more than five proteins), wherein each protein of the plurality comprises a charge-complementary peptide (e.g., a positively charged peptide or a negatively charged peptide) and an antigen. In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises a positively charged peptide and a negatively charged peptide that are capable of co-assembling (e.g., under one or more conditions described herein) into an assembly (e.g., a supramolecular structure, such as a granule, particle, or hydrogel), wherein the positively charged peptide and / or the negatively charged peptide is fused to an antigen (e.g., fused at the N-and / or C-terminus of the charge-complementary peptide), such as directly fused or indirectly fused (e.g., fused via a linker). In some embodiments, at least 10% of the total amount of a charge-complementary peptide in a composition (e.g., at least 10% of the positively charged peptide or at least 10% of the negatively charged peptide in a composition) is fused to an antigen. In some embodiments, 10-100% of the total amount of a charge-complementary peptide in a composition (e.g., 10%-25%, 25%-50%, 50%-75%, or 75%-100% of the total amount of a positively charged peptide or a negatively charged peptide in a composition) is fused to an antigen. In some embodiments, 100% of the total amount of a charge-complementary peptide in a composition is fused to an antigen.

[0088] In some embodiments, an antigen comprises a peptide or protein. Antigens induce or elicit an immune response in a subject. In some embodiments, the antigen is a foreign body relative to the subject. In some embodiments, an antigen comprises one or more epitopes (e.g., one, two, three, four, five, or more than five epitopes). In some embodiments, an antigen comprises one or more epitopes that are recognized by an antibody or an antigen-binding fragment thereof. In some embodiments, an antigen comprises a peptide, wherein the peptide comprises at least 3 amino acids (e.g., 3-50 amino acids, such as 3-10, 10-20, 20-30, 30-40, or 40-50 amino acids). In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises a protein comprising a positively charged peptide and an antigen that comprises a peptide. In some embodiments, an antigen comprises a protein, wherein the

[0089] #14496674v1 protein comprises more than 50 amino acids (e.g., at least 60 amino acids, such as 60-100, 100-250, 250-500, 500-1,000, or more than 1,000 amino acids). In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises a protein comprising a negatively charged peptide and an antigen that comprises a protein.

[0090] In some embodiments, an antigen comprises a peptide or protein, or a fragment thereof (e.g., an immunogenic peptide), located on a pathogen. In some embodiments, a pathogen is pathogenic (e.g., infects and / or causes one or more symptoms of a disease, disorder, or condition) to mammals (e.g., humans). Examples of pathogens and pathogen-associated conditions include, but are not limited to, Adenoviridae, Picornav iridae, Herpesviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Poxviridae, Rhabdoviridae, Togaviridae, Mycobacterium tuberculosis. Streptococcus, Pseudomonas, Shigella, Campylobacter, Salmonella, Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocytis, Stachybotrus, Bacillus anthracis, Clostridium botulinum, Mycobacterium leprae, Yersinia pestis, Rickettsia prowazekii, Bartonella spp., malaria, amoebiasis, babesiosis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, Chagas disease, leishmaniasis, African trypanosomiasis (sleeping sickness), Acanthamoeba keratitis, or primary amoebic meningoencephalitis (naegleriasis). In some embodiments, a pathogen is a vims, a bacterium, a fungus, or a parasite. In some embodiments, the pathogen is pathogenic (e.g., infects and / or causes one or more symptoms of a disease, disorder, or condition) to mammals (e.g., humans).

[0091] In some embodiments, an antigen comprises a tumor antigen. In some embodiments, a tumor antigen is associated with a cancer selected from: acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple

[0092] #14436674' / : idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g,, B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms’ tumor, renal cell carcinoma); liver cancer (e g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma;

[0093] #14496674v1 myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva).

[0094] In some embodiments, an antigen comprises an OT-I restricted Ovalbumin peptide, such as amino acids 257-264 of Ovalbumin(e.g., wherein the antigen comprises the amino acid sequence SIINFEKL (SEQ ID NO: 13)). In some embodiments, an antigen comprises an MHC1I (I-A / I-E) restricted Ovalbumin, such as amino acids 323-339 of Ovalbumin (e.g., wherein the antigen comprises the amino acid sequence ISQAVHAAHAEINEAGR (SEQ ID NO: 15)). In some embodiments, an antigen comprises an MHCI (H2-Db) restricted GP100, such as amino acids 25-33 of GP100 (e.g., wherein the antigen comprises the amino acid sequence EGSRNQDWL (SEQ ID NO: 16)). In some embodiments, an antigen comprises a full-length Ovalbumin protein (e.g., w'herein the full-length Ovalbumin protein is a recombinant protein). In some embodiments, an antigen comprises a full-length GP100 protein (e.g., wherein the full-length GP100 protein is a recombinant protein). In some aspects, the application relates to nucleic

[0095] #14436674' / : acids. For example, in some embodiments, a nucleic acid (e.g., a nucleic acid comprising DNA, RNA, or a combination thereof) comprising a nucleotide sequence (e.g., a gene sequence) encoding a protein described herein (e.g., a protein comprising a charge-complementary peptide and an antigen, such as wherein the charge-complementary peptide and the agent are connected directly or via a linker). In some embodiments, a nucleic acid comprises a vector (e.g., a plasmid).

[0096] Further aspects of the application relate to compositions (e.g., pharmaceutical compositions, such as vaccines) comprising at least one charge-complementary peptide. In some embodiments, a composition comprises a positively charged peptide and a negatively charged peptide, wherein one or both of the peptides are fused to an antigen. In some embodiments, a composition comprises a supramolecular structure formed by co-assembly of proteins comprising a charge-complementary peptide (e.g., a granule, a particle, and / or a hydrogel). In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) that comprises a charge-complementary peptide fused to an antigen comprises one or more pharmaceutically acceptable additives (e.g., an adjuvant and / or an excipient).

[0097] In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises one or more adjuvants, such as cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a single-stranded, unmethylated DNA (e.g., a CpG nucleic acid), and / or a double- stranded RNA (e.g., PolyLC). In some embodiments, the one or more adjuvants comprise an adjuvant that targets and / or affects a cytosolic process (e.g., a process associated with the STING pathway). In some embodiments, the one or more adjuvants comprise an adjuvant targets and / or affects an endosomal process (e.g., aTLR-dependent process, such as a TLR3-depedent process or a TRL9 -dependent process). In some embodiments, an adjuvant that targets and / or affects a TLR3-depedent process comprises a single-stranded, unmethylated DNA, such as a CpG nucleic acid. In some embodiments, an adjuvant that targets and / or affects a TLR9-depedent process comprises a TRL9-dependent process comprises a double-stranded RNA, such as PolyLC. In some embodiments, the molar ratio of charge-complementary peptide to adjuvant in a composition is equal (e.g., wherein the molar ratio of positively charged peptide to adjuvant is equal or wherein the molar ratio of negatively charged peptide to adjuvant is equal). In some embodiments, molar the ratio of charge-complementary peptide to adjuvant in a composition is non-equal (e.g., wherein the molar ratio of positively charged peptide to adjuvant

[0098] #14496674v1 is non-equal or wherein the molar ratio of negatively charged peptide to adjuvant is non-equal). In some embodiments, the molar ratio of charge-complementary peptide to adjuvant is between 1:1 and 1:200 (e.g., wherein the molar ratio of charge-complementary peptide to adjuvant is 1:1, 1:2, 1:5, 1:10, 1:25, 1:50, 1:75, or 1:100).

[0099] Further aspects of the application also relate to methods. For example, in some aspects, the application relates to a method comprising contacting a cell with a charge-complementary peptide comprising an antigen with a cell (e.g., a mammalian cell, such as a human cell), which includes, but is not limited to, an ex vivo or in vitro cell. In some embodiments, a mammalian cell (e.g., a human cell) is an immune cell. Non-limiting examples of immune cells include dendritic cells, NK cells, macrophages, neutrophils, mast cells, T-cells, B-cells, eosinophils, and basophils. In some embodiments, an immune cell is a dendritic cell. In some embodiments, a dendritic cell is a bone marrow -derived dendritic cell.

[0100] As a further example, in some aspects the application relates to a method comprising administering a charge-complementary peptide comprising an antigen to a subject (e.g., a mammalian subject, such as a human subject), such as wherein the method comprises administering a composition (e.g., a pharmaceutical composition, such as a vaccine) to a subject in need thereof. In some embodiments, the subject is characterized as having or at risk of having a disease, disorder, or condition (e.g., wherein the antigen is selected to promote an immune response in the subject to treat, prevent, assist with the prevention of, or assist with the treatment of the disease, disorder, or condition, such as wherein the antigen is a peptide or protein located on a pathogen or wherein the antigen is a tumor antigen). In some aspects, the disclosure provides methods of inducing an antigen-specific immune response in the subject (e.g., by administering a vaccine as described herein). In some embodiments, the vaccine produces an anti-antigen antibody titer that is increased relative to a control (e.g., an untreated subject, or the subject before they were administered the vaccine). In some embodiments, the subject is characterized as having or at risk of having a disease, disorder, or condition associated with reduced immunity or lack of immunity to an antigen described herein (e.g., wherein the antigen is a peptide or protein located on a pathogen or wherein the antigen is a tumor antigen). In some embodiments, the subject is characterized as having or at risk of having a cancer associated with expression of a tumor antigen described herein. In some embodiments, the subject is characterized as ha ving been exposed to a pathogen or is at risk of exposure to a pathogen

[0101] #14496674v1 described herein. In some embodiments, administration of a charge-complementary peptide comprising an antigen (e.g., wherein the charge-complementary peptide comprising the antigen is provided in a composition, such as a pharmaceutical composition, including a vaccine) to a subject is performed subcutaneously, subcutaneously, intravenously, intramuscularly, or by direct injection to one or more cells, tissues, or organs. In some embodiments, administration of a charge-complementary peptide comprising an antigen (e.g., wherein the charge-complementary peptide comprising the antigen is provided in a composition, such as a pharmaceutical composition, including a vaccine) is performed a plurality of times (e.g., two, three, four, or more than four times, such as wherein one or more booster doses are administered to the subject). In some embodiments, a charge-complementary peptide comprising an antigen is administered to a subject for the purposes of vaccinating the subject. In some embodiments, a charge-complementary peptide comprising an antigen is administered to a subject for the purposes of cancer immunotherapy (e.g., for the purposes of inducing an antigen-specific antitumor immune response in the subject).

[0102] In some embodiments, a method comprises contacting charge-complementary peptides, including at least one positively charged peptide and at least one negatively charged peptide, thereby forming a supramolecular structure (e.g., a particle, a granule, or a hydrogel), wherein one or more of the charge-complementary peptides are fused to an antigen. In some embodiments, charge-complementary peptides, wherein one or both of the charge-complementary peptides comprise an antigen, are mixed under stimulating conditions that promote co-assembly of the charge-complementary peptides in a supramolecular structure (e.g., a granule, a particle, and / or a hydrogel).

[0103] In some embodiments, conditions that promote co-assembly of charge-complementary peptides (e.g., co-assembly of charge-complementary peptides in a composition, such as a pharmaceutical composition, which includes a vaccine) involve a crowding agent (e.g., polyethylene glycol (PEG)), providing the charge-complementary peptides in an aqueous solution (e.g., a neutral aqueous solution), and / or providing the charge-complementary peptides in a non-equal molar ratio of positively charged peptide to negatively charged peptide (see, e.g., the description of stimulating conditions in International Patent Application No.

[0104] PCT / US2022 / 025426 (published as WO 2022 / 225,987) and International Patent Application No. PCT / US2023 / 065823 (published as WO 2023 / 201,370)), which are each incorporated by

[0105] #14496674v1 reference in their entirety, including their description of charge-complementary peptides and conditions that promote co-assembly of charge-complementary peptides and crowding agents that promote co-assembly of charge-complementary peptides.

[0106] For example, in some embodiments, the molarity of the positively charged peptide is greater than the molarity of the negatively charged peptide in a solution (e.g., wherein the solution is a composition, such as a pharmaceutical composition, which includes a vaccine). In some embodiments, the non-equal molar ratio of negatively charged peptide to positively charged peptide in the solution is 1:2, 1:50, or any intervening ratio thereof, such as 1:3, 1:4, 1:5, 1:6, 1:7. 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18. 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29. 1:30, 1:31, 1:32, 1:33, 1:34, 1:35. 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, or 1:49. In some embodiments, the non-equal molar ratio of negatively charged peptide to positively charged peptide in the solution is between 1:15 and 1:30. In some embodiments, the non-equal molar ratio of negatively charged peptide to positively charged peptide in the solution is 1:23.

[0107] In some embodiments, the molarity of the negatively charged peptide is greater than the molarity of the positively charged peptide in a solution (e.g., wherein the solution is a composition, such as a pharmaceutical composition, which includes a vaccine). In some embodiments, the non-equal molar ratio of positively charged peptide to negatively charged peptide in the solution is 1:2, 1:50, or any intervening ratio thereof, such as 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, or 1:49.

[0108] In some embodiments, conditions that promote co-assembly of charge-complementary peptides (e.g., co-assembly of charge-complementary peptides in a vaccine) involve contacting charge-complementary peptides (e.g., a positive peptide and a negative peptide that are in an aqueous solution, which includes, but is not limited to, solutions wherein the positive and negative peptides are in non-equal molar ratios) with one or more crowding agents (e.g., 1, 2, 3, 4, 5, or more than 5 crowding agents). Non-limiting examples of crowding agents include sucrose, glycerol, Polysorbate 20 (IUPAC: Polyoxyethylene (20) sorbitan monolaurate; commercially known as “Tween 20”TM), polysorbate 80 (IUPAC: Polyoxyethylene (20) sorbitan monooleate; commercially known as “Tween 80” TM), (l,l,3,3-Tetramethylbutyl)phenyl-

[0109] #14496674v1 polyethylene glycol, Polyethylene glycol tert-octylphenyl ether (commercially known as “Triton X-114’TM), sodium dodecylsulfate (SDS), deoxycholate sodium, (3-((3-cholamidopropyl) dimethylammonio)-l -propanesulfonate) (commonly known as CHAPS detergent), benzalkonium chloride, polyethylene glycol 1000 (PEG 1000), PEG 1500, PEG 1550, PEG 2000, PEG 3350, PEG 6000, PEG 20000, PEG 50000, Ficoll 70, Ficoll 400, serum albumin, and dextran (50k or 500k). In some embodiments, the one or more crowding agents comprise polyethylene glycol. In some embodiments, the one or more crowding agents comprise PEG 1500. In some embodiments, the one or more crowding agents are provided in a concentration of at least 0.1 g / mL (e.g., at least I g / mL, at least 10 g / mL, at least 100 pg / mL, at least 250 pg / mL, or at least 500 pg / mL). In some embodiments, the one or more crowding agents are provided in a concentration of 0.1 g / mL-1,000 pg / mL (e.g., 0.1 pg / mL-lpg / mL, 1 pg / mL-10 pg / mL, 10 pg / mL-100 pg / mL, 100 pg / mL-200 pg / mL, 200 pg / mL-300 pg / mL, 300 pg / mL-400 pg / mL, 400 pg / mL-500 pg / mL, 500 pg / mL-600 pg / mL, 600 pg / mL-700 pg / mL, 700 pg / mL-800 pg / mL, 800 pg / mL-900 pg / mL, 900 pg / mL-1,000 pg / mL). In some embodiments, the one or more crowding agents are provided in a concentration of 0.1 pg / mL-100 pg / mL (e.g., 0.1 pg / mL-l / mL, 1 pg / mL-10 pg / mL, 10 pg / mL-20 pg / mL, 20 pg / mL-30 pg / mL, 30 pg / mL-40 pg / mL, 40 pg / mL-50pg / mL, 50 pg / mL-60 pg / mL, 60 pg / mL-70 pg / mL, 70 pg / mL-80 pg / mL, 80 pg / mL-90 pg / mL, 90 pg / mL-100 pg / mL). In some embodiments, the one or more crowding agents are provided in a concentration of 10 pg / mL-100 pg / mL.

[0110] In some embodiments, a composition (e.g., a pharmaceutical composition, such as a vaccine) comprises an assembly (e.g., a supramolecular structure, such as a particle, a granule, or a hydrogel) of co-assembling peptides (e.g., charge-complementary peptides), wherein at least one of the co-assembling peptides comprise an antigen and wherein the assembly was formed in the presence of one or more crowding agents. In some embodiments, a composition that comprises an assembly of co-assembling peptides (e.g., a composition for administration to a subject, such as a pharmaceutical composition, which includes, but is not limited to, a vaccine) and one or more crowding agents that were used to form the assembly. In some embodiments, the concentration of one or more crowding agents that were used to form an assembly of coassembling peptides in a composition (e.g., a composition for administration to a subject, such as a pharmaceutical composition, which includes, but is not limited to, a vaccine) are adjusted (e.g., increased or decreased) following formation of the assembly. For example, in some

[0111] #14496674v1 embodiments, the concentration of the one or more crowding agents in the composition are increased or decreased by at least 1.1-fold (e.g., increased or decreased by 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold. 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0-fold, or more than 10.0-fold) following coassembly of charge-complementary peptides (e.g., wherein increasing or decreasing the concentration of the one or more crowding agents in the composition comprises using dialysis, gel filtration chromatography, ultrafiltration, precipitation and re-dissolution, lyophilization and reconstitution, or a combination thereof). In some embodiments, one or more crowding agents are removed from a composition (e.g., a composition for administration to a subject, such as a pharmaceutical composition, which includes, but is not limited to, a vaccine) comprising an assembly of co-assembling peptides, wherein the assembly was formed in the presence of the one or more crowding agents (e.g., wherein the one or more crowding agents were removed from the composition using dialysis, gel filtration chromatography, ultrafiltration, precipitation and redissolution, lyophilization and reconstitution, or a combination thereof).

[0112] In some embodiments, conditions that promote co-assembly of charge-complementary peptides (e.g., co-assembly of positively charged peptides and negatively charged peptides in a composition, such as a pharmaceutical composition, which includes a vaccine) involve incubating the charge-complementary peptides in an aqueous solution having a pH of about 5.0 to about 8.5 (e.g., a pH of 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, or 8.0-8.5). In some embodiments, the aqueous solution has a pH of about 6.0 to about 8.0 (e.g., a pH of 6.0-7.0 or 7.0-8.0). In some embodiments, the aqueous solution has a pH of about 6.5 to about 7.5 (e.g., a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some embodiments, the aqueous solution is a neutral aqueous solution.

[0113] In some aspects, the application relates to a kit comprising one or more compositions (e.g., a pharmaceutical composition, such as a vaccine) that can be used to perform a method described herein. In some embodiments, a kit described herein includes one or more containers housing components of a composition for performing a method described herein. In some embodiments, the components may be prepared sterilely and shipped refrigerated or on ice. In some embodiments, a kit includes other components that can be used in a method described herein, such as syringes or vials comprising a pharmaceutical composition and / or instructions for using a composition described herein. A kit described herein can comprise components provided

[0114] #14496674v1 in liquid form (e.g., in solution) and / or components in solid form (e.g., a dry powder). In some embodiments, some of the components can be reconstituted or otherwise processed (e.g., processed into an active form), for example, by the addition of a suitable solvent (e.g., water), which may or may not be provided with the kit.

[0115] EXAMPLES

[0116] Example 1

[0117] This Example relates to a CATCH supramolecular peptide vaccine that induces endosomal escape and potent cross-presentation in dendritic cells. FIGs. 1A-1B show nonlimiting embodiments of co-assembly tags based on charge (CATCH) complementarity peptides and supramolecular compositions thereof as well as granule preparation methods, and representative transmission electron microscopy images of CATCH granule nanoparticles. FIG. 2 shows CATCH can form nanofibrils or nano-sized granules depending on formation conditions. FIGs. 3A-3B shows CATCH granules undergo endosomal escape following entry into cells. Brightfield (FIG. 3 A) and YFP channel (FIG. 3B) of an MDA (fibroblast) cell line expressing Gal8-YFP (Yellow Fluorescent Protein) following treatment with C ATCH Granules. Gal8 is a cytosolic protein that recruits to endosomes following their disruption. FIG. 4 shows CATCH can be linked to peptides to serve as an antigen delivery vehicle. FIGs. 5A-5D shows CATCH granules transfect a dendritic cell line (DC2.4s). FIGs. 5A and 5C show fluorescence microscopy overlay of GFP and DAPI signal from DC2.4s transfected with CATCH granules. FIGs. 5B and 5D show flow cytometry of GFP intensity of DC2.4s following treatment with CATCH granules. FIG. 6 shows CATCH granules induce significant cross presentation in DC2.4s. Flow cytometry of MHCI-SIINFEKL intensity of DC2.4s that are untreated, treated with CATCH granules, or treated with SIINFEKL (SEQ ID NO: 13) peptide (Positive Control). FIG. 7 shows the presence of a population of GFP+ and MHCI-SIINFEKL+ cells. This data indicates entry of granules into DC2.4s and cross-presentation. FIGs. 8A-8C shows CATCH peptides conjugated to an antigen retain transfection ability. FIG. 8A shows a CATCH peptide fused to an MHC-I restricted ovalbumin epitope. FIGs. 8B-8C show fluorescence microscopy and flow cytometry of DC2.4s treated with CATCH granules containing varying percentages of CATCH(6K+)-SIINFEKL indicated that transfection of dendritic cells is maintained with antigen incorporation. FIG. 9

[0118] #14496674v1 shows contour plots of GFP intensity vs MHCI-SIINFEKL intensity for DC2.4s that were treated with CATCH granules containing 0% (Left) or 100% (Right) of CATCH(6K+)-SIINFEKL.

[0119] Example 2

[0120] This Example relates to antigens incorporated into CATCH assemblies that can induce cross presentation on dendritic cells and to adjuvants that can be incorporated into CATCH assemblies to improve immune activation and antigen presentation.

[0121] FIG. 10 shows dynamic light scattering measurements of CATCH(6E-)-Nanoluciferase (left panel) and CATCH(6E-)-GFP assemblies (right panel) prepared with 0%. 10%, or 100% CATCH-SIINFEKL, wherein the percentage refers to the amount of CATCH(6K+) that was modified to comprise SIINFEKL (SEQ ID NO: 13) antigen and used in the composition for CATCH assembly. CATCH assemblies were formed at a 2:2:1 ratio of 30 uM CATCH(6E-)-Nanoluciferase or CATCH(6E-)GFP, 700 uM CATCH(6K+) and CATCH(6K+)-SIINFEKL, and 0.47 mg / mL (final 94 ug / mL) PEG, and diluted at 1 / 10X immediately following formation. Error bars are replicate measurements of the same sample. This data indicated CATCH assembly size increases with greater percent modification of CATCH(6K+) with SIINFEKL ( SEQ ID NO: 13) peptide. This data indicated that CATCH assemblies can be modified with antigen and still undergo cell entry and endosomal escape. Additionally, fluorescence microscopy of DC2.4s treated with CATCH assemblies containing varying percentages of CATCH(6K+)-SIINFEKL indicated that transfection of dendritic cells is maintained with antigen incorporation.

[0122] FIG. 11 shows MHCI-SIINFEKL presentation of DC2.4s treated with CATCH assemblies with 0, 10, 50, or 100% modification with SIINFEKL (SEQ ID NO: 13) antigen compared to untreated, CATCH-antigen alone, and a SIINFEKL (SEQ ID NO: 13) positive control, wherein the percentage refers to the amount of CATCH(6K+) that was modified to comprise SIINFEKL (SEQ ID NO: 13) antigen and used in the composition for CATCH assembly. SIINFEKL (SEQ ID NO: 13) antigen presentation was measured via an antibody that specifically binds to the presented antigen. “CATCH-SIINFEKL Alone” refers to the SIINFEKL (SEQ ID NO: 13) (antigen) modified CATCH(6K+) peptide which was provided to the cells as a control group. “CATCH-SIINFEKL Assembly” refers to the CATCH-SIINFEKL peptide in combination with a co-assembling CATCH peptide and a crowding agent. This data indicated cross presentation (MHCI-SIINFEKL) of CATCH assemblies by the percentage of CATCH-

[0123] #14496674v1 antigen (SIINFEKL (SEQ ID NO: 13)) modification. This data also demonstrated that CATCH-SIINFEKL assemblies (e.g., granules) can deliver greater amounts of antigen to cells (e.g., immune cells, such as dendritic cells) than the CATCH-SIINFEKL alone control.

[0124] The data in FIGs. 10-11 also indicated that CATCH granules improved antigen presentation relative to a CATCH-SIINFEKL control at each percentage of CATCH modification with SIINFEKL (SEQ ID NO: 13) antigen.

[0125] FIGs. 12A-12B show non-limiting examples of adjuvants that can be used to promote entry of CATCH assemblies into cells.

[0126] FIGs. 13A-13B show data from analyzing samples containing CpG alone and CATCH assemblies formed at 1:1, 1:2, 1:10, 1:100 ratios of CATCH(6K+) to CpG (FIG. 13A) and concentrations of cGAMP within each sample for different starting concentrations or ratios of CATCH(6K+) to cGAMP (FIG. 13B). The gel electrophoresis results in FIG. 13A, from left to right, show CpG alone, and CATCH assemblies formed at 1:1, 1:2, 1:10, 1:100 ratios of CATCH(6K+) to CpG. CpG labelled with SYBR Safe for visualization in gel. Concentration of cGAMP within each sample represented as free cGAMP (upper portion of each histogram bar) or cGAMP incorporated into CATCH assemblies (lower portion of each histogram bar) for different starting concentrations or ratios of CATCH(6K+) to cGAMP. This data indicated concentration of free or incorporated cGAMP (uM) at different starting concentrations / ratios.

[0127] FIG. 14 shows CD86 (left panel) and MHCI-SIINFEKL (right panel) detected in bone marrow-derived dendritic cells treated with CpG incorporated CATCH assemblies at a 1:10 ratio of CpG to CATCH(6K+). This data indicated dendritic cells treated with CpG loaded CATCH assemblies display increased CD86 (activation marker) and MHCI-SIINFEKL (cross presentation) in comparison to cells treated with CATCH assemblies alone. In some embodiments, any antigen may be used (e.g., a viral antigen, a cancer antigen, or a bacterial antigen). As described above, dendritic cells may be screened for activation markers and / or for the presence of antigen on the surface of the dendritic cells.

[0128] Example 3

[0129] This Example relates to CATCH assemblies injected intravenously enter B cell follicles in the spleen.

[0130] #14496674v1 FIG. 15 shows microscopy (left panel) and DLS (right panel) analyses of splenic tissue following intravenous injection of CATCH assemblies comprising CATCH-SIINFEKL. At 30 minutes post-splenic injection, “standard” antigen (leftmost panel) and (CATCH-SIINFEKL) modified CATCH-GFP assemblies (middle panel) were detected in addition CD20. DLS analysis was used to demonstrate the size of these assemblies. This data indicated that CATCH assemblies enter the spleen and tend to cluster around follicles (B cells) following intravenous injection.

[0131] EQUIVALENTS

[0132] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0133] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0134] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0135] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0136] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also

[0137] #14496674v1 including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0138] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0139] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0140] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g..

[0141] #14496674v1 “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0142] #14496674v1

Claims

CLAIMS1. A method comprising administering a vaccine to a subject in need thereof, wherein the vaccine comprises a protein and a co-assembling peptide, wherein:(i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or(ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide.

2. The method of claim 1, wherein the protein comprises the negatively charged coassembling peptide and the antigen, and the co-assembling peptide comprises the positively charged peptide.

3. The method of claim 2, wherein the antigen comprises a protein.

4. The method of claim 1, wherein the protein comprises the positively charged coassembling peptide and the antigen, and the co-assembling peptide comprises the negatively charged peptide.

5. The method of claim 4, wherein the antigen comprises a peptide.

6. The method of any one of claims 1-5, wherein the subject has or is at risk of having a disease or a disorder associated with reduced immunity or lack of immunity to the antigen.

7. The method of any one of claims 1-6, wherein the antigen is a peptide or protein located on a pathogen.

8. The method of claim 7, wherein the pathogen is a mammalian pathogen.#14496674v19. The method of claim 8, wherein the mammalian pathogen is a human pathogen.

10. The method of any one of claims 7-9, wherein the subject is at risk of infection with the pathogen.

11. The method of any one of claims 7-10, wherein the subject has been exposed to the pathogen or is at risk of exposure to the pathogen.

12. The method of any one of claims 1-6, wherein the antigen is a tumor antigen.

13. The method of claim 12, wherein the subject has or is at risk of having cancer.

14. The method of claim 13, wherein the cancer is associated with expression of the tumor antigen.

15. The method of any one of claims 1-14, wherein the subject is a mammalian subject.

16. The method of claim 15, wherein the mammalian subject is a human subject.

17. The method of any one of claims 1-16, wherein the vaccine further comprises a crowding agent.

18. The method of claim 17, wherein the crowding agent comprises polyethylene glycol (PEG).

19. The method of any one of claims 1-18, wherein the vaccine further comprises an adjuvant.

20. The method of claim 19, wherein the adjuvant comprises a nucleic acid.#14496674v121. The method of claim 20, wherein the nucleic acid comprises cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a single-stranded, unmethylated DNA, or a double-stranded RNA.

22. A vaccine comprising:(a) a protein and a co-assembling peptide, wherein the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; and(b) an adjuvant.

23. The vaccine of claim 22, wherein the antigen comprises a peptide.

24. A vaccine comprising:(a) a protein and a co-assembling peptide, wherein the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide; and(b) an adjuvant.

25. The vaccine of claim 24, wherein the antigen comprises a protein.

26. The vaccine of any one of claims 22-25, wherein the antigen is a peptide or protein located on a pathogen.

27. The vaccine of claim 26, wherein the pathogen is a mammalian pathogen.

28. The vaccine of claim 27, w'herein the mammalian pathogen is a human pathogen.

29. The vaccine of any one of claims 22-25, wherein the antigen is a tumor antigen.

30. The vaccine of any one of claims 22-29, wherein the vaccine comprises a crowding agent.#14436674' / :

31. The vaccine of claim 30, wherein the crowding agent comprises polyethylene glycol (PEG).

32. The vaccine of any one of claims 22-31, wherein the adjuvant comprises a nucleic acid.

33. The vaccine of claim 32, wherein the nucleic acid comprises cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a single-stranded, unmethylated DNA, or a double-stranded RNA.

34. The vaccine of any one of claims 22-33, wherein the vaccine comprises a supramolecular structure that comprises the protein.

35. A method of producing the vaccine of any one of claims 22-34, the method comprising contacting the positively charged co-assembling peptide with the negatively co-assembling charged peptide, thereby producing a supramolecular structure, wherein the positively charged peptide and / or the negatively charged peptide comprises an antigen.

36. The method of claim 35, wherein the positively charged peptide and the negatively charged peptide are contacted in the presence of a crowding agent.

37. A protein comprising a co-assembling peptide and an antigen.

38. The protein of claim 37, wherein the co-assembling peptide comprises a positively charged peptide.

39. The protein of claim 37, wherein the co-assembling peptide comprises a negatively charged peptide.

40. The protein of any one of claims 37-39, wherein the antigen is a peptide or protein located on a pathogen.#14496674v141. The protein of claim 40, wherein the pathogen is a mammalian pathogen.

42. The protein of claim 41, wherein the mammalian pathogen is a human pathogen.

43. The protein of any one of claims 37-39, wherein the antigen is a tumor antigen.

44. A composition comprising the protein of any one of claims 37-43.

45. The composition of claim 44, wherein the composition comprises a supramolecular structure that comprises the protein.

46. A method comprising contacting the protein of any one of claims 37-43 or the composition of claim 44 or 45 with a cell.

47. The method of claim 46, wherein the cell is a mammalian cell.

48. The method of claim 47, wherein the mammalian cell is a human cell.

49. A method comprising administering the protein of any one of claims 37-43 or the composition of claim 44 or 45 to a subject.

50. The method of claim 49, wherein the subject is a mammalian subject.

51. The method of claim 50, wherein the mammalian subject is a human subject.

52. A method comprising contacting a positively charged peptide with a negatively charged peptide, thereby producing a supramolecular structure, wherein the positively charged peptide and / or the negatively charged peptide comprises an antigen.#14496674v153. The method of claim 52, wherein the positively charged peptide and the negatively-charged peptide are contacted in the presence of a crowding agent.

54. A composition for use as a vaccine, wherein the composition comprises a protein and a co-assembling peptide, wherein:(i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or(ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide.

55. A composition for use in a method of vaccination, the method comprising administering to a subject a composition comprising a protein and a co-assembling peptide, wherein:(i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or(ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide.

56. Use of a composition in the manufacture of a vaccine, the composition comprising a protein and a co-assembling peptide, wherein:(i) the protein comprises a positively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a negatively charged co-assembling peptide; or(ii) the protein comprises a negatively charged co-assembling peptide and an antigen and the co-assembling peptide comprises a positively charged co-assembling peptide.#14496674v157. The composition for use of claim 54 or 55, or the use of claim 56, wherein the composition comprises an adjuvant and / or a crowding agent.#14496674v1

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