Multi-domain protein vaccine

Abstract

To provide a novel cancer vaccine.SOLUTION: Disclosed herein is a protein fusion technology that allows the combination of one or more cancer vaccine epitopes with scaffold domains. Also disclosed herein are polypeptide and polynucleic acid compositions encompassed by the protein fusion technology and methods of using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 912,903, filed October 9, 2019, the entire contents of which are incorporated herein by this reference in their entirety. [Background technology]

[0002] Cancer immunotherapy aims to harness a patient's immune system to treat cancer. It takes advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor antigens, which are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing antitumor responses and involves the use of monoclonal antibodies, lymphocytes, and cytokines. Active immunotherapy aims to induce new immune responses in patients. Tumor vaccines typically consist of tumor antigens and immune stimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Some of the key barriers to developing curative and tumor-specific immunotherapy are efficiently identifying and selecting highly specific and restricted tumor antigens and delivering them to subjects in need in a way that maximizes T cell activation in the subject for the generation of high antitumor immunogenicity.

[0003] Tumor neoantigens, which arise as a result of genetic alterations in malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific class of antigens and can be patient-specific or shared. Tumor neoantigens are unique to tumor cells because the mutations and their corresponding proteins are present only in tumors. They also evade central tolerance and are therefore likely to be immunogenic. Therefore, tumor neoantigens provide excellent targets for immune recognition, including by both humoral and cellular immunity. However, tumor neoantigens are rarely used in cancer vaccines or immunogenic compositions due to the technical difficulties involved in identifying them, optimizing antigen selection, and producing neoantigens for use in vaccines or immunogenic compositions. Therefore, there remains a need for the development of additional cancer therapeutics.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned in this application are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0005] In some aspects, provided herein are compositions comprising a nucleic acid molecule encoding a fusion polypeptide, wherein the fusion polypeptide comprises a polypeptide sequence comprising: (a) one or more antigen polypeptide sequences; and (b) two or more scaffold polypeptide sequences, wherein each of the two or more scaffold polypeptide sequences comprises a human polypeptide sequence, a fragment thereof, or a variant thereof, and wherein (i) the molecular weight of the two or more scaffold polypeptide sequences is greater than 11 kDa, or (ii) each of the two or more scaffold polypeptide sequences comprises at least 21 amino acid residues.

[0006] In some aspects, provided herein are compositions comprising a fusion polypeptide, wherein the fusion polypeptide comprises a polypeptide sequence comprising: (a) one or more antigen polypeptide sequences; and (b) one or more scaffold polypeptide sequences, wherein the scaffold polypeptide is selected from Stefin A, titin-I27, fragments thereof, and variants thereof.

[0007] In some embodiments, at least one of the scaffold polypeptide sequences is connected to at least one of the one or more antigen polypeptide sequences via one or more linker sequences, hi some embodiments, the fusion polypeptide is configured to facilitate cleavage of the linker, cleavage of at least one of the one or more antigen polypeptides, or presentation of at least one of the one or more antigen polypeptides.

[0008] In some aspects, provided herein are compositions comprising a fusion polypeptide or a nucleic acid molecule encoding the fusion polypeptide, wherein the fusion polypeptide comprises a polypeptide sequence comprising: (a) one or more antigen polypeptide sequences; and (b) one or more scaffold polypeptide sequences, wherein at least one of the scaffold polypeptide sequences is connected to at least one of the one or more antigen polypeptide sequences via one or more linker sequences, and the fusion polypeptide is configured to facilitate cleavage of the linker, cleavage of at least one of the one or more antigen polypeptides, or presentation of at least one of the one or more antigen polypeptides. In some embodiments, the antigen polypeptide is a cancer antigen. In some embodiments, each of the scaffold polypeptide sequences comprises a human polypeptide sequence, a fragment thereof, or a variant thereof.

[0009] In some embodiments, the scaffold polypeptide comprises a recombinant human polypeptide.

[0010] In some embodiments, the scaffold polypeptide is not configured to have target binding properties.

[0011] In some embodiments, the scaffold polypeptides are each independently selected from titin I27, ubiquitin, Stefin A, 10FN-III, Ig-L filamin A, tenascin, fragments thereof, and variants thereof.

[0012] In some embodiments, the scaffold polypeptides are each independently selected from Stefin A, titin I27, fragments thereof, and variants thereof.

[0013] In some embodiments, the scaffold polypeptide lacks (i) post-translational modifications, (ii) intrapeptide disulfide bonds, or both.

[0014] In some embodiments, the scaffold polypeptide is non-immunogenic.

[0015] In some embodiments, the scaffold polypeptide is not configured to have enzymatic activity.

[0016] In some embodiments, each of the scaffold polypeptides is Stefin A, a fragment thereof, or a variant thereof.

[0017] In some embodiments, the scaffold polypeptide sequences are the same or different.

[0018] In some embodiments, the fusion polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 scaffold polypeptides.

[0019] In some embodiments, the fusion polypeptide comprises at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 scaffold polypeptides.

[0020] In some embodiments, the fusion polypeptide comprises six scaffold polypeptides.

[0021] In some embodiments, the molecular weight of the scaffold polypeptide sequence is at least 11 kDa, 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, or 200 kDa.

[0022] In some embodiments, the molecular weight of the scaffold polypeptide sequence is at most 15 kDa, 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, 200 kDa, or 500 kDa.

[0023] In some embodiments, the molecular weight of each of the scaffold polypeptide sequences is at least 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, or 100 kDa.

[0024] In some embodiments, the molecular weight of each of the scaffold polypeptide sequences is at most 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, or 250 kDa.

[0025] In some embodiments, each of the scaffold polypeptide sequences comprises at least 21, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 amino acid residues.

[0026] In some embodiments, each of the scaffold polypeptide sequences comprises at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75, at most 80, at most 85, at most 90, at most 100, at most 125, at most 150, at most 175, or at most 200 amino acid residues.

[0027] In some embodiments, the molecular weight of the fusion polypeptide is greater than 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, or 200 kDa.

[0028] In some embodiments, the molecular weight of the fusion polypeptide is greater than 75 kDa.

[0029] In some embodiments, the molecular weight of the fusion polypeptide is less than or equal to 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, 200 kDa, or 500 kDa.

[0030] In some embodiments, each of the one or more antigenic polypeptide sequences comprises fewer than 100, fewer than 75, fewer than 50, or fewer than 35 amino acid residues.

[0031] In some embodiments, each of the one or more antigenic polypeptide sequences comprises more than 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues.

[0032] In some embodiments, at least one of the scaffold polypeptide sequences is linked via its N-terminus to the remainder of the fusion polypeptide.

[0033] In some embodiments, at least one of the scaffold polypeptide sequences is linked via its C-terminus to the remainder of the fusion polypeptide.

[0034] In some embodiments, at least two of the scaffold polypeptide sequences are uninterrupted by antigen sequences or linker sequences.

[0035] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (Ia): A n -S m , or S m -A n Formula (Ia), wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, m is an integer greater than or equal to 2, and n is an integer greater than or equal to 1. In some embodiments, n is an integer selected from 1 to 5, and m is 2.

[0036] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (Ib): S O -A n -S m Formula (Ib) wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, and each of o, n, and m independently is an integer greater than or equal to 1. In some embodiments, o is 1 or 2, m is 1 or 2, and n is an integer selected from 1 to 5. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having the structure SAS, ASAS, SASA, SASAS, SASASAS, SASASASAS, or SASASASASAS.

[0037] In some embodiments, the fusion polypeptide sequence comprises two or more linker sequences.

[0038] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (IIa): (LAL) n -S m , or S m -(LAL) n Formula (IIa), wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, each L independently represents a linker sequence or is absent, m is an integer greater than or equal to 2, and n is an integer greater than or equal to 1. In some embodiments, n is an integer selected from 1 to 5, and m is 2.

[0039] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (IIb): S o --(LAL) n -S m Formula (IIb), wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, each L independently represents a linker sequence or is absent, and each of o, n, and m independently is an integer greater than or equal to 1. In some embodiments, o is 1 or 2, m is 1 or 2, and n is an integer selected from 1 to 5. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having the structure SLALS, SALSLALS, SLALSLA, SLALSLALS, SLALSLALSLALS, SLALSLALSLALSLAS, or SLALSLALSLALSLASLALS.

[0040] In some embodiments, the scaffold polypeptide and the antigen polypeptide are positioned within the fusion polypeptide in an alternating manner.

[0041] In some embodiments, the fusion polypeptide is in a linear format.

[0042] In some embodiments, the fusion polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 antigenic polypeptides.

[0043] In some embodiments, the fusion polypeptide comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 antigen polypeptides.

[0044] In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5, or 6 antigen polypeptides.

[0045] In some embodiments, each of the scaffold polypeptide sequences is connected to one or two of the antigen polypeptide sequences via at least one of the linker sequences.

[0046] In some embodiments, the fusion polypeptide is configured to facilitate cleavage of the linker or at least one of the one or more antigen polypeptides.

[0047] In some embodiments, at least one of the linker sequences comprises a cleavage site.

[0048] In some embodiments, the cleavage site is cleavable by a peptidase or protease.

[0049] In some embodiments, the cleavage site is cleavable by a cellular peptidase or protease.

[0050] In some embodiments, the fusion polypeptide is configured to promote presentation of at least one of the one or more antigenic polypeptides.

[0051] In some embodiments, at least one of the linker sequences comprises a lysine residue, an arginine residue, a serine residue, a threonine residue, an asparagine residue, a histidine residue, an alanine residue, a glutamine residue, an aspartic acid residue, a methionine residue, a tyrosine residue, a glycine residue, a proline residue, a glutamic acid residue, a tryptophan residue, a phenylalanine residue, a valine residue, an isoleucine residue, a cysteine, a leucine residue, or any combination thereof.

[0052] In some embodiments, at least one of the linker sequences comprises a lysine, arginine, or alanine residue directly connected to the N-terminus of at least one of the antigenic polypeptides.

[0053] In some embodiments, at least one of the linker sequences comprises a serine, lysine, arginine, or alanine residue directly connected to the C-terminus of at least one of the antigenic polypeptides.

[0054] In some embodiments, the one or more antigen polypeptides are HLA class I antigen polypeptides, and at least one of the linker sequences comprises a lysine residue, an arginine residue, a serine residue, a threonine residue, an asparagine residue, a histidine residue, an alanine residue, a glutamine residue, an aspartic acid residue, a methionine residue, a tyrosine residue, a glycine residue, a proline residue, a glutamic acid residue, a tryptophan residue, a phenylalanine residue, a valine residue, an isoleucine residue, a leucine residue, a cysteine ​​residue, or any combination thereof.

[0055] In some embodiments, one or more antigen polypeptides are HLA class I antigen polypeptides, and at least one of the linker sequences comprises a lysine, arginine, or alanine residue directly connected to the N-terminus of at least one of the antigen polypeptides. In some embodiments, one or more antigen polypeptides are HLA class I antigen polypeptides, and at least one of the linker sequences comprises a serine, lysine, arginine, or alanine residue directly connected to the C-terminus of at least one of the antigen polypeptides. In some embodiments, one or more antigen polypeptides are HLA class II antigen polypeptides, and at least one of the linker sequences comprises an aspartic acid, methionine, leucine, valine, isoleucine, tyrosine, phenylalanine, tryptophan residue, or any combination thereof. In some embodiments, one or more antigen polypeptides are HLA class II antigen polypeptides, and at least one of the linker sequences comprises an aspartic acid, methionine, leucine, tyrosine, or phenylalanine residue directly connected to the N-terminus of at least one of the antigen polypeptides. In some embodiments, one or more antigen polypeptides are HLA class II antigen polypeptides, and at least one of the linker sequences comprises a methionine residue, a leucine residue, a valine residue, an isoleucine residue, a tyrosine residue, a phenylalanine residue, or a tryptophan residue directly connected to the C-terminus of at least one of the antigen polypeptides.

[0056] In some embodiments, at least one of the linker sequences comprises an aspartic acid residue, a methionine residue, a leucine residue, a valine residue, an isoleucine residue, a tyrosine residue, a phenylalanine residue, a tryptophan residue, or any combination thereof.

[0057] In some embodiments, at least one of the linker sequences comprises an aspartic acid residue, a methionine residue, a leucine residue, a tyrosine residue, or a phenylalanine residue directly connected to the N-terminus of at least one of the antigenic polypeptides.

[0058] In some embodiments, at least one of the linker sequences comprises a methionine, leucine, valine, isoleucine, tyrosine, phenylalanine, or tryptophan residue directly connected to the C-terminus of at least one of the antigenic polypeptides.

[0059] In some embodiments, each of the linker sequences comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 amino acid residues.

[0060] In some embodiments, each of the linker sequences comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or 100 amino acid residues.

[0061] In some embodiments, the linker is flexible.

[0062] In some embodiments, the linker sequences are the same or different.

[0063] In some embodiments, the solubility of a fusion polypeptide in an aqueous solvent is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the solubility of its corresponding polypeptide lacking the scaffold polypeptide, measured in the same solvent.

[0064] In some embodiments, the solubility of the fusion polypeptide in an aqueous formulation is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold greater than the solubility of its corresponding polypeptide lacking the scaffold polypeptide in the aqueous formulation.

[0065] In some embodiments, the serum solubility of the fusion polypeptide is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the serum solubility of its corresponding polypeptide lacking the scaffold polypeptide.

[0066] In some embodiments, the serum half-life of the fusion polypeptide is at least 1.1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times longer than the serum half-life of its corresponding polypeptide lacking the scaffold polypeptide. In some embodiments, the serum half-life of the fusion polypeptide is at least 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, or 10,000 times longer than the serum solubility of its corresponding polypeptide lacking the scaffold polypeptide.

[0067] In some embodiments, accumulation of the fusion polypeptide in lymph nodes after administration to a subject is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 5000-fold, or 10000-fold greater than the accumulation of its corresponding polypeptide lacking the scaffold polypeptide, as measured by mean radiant efficiency.

[0068] In some embodiments, the antigen-specific T cell response to the fusion polypeptide after administration to a subject is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, or 300-fold greater than the antigen-specific T cell response to the corresponding polypeptide lacking the scaffold polypeptide, as measured by an increase in the frequency of antigen-specific T cells or in the secretion of IFNγ.

[0069] In some embodiments, the fusion polypeptide comprises one or more binding moieties configured to bind to an antigen-presenting cell, an adjuvant, or a reagent.

[0070] In some embodiments, the multivalent fusion polypeptide is functionalized by mixing with an anti-scaffold antibody or fragment thereof coupled to a functionalizing agent such as a dendritic cell (DC) targeting domain, an adjuvant, or an immunomodulatory agent.

[0071] In some embodiments, the antigen-presenting cell is a dendritic cell (DC), a macrophage, a Langerhans cell, or a B cell.

[0072] In some embodiments, the one or more binding moieties are configured to bind to one or more receptors expressed on dendritic cells.

[0073] In some embodiments, the one or more receptors comprise a C-type lectin receptor, a scavenger receptor, an F4 / 80 receptor, a DC-specific transmembrane protein (e.g., DC-STAMP), an Fc receptor, or any combination thereof.

[0074] In some embodiments, the one or more receptors include Clec9a or XCR1.

[0075] In some embodiments, at least one of the binding moieties is comprised in a scaffold polypeptide.

[0076] In some embodiments, at least one of the binding moieties is attached directly to the N-terminus or C-terminus of one of the scaffold polypeptides.

[0077] In some embodiments, at least one of the binding moieties is directly attached to the N-terminus or C-terminus of one of the antigenic polypeptides.

[0078] In some embodiments, at least one of the binding moieties is connected to a linker polypeptide.

[0079] In some embodiments, at least one of the binding moieties is terminally linked to the first or last scaffold polypeptide in the N-terminal to C-terminal direction. In some embodiments, the composition comprises one or more binding moieties capable of binding to a fusion polypeptide. In some embodiments, one or more binding moieties bind to the fusion polypeptide. In some embodiments, one or more binding moieties are conjugated to the fusion polypeptide.

[0080] In some embodiments, the one or more cancer antigen polypeptides comprise a plurality of antigen polypeptides. In some embodiments, the one or more cancer antigen polypeptides are autoimmune peptides.

[0081] In some embodiments, the antigenic polypeptide is a neoantigenic peptide.

[0082] In some embodiments, the neoepitope of each peptide is unique. In some embodiments, each of the cancer neoantigen peptides or portions thereof binds to a protein encoded by an HLA allele expressed by the subject, is encoded by an expressed gene in at least one of the subject's cancer cells, and at least one of the cancer neoantigen peptides or portions thereof contains one or more mutations that are not present in the subject's normal tissue.

[0083] In some embodiments, at least one of the one or more mutations is (A) a point mutation, wherein the cancer neoantigen peptide binds to a protein encoded by an HLA allele expressed by the subject with an IC50 of less than 500 nM and with higher affinity than the corresponding wild-type peptide, (B) a splice site mutation, (C) a frameshift mutation, (D) a read-through mutation, or (E) a gene fusion mutation.

[0084] In some embodiments, a first cancer neo-antigen peptide of the plurality binds to a protein encoded by a first HLA allele expressed by the subject, and a second cancer neo-antigen peptide of the plurality binds to a protein encoded by a second HLA allele expressed by the subject, wherein the first and second HLA alleles expressed by the subject are different HLA alleles.

[0085] In some embodiments, at least one of the cancer neoantigen peptides binds to a protein encoded by an HLA allele expressed by the subject with an IC50 of less than 250 nM.

[0086] In some embodiments, the fusion polypeptide is expressed in a host cell. In some embodiments, the fusion polypeptide is a synthetic construct.

[0087] In some embodiments, the nucleic acid molecule encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 different fusion polypeptides.

[0088] In some embodiments, the nucleic acid molecule encodes at most 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 different fusion polypeptides.

[0089] In some embodiments, the nucleic acid molecule is RNA or DNA.

[0090] In one aspect, disclosed herein are fusion polypeptides encoded by the described nucleic acid molecules.

[0091] In one aspect, disclosed herein is a composition comprising the fusion polypeptide described. In some embodiments, the composition comprises one or more binding moieties bound to the fusion polypeptide. In some embodiments, one or more binding moieties are conjugated to the fusion polypeptide.

[0092] In one aspect, disclosed herein is a nucleic acid molecule encoding two or more scaffold polypeptides spaced by one or more linkers and one or more restriction sites located in at least one of the linkers, wherein (i) the two or more scaffold polypeptide sequences have a molecular weight of greater than 11 kDa, or (ii) each of the two or more scaffold polypeptide sequences comprises at least 21 amino acid residues. In some embodiments, the scaffold polypeptides are encoded by RNA or DNA.

[0093] In one aspect, disclosed herein is a plurality of nucleic acid molecules comprising: a first nucleic acid molecule comprising a nucleic acid sequence encoding a first antigenic polypeptide and a nucleic acid sequence encoding a first scaffold polypeptide; and a second nucleic acid molecule comprising a nucleic acid sequence comprising a sequence complementary to the nucleic acid sequence encoding the first antigenic polypeptide and a nucleic acid sequence encoding a second scaffold polypeptide.

[0094] In another aspect, disclosed herein is a plurality of nucleic acid molecules comprising: a first nucleic acid molecule comprising a nucleic acid sequence encoding a first antigenic polypeptide, a nucleic acid sequence encoding a first scaffold polypeptide, and a nucleic acid sequence encoding a second antigenic polypeptide; and a second nucleic acid molecule comprising a nucleic acid sequence comprising a sequence complementary to the nucleic acid sequence encoding the second antigenic polypeptide, and a nucleic acid sequence encoding the second scaffold polypeptide.

[0095] In yet another aspect, disclosed herein is a plurality of nucleic acid molecules comprising: a first nucleic acid molecule comprising a nucleic acid sequence encoding a first antigenic polypeptide, a nucleic acid sequence encoding a first scaffold polypeptide, and a nucleic acid sequence encoding a second antigenic polypeptide; and a second nucleic acid molecule comprising a nucleic acid sequence comprising a sequence complementary to the nucleic acid sequence encoding the second antigenic polypeptide, a nucleic acid sequence encoding the second scaffold polypeptide, and a nucleic acid sequence encoding a third antigenic polypeptide.

[0096] In some embodiments, the first scaffold sequence and the second scaffold sequence are the same.

[0097] In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are different.

[0098] In some embodiments, the first, second, and third antigenic polypeptides are different.

[0099] In some embodiments, the nucleic acid molecule is RNA or DNA.

[0100] In one aspect, disclosed herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent and a described composition or a described fusion polypeptide.

[0101] In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0102] In some embodiments, the adjuvant is PolyIC:LC.

[0103] In some embodiments, the pharmaceutical composition comprises a pH adjuster.

[0104] In some embodiments, the pharmaceutical composition comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is an immunomodulator, a cytokine or chemokine, or a checkpoint inhibitor. In some embodiments, the second therapeutic agent is administered to a subject in need thereof before administering a pharmaceutical composition comprising a fusion polypeptide or a nucleic acid encoding the fusion polypeptide. In some embodiments, the second therapeutic agent is administered simultaneously with the administration of a pharmaceutical composition comprising a fusion polypeptide or a nucleic acid encoding the fusion polypeptide. In some embodiments, the second therapeutic agent is administered after administering a pharmaceutical composition comprising a fusion polypeptide or a nucleic acid encoding the fusion polypeptide.

[0105] In one aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising expressing a described nucleic acid molecule in a genetically modified cell, thereby producing the immunogenic fusion polypeptide.

[0106] In another aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising providing a nucleic acid molecule described herein; inserting one or more nucleic acid molecules encoding one or more antigenic polypeptides into at least one of the restriction sites, thereby producing a new nucleic acid molecule; and expressing the new nucleic acid molecule in a genetically modified cell, thereby producing the immunogenic fusion polypeptide.

[0107] In some embodiments, one or more nucleic acid molecules encoding one or more antigenic polypeptides are inserted through an isothermal reaction or by restriction enzyme-based cloning.

[0108] In one aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising providing a plurality of nucleic acid molecules described herein, joining the nucleic acid molecules by hybridization, and expressing the joined nucleic acid molecules in a genetically modified cell, thereby producing the immunogenic fusion polypeptide.

[0109] In some embodiments, the fusion polypeptide is expressed in a bacterial expression system.

[0110] In some embodiments, the bacterial expression system is an Escherichia coli expression system.

[0111] In one aspect, provided herein is a method of producing an immunogenic fusion polypeptide, the method comprising: (a) providing a nucleic acid molecule described in any of the above paragraphs, or specifically paragraphs 89-96; (b) inserting one or more nucleic acid molecules encoding one or more antigenic polypeptides into at least one of the restriction sites, thereby producing a new nucleic acid molecule; and (c) expressing the new nucleic acid molecule by in vitro translation or in a genetically modified cell, thereby producing an immunogenic fusion polypeptide.

[0112] In one aspect, provided herein are methods for producing an immunogenic fusion polypeptide, the method comprising: (a) providing a plurality of nucleic acid molecules described in the preceding paragraph, or specifically in any one of paragraphs 89-96; (b) joining the nucleic acid molecules by hybridization; and (c) expressing the joined nucleic acid molecules by in vitro translation or in a genetically modified cell, thereby producing the immunogenic fusion polypeptide. In some embodiments, the fusion polypeptide is expressed in a bacterial expression system. In some embodiments, the bacterial expression system is an E. coli expression system.

[0113] In some embodiments, the fusion polypeptide is expressed by in vitro translation. In some embodiments, the method further comprises a gap filling step and / or a ligation step. In some embodiments, the gap filling step comprises a polymerase-mediated gap filling step.

[0114] In one aspect, disclosed herein is a method of treating or preventing cancer in a human subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0115] In some embodiments, the pharmaceutical composition comprises multiple neoantigenic peptides.

[0116] In some embodiments, the pharmaceutical composition comprises multiple fusion polypeptides.

[0117] In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously.

[0118] In some embodiments, the dose of the fusion polypeptide is divided into at least two, at least three, at least four, or at least five subdoses.

[0119] In some embodiments, each sub-dose of fusion polypeptide comprises 1, 2, 3, 4, 5, or more fusion polypeptides.

[0120] In some embodiments, each fusion polypeptide is administered at a dose of 0.01 to 100 μg.

[0121] In some embodiments, each fusion polypeptide is administered at a dose of 100 μg to 10 mg.

[0122] In some embodiments, the total dose of fusion polypeptide administered is between 0.01 and 100 mg.

[0123] In some embodiments, the cancer is a solid tumor.

[0124] In some embodiments, the cancer is melanoma, lung cancer, or bladder cancer.

[0125] In one aspect, provided herein is a library comprising a plurality of recombinant expression constructs, each expression construct of the plurality comprising: (a) a promoter sequence; and (b) (i) a start codon downstream of the promoter sequence; (ii) a first polynucleotide sequence downstream of the start codon, the first polynucleotide sequence comprising different template polynucleotide sequences, the different templates from which are (A) derived from a sample comprising diseased cells from a subject having a disease and (B) encoding a peptide sequence of a protein encoded by diseased cells from the subject; and (iii) a second polynucleotide sequence downstream of the first polynucleotide sequence, the second polynucleotide sequence comprising (A) a frame check sequence and a sequence encoding one or more affinity tags downstream of the frame check sequence, or (B) a frame check sequence comprising a sequence encoding an affinity tag. In some embodiments, the frame check sequence operates to terminate translation of the fusion polypeptide when the different template polynucleotide sequence, or a copy thereof, is out of frame with the sequence encoding the affinity tag. In some embodiments, the frame check sequence is: N1-N2-N3-N4-N5-N6-N7-N8-N9 wherein each N is independently a nucleic acid selected from the group consisting of A, T, U, C, and G, and wherein each of N1-N2-N3, N4-N5-N6, and N7-N8-N9 is not a stop codon, and each of N2-N3-N4 and N6-N7-N8 is a stop codon. In some embodiments, the frame check sequence encodes Val-Gly-Ser. In some embodiments, the frame check sequence encodes a linker that connects the first polynucleotide sequence to the sequence encoding the affinity tag.

[0126] In some embodiments, the sequence encoding the affinity tag is a frame check sequence. In some embodiments, the affinity tag is a fragment crystallizable region (Fc region) or peptide sequence that binds to an Fc receptor, a GST tag, a His tag, a peptide sequence that binds to protein A, a peptide sequence that binds to protein G, or an epitope of an antibody, or a binding fragment thereof. In some embodiments, the affinity tag is non-immunogenic. In some embodiments, the affinity tag is human. In some embodiments, the sequence encoding the affinity tag encodes a size-enhancing polypeptide, and / or (ii) each expression construct of the library comprises a sequence encoding a size-enhancing polypeptide. In some embodiments, the sequence encoding the size-enhancing polypeptide is downstream of the first polynucleotide sequence, and / or at least one of the sequences encoding the size-enhancing polypeptide is upstream of a different template polynucleotide sequence. In some embodiments, the affinity tag is an epitope of a size-enhancing polypeptide. In some embodiments, the sequence encoding the size-enhancing polypeptide encodes a plurality of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more size-enhancing polypeptides. In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, or more size-enhancing polypeptides of the plurality of size-enhancing polypeptides are the same. In some embodiments, the sequence encoding the size-enhancing polypeptides encodes one or more linkers between two or more of the size-enhancing polypeptides. In some embodiments, the molecular weight of the plurality of size-enhancing polypeptides is at least 15 kDa. In some embodiments, the molecular weight of the plurality of size-enhancing polypeptides is 200 kDa or less. In some embodiments, the molecular weight of the plurality of size-enhancing polypeptides is 40 kDa to 80 kDa or 50 kDa to 70 kDa.

[0127] In some embodiments, the different template polynucleotide sequences encode peptide sequences of proteins expressed by diseased cells from a subject, the subject is human, and the disease is cancer, hi some embodiments, the cancer is melanoma, bladder cancer, or lung cancer.

[0128] In some embodiments, the disease is an autoimmune disease.

[0129] In some embodiments, the sample comprises a biopsy, a blood sample, or a peripheral blood mononuclear cell (PBMC) sample, and the sample comprises diseased cells. In some embodiments, each expression construct in the plurality comprises the same promoter, the same start codon, the same frame check sequence, the same sequence encoding a size-enhanced polypeptide, or any combination thereof. In some embodiments, each of the different template polynucleotide sequences is at least 24 bps in length, at least 45 bps in length, at most 450 bps in length, at most 25 0 bps length, 24-300 bps length, 45-450 bps length, or any of these Any combination is possible.

[0130] In some embodiments, each of the different template polynucleotide sequences is cDNA.

[0131] In some embodiments, each of the different template polynucleotide sequences is derived from an RNA molecule.

[0132] In some embodiments, each of the different template polynucleotide sequences is derived from genomic DNA (gDNA). In some embodiments, the library comprises a plurality of different template polynucleotides representing at least 5% and up to 100% of the exome.

[0133] In some embodiments, the library comprises a plurality of different template polynucleotides encoding at least 5% to up to 100% of the peptide sequences of a proteome. In some embodiments, the library comprises a plurality of different template polynucleotides encoding peptide sequences derived from at least 5% of the proteins of a proteome. In some embodiments, the exome or proteome is the exome or proteome of a diseased cell. In some embodiments, about 5% to about 25% of the polypeptides expressed from the library comprise an affinity tag. In some embodiments, about 20% to about 40% of the polypeptides expressed from the library comprise an affinity tag. In some embodiments, about 50% to about 90% of the polypeptides expressed from a library comprising at least 60 amino acid residues do not comprise an affinity tag. In some embodiments, about 85% to about 95% of the polypeptides expressed from an in-frame expression construct comprise a polypeptide attached to an affinity tag. In some embodiments, the library comprises at least 100, 1000, 10,000, 100,000, 1 x 10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 pieces or up to 1 x 10 10 In some embodiments, the different template polynucleotides encode the peptide sequences of at least 10, 50, 100, 500, 1000, 10,000, or 100,000 different proteins.

[0134] In some embodiments, at least about 10, 100, 500, 1000, 5000, or 10000 of the different template polynucleotides encode variant peptide sequences or mRNA translation products derived from aberrantly transcribed, spliced, or translated mRNA or derived from gene rearrangements in the diseased cell.

[0135] In some embodiments, the variant peptide sequence is a cancer cell-specific variant peptide sequence. In some embodiments, the variant peptide sequence comprises a point mutation or an indel.

[0136] In some embodiments, the expression construct further comprises one or more scaffold polypeptide sequences upstream of the one or more antigen polypeptide sequences, hi some embodiments, the expression construct further comprises a linker sequence downstream of the one or more scaffold polypeptide sequences and upstream of the one or more antigen polypeptide sequences.

[0137] In some embodiments, the first polynucleotide sequence further comprises an upstream adaptor sequence immediately upstream of the different template polynucleotide sequence and / or a downstream adaptor sequence immediately downstream of the different template polynucleotide sequence.

[0138] In some embodiments, the fusion protein is multivalent.

[0139] Provided herein is a polypeptide library encoded by any one of the preceding claims.

[0140] In some embodiments, the polypeptides of the polypeptide library are expressed in a host cell, expressed using an in vitro translation system, and / or expressed from a phage vector, in some embodiments, the phage vector is a filamentous phage vector, such as an M13 phage vector, an f1 phage vector, or an fd phage vector.

[0141] In some embodiments, the polypeptides of the library are expressed as part of a virus-like particle (VLP). In some embodiments, the VLP-like particle can be expressed in a host cell, e.g., a bacterial cell, e.g., an Escherichia coli cell. In some embodiments, the VLP self-assembles within the host cell. In some embodiments, the host cell is a bacterium.

[0142] In some embodiments, the polypeptides of the library are in vitro translated polypeptides.

[0143] In some embodiments, the polypeptide library comprises a plurality of isolated polypeptides.

[0144] In some embodiments, the polypeptides of the library are isolated or purified or enriched via at least an affinity tag.

[0145] In one aspect, provided herein is an individualized recombinant proteome library comprising a plurality of recombinant fusion polypeptides expressed in a host cell, wherein the plurality of recombinant fusion polypeptides comprises a plurality of polypeptide sequences encoded by a plurality of at least 10 different template polynucleotide sequences from a sample comprising diseased cells from a subject having a disease, wherein each of the polypeptide sequences encoded by the plurality of at least 10 different template polynucleotides comprises, in an N to C direction, (i) a polypeptide sequence encoded by a different template polynucleotide of the at least 10 different template polynucleotide sequences, and (ii) (A) a frame check sequence and a sequence encoding an affinity tag downstream of the frame check sequence, or (B) a frame check sequence including a sequence encoding an affinity tag, and / or a size-enhanced polypeptide sequence that is at least 40 kDa.

[0146] In one aspect, provided herein is an individualized recombinant proteome library comprising a plurality of recombinant fusion polypeptides expressed in a host cell, wherein the plurality of recombinant fusion polypeptides comprises a plurality of polypeptide sequences encoded by a plurality of at least 1000 different template polynucleotide sequences from a sample comprising diseased cells from a subject having a disease.

[0147] In one aspect, provided herein is a vaccine composition comprising the polypeptide library or personalized recombinant proteome library described above.

[0148] In one aspect, provided herein is a method of treatment comprising administering to a subject the above-described polypeptide library or personalized recombinant proteome library.

[0149] In one aspect, provided herein is a pharmaceutical composition comprising the above-described polypeptide library or personalized recombinant proteome library and a pharmaceutically acceptable excipient.

[0150] In one aspect, provided herein is a method of treatment comprising administering to a subject the pharmaceutical composition described above.

[0151] In one aspect, provided herein is a cell population comprising the polypeptide library or personalized recombinant proteome library described herein. In some embodiments, each cell of the cell population expresses a single different polypeptide sequence encoded by a plurality of different template polynucleotides.

[0152] Provided herein is a method of constructing expression vectors, the method comprising: (a) providing a plurality of different template polynucleotides from a sample comprising diseased cells from a subject having a disease; (b) attaching adapter sequences to the plurality of different template polynucleotides, thereby forming a plurality of different adapter-tagged template polynucleotides; (c) amplifying the different adapter-tagged template polynucleotides; (d) inserting the amplified different adapter-tagged template polynucleotides into a vector, thereby forming a library of recombinant expression constructs; and (e) expressing polypeptides encoded by the library of recombinant expression constructs; and enriching the expressed polypeptides.

[0153] In some embodiments, the methods comprise contacting a plurality of different polynucleotides with a library of exome capture oligonucleotides, wherein the capture oligonucleotides comprise a target sequence.

[0154] In some embodiments, the exome capture oligonucleotides are immobilized on a surface.

[0155] In some embodiments, amplifying comprises amplifying with random primers.

[0156] In some embodiments, amplifying includes amplifying without bias or is not target specific.

[0157] In some embodiments, the methods involve hybridizing a plurality of different template polynucleotide sequences to a plurality of reference polynucleotide sequences from a reference sample or non-diseased cells from a subject with the disease.

[0158] In some embodiments, the method further comprises selectively enriching double-stranded polynucleotides that contain mismatches from double-stranded polynucleotides that do not contain mismatches.

[0159] In some embodiments, selectively enriching comprises contacting the double-stranded polynucleotide with an agent that specifically binds to double-stranded polynucleotides that contain mismatches.

[0160] In some embodiments, the agent is a DNA base mismatch recognition agent.

[0161] In some embodiments, the agent is a MutS protein or a functional fragment thereof. In some embodiments, the MutS protein is a MutS protein found in, among others, E. coli, Salmonella sp., Haemophilus sp., Azotobacter sp., Acinetobacter sp., Bacillus sp., Borrelia sp., Chlamydia sp., Helicobacter sp., Neisseria sp., Deinococcus radiodurans, and Streptococcus sp., derived from bacterial species, including, but not limited to:

[0162] In some embodiments, the mismatch comprises a single nucleotide variant, insertion or deletion.

[0163] In some embodiments, the method further comprises combining (i) the selectively enriched double-stranded polynucleotides containing mismatches with (ii) a plurality of different template polynucleotides from a sample comprising diseased cells from a subject with the disease that are not selectively enriched.

[0164] In some embodiments, the plurality of different target polynucleotides comprises sequences derived from the entire exome and / or exome-intron boundary sequences.

[0165] In some embodiments, the plurality of different target polynucleotides comprises sequences derived from a subset of the exome based on expression data for one or more cancer types.

[0166] In some embodiments, the plurality of different target polynucleotides comprises sequences from an entire genome.

[0167] In some embodiments, the plurality of different target polynucleotides comprises sequences from a set of whole genome sequences enriched in a library of exome capture oligonucleotides.

[0168] In some embodiments, the method further comprises cloning a plurality of different target polynucleotides into an expression vector.

[0169] In some embodiments, the set of reference polynucleotides comprises a reference genomic exon capture probe set or polynucleotides from a non-tumor sample from the subject.

[0170] In some embodiments, the library of exome capture oligonucleotides comprises frequently occurring human polymorphic sequences.

[0171] In some embodiments, the reference sample comprises a non-tumor sample from the subject.

[0172] In some embodiments, the method further comprises performing reverse transcription.

[0173] In some embodiments, the method does not further comprise sequencing.

[0174] In some embodiments, the method does not further comprise predicting or determining binding of the epitope to a protein encoded by an HLA allele and / or presentation of the epitope by a protein encoded by an HLA allele.

[0175] In some embodiments, attaching adapter sequences to the plurality of different template polynucleotides comprises attaching strand-specific adapter sequences.

[0176] In some embodiments, the different target polynucleotide sequences are derived from gDNA.

[0177] In some embodiments, the different target polynucleotide sequences are derived from mRNA or exome sequences.

[0178] In some embodiments, enriching comprises enriching for affinity tag containing expressed polypeptides.

[0179] In some embodiments, the method further comprises fragmenting the polynucleic acids of the sample.

[0180] In some embodiments, the method further comprises shearing genomic DNA of the sample.

[0181] In some embodiments, the sample is an FFPE sample.

[0182] Provided herein is a method of treatment comprising performing the above method and administering the enriched expressed polypeptide to a subject. [Brief explanation of the drawings]

[0183] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0184] [Figure 1] Option A, a polypeptide construction option, is shown. [Figure 2] Another option for polypeptide construction, option B, is shown. [Figure 3] Yet another option for polypeptide construction, option C, is shown. [Figure 4] Some of the considerations for scaffold domain selection and six exemplary scaffold domains are shown. [Figure 5A] 1 shows the results of testing an exemplary scaffold domain in a solubility challenge test and expression level test as a hexameric polybody. [Figure 5B] 1 shows the results of a solubility challenge study using the scaffold domains shown to solubilize Alg8 peptides. [Figure 5C] Figure 1 shows the results of a solubility challenge study using Stefin A as a scaffold domain to solubilize the indicated peptides. S: soluble fraction, I: insoluble fraction. [Figure 6] Polybody constructs cloned for expression level testing are shown. [Figure 7A]Figure 7 shows lymph node accumulation studies for polybody constructs with 1 to 6 scaffold domains. Figure 7A shows an image of the banding pattern of the polybody constructs stained with Coomassie blue, Figure 7B shows the injection and measurement schedule for the accumulation study, and Figure 7C shows the injection site in a mouse. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8A-B] Figure 8 shows lymph node accumulation of fluorophore-labeled polybody constructs compared to synthetic long peptides. Figure 8A shows fluorescence accumulation in the inguinal lymph nodes of mice, Figure 8B shows fluorescence accumulation in the axillary lymph nodes of mice, and Figure 8C shows the intensity of fluorescence accumulation of hexameric polybodies compared to synthetic long peptides. Figure 8D shows FACS analysis of lymph nodes. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 9] Fluorophore accumulation of polybody constructs compared to synthetic long peptides in mouse left and right inguinal lymph nodes (iLN) and axillary lymph nodes (aLN) is shown. [Figure 10] Figure 10 shows the in vivo immunogenicity of polybody constructs versus synthetic long peptides using MC38 tumor model epitopes. Figures 10A and 10B show the immune response to polybody constructs and synthetic long peptides, respectively, containing the Reps1 and Adpgk epitopes. Figure 10C shows the construction of polybody 1 (PB1), polybody 2 (PB2), and polybody 3 (PB3). [Figure 11] Exemplary polybody constructs are shown in Figure 11A, where Figure 11B shows an exemplary polybody construct comprising a non-functional scaffold domain, and Figure 11B, where Figure 11B shows an exemplary polybody construct comprising functional and non-functional scaffold domains. [Figure 12] Exemplary polybody constructs are shown: Figure 12A shows an exemplary polybody construct that includes a non-functional scaffold domain, and Figure 12B shows an exemplary polybody construct that includes a functionalized peptide tag. [Figure 13]1 shows a manufacturing pipeline for a fusion polypeptide. [Figure 14] A schematic representation of the method for preparing an exome-capture library of randomly primed cDNA prepared from RNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples to generate an artificial miniproteome vaccine (AMPVax) is shown. [Figure 15A] A diagrammatic representation of the AmpVax enrichment exome capture method is shown. [Figure 15B] A schematic representation of the AmpVax enrichment exome capture method using MutS enrichment is shown. [Figure 15C] A schematic representation of the AmpVax enriched exome capture method using normal genomic DNA for MutS bait is shown. [Figure 15D] A diagrammatic representation of the workflow for the AmpVax enrichment exome capture method and the AmpVax enrichment exome capture method with MutS enrichment is shown. [Figure 16A] FIG. 1 shows a diagrammatic representation of exemplary polybody fusion constructs for exome capture libraries. [Figure 16B] FIG. 1 shows a diagrammatic representation of exemplary polybody fusion constructs for exome capture libraries. [Figure 16C] FIG. 1 shows a diagrammatic representation of exemplary polybody fusion constructs for exome capture libraries. [Figure 16D] FIG. 1 shows a diagrammatic representation of exemplary polybody fusion constructs for exome capture libraries. [Figure 16E] FIG. 1 shows a diagrammatic representation of exemplary polybody fusion constructs for exome capture libraries. [Figure 17] FIG. 1 shows a diagrammatic representation of an exemplary method for constructing a plasmid for expressing polybody fusions. [Figure 18A]Figure 18A shows a graphical representation of the distribution / uniformity after 50-fold enrichment with 1 / 5 SNP and reference exome MutS capture. Figure 18B shows a graphical representation of the distribution / uniformity after 50-fold enrichment with 1 / 5 SNP and normal DNA MutS capture. Figure 18C shows a graphical comparison of the reference exome vs. normal DNA with no SNP design for MutS capture. [Figure 18B] Same as above. [Figure 18C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0185] The present disclosure focuses on important aspects of therapeutic development, including peptide or nucleic acid encoding therapeutic peptides, including peptide modifications for stability and targeted delivery within the recipient of the therapeutic. In some embodiments, the peptide is modified for increased immunogenicity. In some embodiments, the peptide is a neoantigen peptide. In some embodiments, the peptide is a neoantigen for treating a disease in a subject, the disease being an immune disease. In some embodiments, the disease is cancer. In some embodiments, the modification comprises a peptide fusion. Provided herein are methods and pharmaceutical compositions for delivering neoantigens in fusion form. Furthermore, neoantigen peptides can be efficiently delivered to lymph nodes in fusion form, thereby exposing and priming a larger population of naive T lymphocytes to the neoantigen peptide.

[0186] In one aspect, described herein is a highly modular protein fusion technology, termed polybodies, that enables efficient delivery, tissue uptake, and functionality of neoantigenic peptides. In some embodiments, polybodies protect neoantigenic peptides from enzymatic digestion in serum after subcutaneous injection. In some embodiments, polybodies aid in targeting neoantigenic peptides to lymph nodes. In some embodiments, polybodies aid in lymph node retention of neoantigenic peptides. In some embodiments, polybodies aid in effective priming and activation of T lymphocytes.

[0187] In some embodiments, modular protein fusion technology allows for the combination of multiple cancer vaccine epitopes (e.g., neoantigens, tumor-associated antigens) on a single protein construct. In some embodiments, the epitopes are surrounded by scaffolding domains. In some embodiments, these scaffolding domains keep the antigenic peptides more soluble and prevent premature degradation caused by circulating proteases and peptidases in the patient. In some embodiments, these scaffolding domains are functionalized to add desired functionality to the polybody to further increase the immunogenicity and anti-tumor efficacy of the vaccine.

[0188] Described herein are new immunotherapeutic agents and their uses based on the discovery of neoantigens that arise from mutational events unique to an individual's tumor. Thus, the disclosure described herein provides peptides, polynucleotides encoding the peptides, and peptide-binding agents that can be used, for example, to stimulate an immune response against tumor-associated antigens or neoepitopes and to generate immunogenic compositions or cancer vaccines for use in treating disease.

[0189] The following description and examples illustrate in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and can therefore be varied. Those skilled in the art will recognize that there are many variations and modifications of the present disclosure that fall within the scope of the present disclosure.

[0190] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0191] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0192] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0193] Various terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless otherwise specified. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well, unless the context clearly dictates otherwise. As understood herein, "or" means "and / or" unless otherwise stated. The terms "and / or" and "any combinations thereof," and their grammatical equivalents used herein, may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the phrase "A, B, and / or C" or "A, B, C, or any combinations thereof" below may mean "individually A, individually B, individually C, A and B, B and C, A and C, and A, B, and C." The term "or" may be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0194] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or more preferably within 2-fold of a value. When specific values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0195] As used in the specification and claim(s), the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0196] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0197] The major histocompatibility complex, or "MHC," is a cluster of genes that plays a role in controlling the cellular interactions responsible for physiological immune responses. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, 3 rd Ed., Raven Press, New York (1993). "Major histocompatibility complex (MHC) proteins or molecules," "MHC molecules," "MHC proteins," or "HLA proteins" should be understood to mean proteins capable of representing potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes) that bind peptides resulting from proteolytic cleavage of protein antigens, transport them to the cell surface, and present them there to specific cells, particularly cytotoxic T lymphocytes, T helper cells, or B cells. The major histocompatibility complex in the genome contains gene regions whose gene products, expressed on the cell surface, are important for binding and presentation of endogenous and / or foreign antigens and thus for regulating immunological processes. The major histocompatibility complex is divided into two groups of genes encoding different proteins: MHC class I molecules and MHC class II molecules. The cellular biology and expression patterns of the two MHC classes are adapted to these different roles.

[0198] Human leukocyte antigens or "HLA" are human class I or class II major histocompatibility complex (MHC) proteins (see, e.g., Stites, et al., Immunology, 8 th Ed., Lange Publishing, Los Altos, Calif. (1994).

[0199] As used herein, "polypeptide," "peptide," and their grammatical equivalents refer to a polymer of amino acid residues. A polymer of amino acid residues is typically a series of amino acid residues connected one to the other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. Examples of amino acids that may be used include β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The present disclosure further contemplates that expression of the polypeptides described herein in engineered cells may be associated with post-translational modification of one or more amino acids of the polypeptide construct.Non-limiting examples of post-translational modifications include phosphorylation, acylation, including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0200] An immunogenic peptide or immunogenic epitope or peptide epitope is one in which the peptide binds to an HLA molecule and elicits a cell-mediated or humoral response, such as the activation of cytotoxic T lymphocytes (CTLs, e.g., CD8 + )), helper T lymphocytes (Th (e.g., CD4 + ) and / or peptides containing allele-specific motifs to induce B lymphocyte responses. Thus, the immunogenic peptides described herein can bind to the appropriate HLA molecule and subsequently induce a CTL (cytotoxic) response, or an HTL (and humoral) response against the peptide.

[0201] Neoantigens are a class of tumor antigens that arise from tumor-specific protein changes.Neoantigens include, but are not limited to, tumor antigens that arise from cancer-specific mutations in subjects, including, but not limited to, substitution mutations, frameshift mutations, gene fusions, in-frame deletions or insertions.Neoantigens can also include endogenous retroviral polypeptides and polypeptides that contain overexpressed tumor-specific mutations.

[0202] The terms "peptide" and "polypeptide" are used interchangeably and refer to a series of L-amino acid residues, typically connected one to the other by a peptide bond between the α-amino and carboxyl groups of adjacent amino acids. In some embodiments, a polypeptide comprises a single translation product containing multiple distinct epitopes. In some embodiments, a polypeptide comprises a single translation product containing a single distinct peptide, such as an epitope or neo-epitope, and one or more additional non-distinct elements, such as one or more additional scaffold proteins. In some embodiments, a polypeptide comprises multiple epitopes. In some embodiments, a polypeptide may be a branched multi-peptide structure. Polypeptides or peptides, in either their neutral (uncharged) or salt form, may be of various lengths, free of or containing modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptides described herein. In some embodiments, a peptide or polypeptide comprises at least one flanking sequence. As used herein, the term "flanking sequence" refers to a fragment or region of a neo-antigenic peptide that is not part of a neo-epitope. "Variant polypeptide," "neoantigenic polypeptide," "neoantigenic polypeptide," "variant peptide," "neoantigenic peptide," and "neoantigenic peptide" are used interchangeably and refer to peptides or polypeptides that contain mutations.

[0203] The term "residue" refers to an amino acid residue or amino acid mimetic residue that is incorporated into a peptide or protein by means of an amide bond or amide bond mimetic, or a nucleic acid (DNA or RNA) that encodes the amino acid or amino acid mimetic.

[0204] A "neoepitope," "tumor-specific neoepitope," or "tumor antigen" refers to an epitope or antigenic determinant region that is not present in reference non-diseased cells, such as non-cancer cells or germline cells, but is found in diseased cells, such as cancer cells. As used herein, the term "neoepitope" refers to an antigenic determinant region within a peptide or neoantigenic peptide. A neoepitope may contain at least one "anchor residue" and at least one "anchor residue-adjacent region." A neoepitope may further contain a "separation region." The term "anchor residue" refers to an amino acid residue that binds to a specific pocket on HLA and provides specificity for the interaction with HLA. In some cases, the anchor residue may be in a canonical anchor position. In other cases, the anchor residue may be in a non-canonical anchor position. A neoepitope may bind to an HLA molecule through primary and secondary anchor residues that protrude into a pocket within the peptide-binding groove. In the peptide-binding groove, specific amino acids form a pocket that accommodates the corresponding side chains of the anchor residues of the presented neoepitope. Peptide binding preferences exist between different alleles of both HLA I and HLA II molecules. HLA class I molecules bind short neoepitopes, whose N- and C-termini are anchored in pockets located at the ends of the neoepitope-binding groove. Most HLA class I-binding neoepitopes are approximately 9 amino acids long, but longer neoepitopes can be accommodated by their central protuberances, resulting in binding neoepitopes of approximately 8–12 amino acids. Neoepitopes binding to HLA class II proteins are not constrained in size and can vary from approximately 16–25 amino acids. The neoepitope-binding groove in HLA class II molecules is open at both ends, allowing peptides of relatively long lengths to bind. While the approximately 9-amino acid core segment contributes most to neoepitope recognition, anchor residues flanking this region are also important for peptide specificity for HLA class II alleles. In some cases, anchor residues adjacent to the core region that contribute to peptide specificity are present N-terminal to the core region residues. In other cases, the anchor residues adjacent to the core region are C-terminal to the core region residues.In yet other cases, the anchor residues adjacent to the core region that contribute to the specificity of the peptide for HLA are both N-terminal and C-terminal to the core region residues.

[0205] " Reference " can be used to correlate and compare the results obtained by the method of the present disclosure from tumor specimen. Typically, " reference " can be obtained based on one or more normal specimens, particularly specimens that are not affected by cancer disease, obtained from a patient or one or more different individuals, for example, healthy individuals, particularly individuals of the same species. " Reference " can be experimentally determined by testing a sufficient number of normal specimens.

[0206] An "epitope" is a collective molecular feature, such as primary, secondary, and tertiary peptide structure and charge, that together form the site recognized by, for example, an immunoglobulin, a T cell receptor, an HLA molecule, or a chimeric antigen receptor. Alternatively, an epitope can be defined as the set of amino acid residues involved in recognition by a specific immunoglobulin, or, in the context of T cells, the set of amino acid residues required for recognition by a T cell receptor protein, a chimeric antigen receptor, and / or a major histocompatibility complex (MHC) receptor. A "T cell epitope" should be understood to mean a peptide sequence that can be bound by a class I or II MHC molecule in the form of a peptide-presenting MHC molecule or MHC complex, and then, in this form, can be recognized and bound by T cells, such as T lymphocytes or T helper cells. Epitopes can be prepared by isolation from natural sources, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can include "amino acid mimetics," which are artificial amino acid residues, such as D-isomers of naturally occurring L-amino acid residues or non-naturally occurring amino acid residues such as cyclohexylalanine. Throughout this disclosure, epitopes may sometimes be referred to as peptides or peptide epitopes. It should be understood that proteins or peptides comprising the epitopes or analogs described herein and additional amino acid(s) remain within the scope of this disclosure. In certain embodiments, peptides comprise fragments of antigens. In certain embodiments, the peptides of the present disclosure are limited in length. Length-limited embodiments occur when a protein or peptide comprising an epitope described herein contains a region (i.e., a continuous series of amino acid residues) that is 100% identical to a native sequence.

[0207] The nomenclature used to describe peptides or proteins follows the conventional practice of presenting the amino group at the left side of each amino acid residue (the amino-terminus or N-terminus) and the carboxyl group at the right side of each amino acid residue (the carboxyl-terminus or C-terminus). When amino acid residue positions are referred to in a peptide epitope, they are numbered from amino to carboxyl, with position 1 being the residue located at the amino terminus of the epitope or peptide or protein that may be part of it. Although not specifically shown in formulas representing selected specific embodiments of the present disclosure, the amino and carboxyl terminal groups are in the form they assume at physiological pH values ​​unless otherwise specified. In amino acid structural formulas, each residue is generally represented by a standard three-letter or single-letter designation, although when the three-letter designation or full name is used without capital letters, it may refer to an L-amino acid residue. Glycine, which does not have an asymmetric carbon atom, is simply referred to as "Gly" or "G." The amino acid sequences of peptides presented herein are generally designated using standard single-letter designations. (A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.)

[0208] The term mutation refers to a change in a nucleic acid sequence (nucleotide substitution, addition, or deletion) compared to a reference. A somatic mutation is a genetic change acquired by a cell that can be passed on to the descendants of the mutant cell during cell division. A somatic mutation is different from a germline mutation, which is an inherited genetic change that occurs in germ cells (i.e., sperm and eggs). In some embodiments, the mutation is a non-synonymous mutation. The term non-synonymous mutation refers to a mutation, e.g., a nucleotide substitution, that does not result in an amino acid change, such as an amino acid substitution in the translation product. A frameshift occurs when a mutation disrupts the normal phase of the codon periodicity of a gene (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. Different mutations in a gene can achieve the same altered reading frame.

[0209] Conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue with a similar side chain.Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).For example, the substitution of phenylalanine with tyrosine is a conservative substitution.Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate peptide function are well known in the art.

[0210] As used herein, the term affinity refers to a measure of the strength of binding between two members of a binding pair, e.g., an HLA-binding peptide and a class I or II HLA. Dis the dissociation constant and has units of molar concentration. The affinity constant is the reciprocal of the dissociation constant. The affinity constant is sometimes used as a general term to describe the chemical entity. It is a direct measure of the energy of binding. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using a commercially available Biacore SPR unit. Affinity can also be measured as the inhibitory concentration (IC 50 ) at which 50% of the peptide is displaced. Similarly, ln(IC 50 ) is IC 50 It refers to the natural logarithm of K. off refers to, for example, the off-rate constant for the dissociation of an HLA-bound peptide and class I or II HLA. Throughout this disclosure, the results of "binding data" are referred to as "IC 50 " can be expressed in terms of IC 50 is the concentration of the tested peptide in the binding assay at which 50% inhibition of binding of the labeled reference peptide is observed. Given the conditions under which the assay is performed (i.e., limiting HLA protein and labeled reference peptide concentrations), these values ​​are D The binding assay is close to the value. Assays for determining binding are well known in the art and are described in detail in, for example, PCT Publication No. WO 94 / 20127 and WO 94 / 03205, and other publications such as Sidney et al., Current Protocols in Immunology 18.3.1 (1998), Sidney, et al., J. Immunol. 154: 247 (1995), and Sette, et al., Mol. Immunol. 31: 813 (1994). Alternatively, binding can be expressed relative to the binding of a reference standard peptide. For example, the IC of the reference standard peptide 50 Compared to that, the IC 50Binding can also be determined using other assay systems, including those using live cells (e.g., Ceppellini et al., Nature 339:392 (1989), Christnick et al., Nature 352:67 (1991), Busch et al., Int. Immunol. 2:443 (1990), Hill et al., J. Immunol. 147:189 (1991), del Guercio et al., J. Immunol. 154:685 (1995)), cell-free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152,2890 (1994), Marshall et al. al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)), high-flow soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurements of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990), Schumacher et al., Cell 62:563 (1990), Townsend et al., Cell 62:285 (1990), Parker et al. al., J. Immunol. 149:1896 (1992)). "Cross-reactive binding" is evident in some peptides, ie, peptides exhibit promiscuous binding to more than one HLA molecule.

[0211] When used to discuss epitopes, the term "derived" and its grammatical equivalents are synonymous with "prepared" and its grammatical equivalents. Derived epitopes can be isolated from natural sources, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can contain "amino acid mimetics," which are artificial amino acid residues, such as D isomers of naturally occurring L-amino acid residues or unnatural amino acid residues such as cyclohexylalanine. Derived or prepared epitopes can be analogs of natural epitopes.

[0212] "Diluents" include sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is also a diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as diluents, for example, in injectable solutions.

[0213] A "native" or "wild-type" sequence refers to a sequence found in nature. Such a sequence can inherently include a longer sequence.

[0214] The term "receptor" should be understood to mean a biological molecule or group of molecules that can bind to a ligand. Receptors can play a role in transmitting information in cells, cell formations, or organisms. A receptor includes at least one receptor unit, where each receptor unit can be, for example, a protein molecule. The receptor has a structure complementary to that of the ligand and can complex with the ligand as a binding partner. The information is transmitted, in particular, by a conformational change of the receptor after complexation of the ligand on the surface of the cell. In some embodiments, receptors should be understood to mean specific proteins of MHC class I and II that can form receptor / ligand complexes with ligands, in particular peptides or peptide fragments of a suitable length.

[0215] A "ligand" should be understood to mean a molecule that has a structure complementary to that of a receptor and is capable of forming a complex with the receptor. In some embodiments, a ligand should be understood to mean a peptide or peptide fragment that has a suitable length and a suitable binding motif within its amino acid sequence so that the peptide or peptide fragment can form a complex with an MHC class I or MHC class II protein.

[0216] In some embodiments, "receptor / ligand complex" should also be understood to mean a "receptor / peptide complex" or "receptor / peptide fragment complex" comprising a class I or class II peptide- or peptide fragment-presenting MHC molecule.

[0217] "Synthetic peptide" refers to a peptide obtained from a non-natural source, e.g., an artificial peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptide" includes "fusion proteins."

[0218] The term "motif" refers to a pattern of residues in a peptide of a defined length, e.g., less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, e.g., about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for class I HLA motifs, and about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for class II HLA motifs, that is recognized by a specific HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of primary and secondary anchor residues. In some embodiments, MHC class I motifs specify peptides 9, 10, or 11 amino acid residues in length.

[0219] As used herein, the term "naturally occurring" and its grammatical equivalents refer to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0220] According to the present disclosure, the term "vaccine" relates to a pharmaceutical preparation (composition) or product that, upon administration, induces an immune response, e.g., a cellular or humoral immune response that recognizes and attacks pathogens or diseased cells, such as cancer cells. Vaccines can be used for the prevention or treatment of diseases. The term "individualized cancer vaccine" or "personalized cancer vaccine" refers to a specific cancer patient, meaning that the cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0221] A "protective immune response" or "therapeutic immune response" refers to a CTL and / or HTL response against an antigen derived from a pathogenic antigen (e.g., a tumor antigen) that in some way prevents or at least partially arrests disease symptoms, side effects, or progression. The immune response can also include an antibody response promoted by stimulation of helper T cells.

[0222] "Antigen processing" or "processing," and its grammatical equivalents, refer to the degradation of a polypeptide or antigen into process products, which are fragments of the polypeptide or antigen (e.g., degradation of a polypeptide into peptides), and the association of one or more of these fragments (e.g., by binding) with an MHC molecule for presentation by a cell, e.g., an antigen-presenting cell, to specific T cells.

[0223] Antigen-presenting cells (APCs) are cells that present peptide fragments of protein antigens in association with MHC molecules on their cell surface. Some APCs can activate antigen-specific T cells. Professional antigen-presenting cells are highly efficient at internalizing antigens by either phagocytosis or receptor-mediated endocytosis and then displaying antigen fragments bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. Further costimulatory signals are then generated by the antigen-presenting cell, leading to T cell activation. The expression of costimulatory molecules is a defining characteristic of professional antigen-presenting cells. The main types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, which have the broadest range of antigen presentation and are perhaps the most important antigen-presenting cells. Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via both MHC class II and class I antigen presentation pathways. Dendritic cells are well known to be potent inducers of immune responses, and activation of these cells is a critical step for the induction of antitumor immunity. Dendritic cells are conveniently classified as "immature" and "mature" cells, which can be used as a simple method to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells, with a high capacity for antigen uptake and processing, which correlates with high expression of Fc receptors (FcRs) and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers but high expression of cell surface molecules responsible for T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and costimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0224] As used herein, in the context of two nucleic acid or amino acid sequences of a polypeptide, the term "identical" and its grammatical equivalents, or "sequence identity," refers to residues in two sequences that are the same when aligned for maximum correspondence over a particular comparison window. As used herein, an exemplary "comparison window" refers to a segment of at least about 20 contiguous positions, typically about 50 to about 200, more typically about 100 to about 150, and a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Nat. Acad. Sci. USA, 85:2444 (1988), or by computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program by Intelligentics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA). The CLUSTAL program is described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988), Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992), and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein have at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference polypeptide, or a fragment thereof, as measured, for example, by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% sequence identity to a reference nucleic acid or a fragment thereof, as measured, for example, by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, that percentage of residues in the smaller molecule will find matching residues in the larger molecule in the order in which the two molecules are optimally aligned.

[0225] "Substantially identical" and its grammatical equivalents, as applied to nucleic acid or amino acid sequences, means that the nucleic acid or amino acid sequence contains a sequence having at least 90% or more sequence identity, at least 95%, at least 98%, and at least 99%, when compared to a reference sequence using the above-mentioned programs, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In embodiments, substantial identity exists over a region of the sequences that is at least about 50 residues long, over a region of at least about 100 residues, and in embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding region.

[0226] Vector generally refers to the construct that can deliver and usually express one or more gene(s) or sequence(s) of interest in host cell.Examples of vector include but are not limited to virus vector, naked DNA or RNA expression vector, plasmid, cosmid or phage vector, DNA or RNA expression vector associated with cationic condensing agent, and DNA or RNA expression vector encapsulated in liposome.

[0227] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, an isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. In some embodiments, an "isolated polynucleotide" encompasses a PCR or quantitative PCR reaction, including a polynucleotide amplified in a PCR or quantitative PCR reaction.

[0228] The terms "isolated," "biologically pure," and their grammatical equivalents refer to a material that is substantially or essentially free from components that normally accompany the material as found in its natural state. Thus, the isolated peptides described herein do not contain some or all of the materials normally associated with the peptide in their native environment. An "isolated" epitope refers to an epitope that does not contain the entire sequence of the antigen from which the epitope is derived. Typically, an "isolated" epitope does not have additional amino acid residues attached to it that result in a sequence that is 100% identical over the entire length of the native sequence. The native sequence may be a sequence such as a tumor-associated antigen from which the epitope is derived. Thus, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs in nature). An "isolated" nucleic acid is a nucleic acid that has been removed from its natural environment. For example, a naturally occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide separated from some or all of the coexisting materials in the natural system is isolated. Such polynucleotides may be part of a vector, and / or such polynucleotides or peptides may be part of a composition, which may still be "isolated" in that such vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically.

[0229] As used herein, the term "substantially purified," and its grammatical equivalents, refers to a nucleic acid sequence, polypeptide, protein, or other compound that is essentially free, i.e., more than about 50% free, more than about 70% free, or more than about 90% free, from polynucleotides, proteins, polypeptides, and other molecules with which the nucleic acid sequence, polypeptide, protein, or other compound is naturally associated.

[0230] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0231] The terms "polynucleotide," "nucleotide," "nucleic acid," "polynucleic acid," or "oligonucleotide," and their grammatical equivalents, are used interchangeably herein to refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. Thus, these terms include double- and single-stranded DNA, triple-stranded DNA, and double- and single-stranded RNA. They also include modifications, such as by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also meant to encompass molecules containing non-natural or synthetic nucleotides and nucleotide analogs. The nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into cells, for example, by transfection, transformation, or transduction. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, polynucleotides and nucleic acids can be in vitro transcribed mRNA. In some embodiments, the polynucleotide administered using the methods of the present disclosure is mRNA.

[0232] Generally, transfection, transformation, or transduction refers to the introduction of one or more exogenous polynucleotides into host cells by using physical or chemical methods.Many transfection techniques are known in the art, including, for example, calcium phosphate DNA coprecipitation (e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)), DEAE-dextran, electroporation, cationic liposome-mediated transfection, tungsten particle-promoted microprojectile bombardment (see Johnston, Nature, 346: 776-777 (1990)), and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).Phage or viral vectors can be introduced into host cells after the propagation of infectious particles in suitable packaging cells, many of which are commercially available.

[0233] Nucleic acids and / or nucleic acid sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are "homologous" if their encoding DNA is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules can be referred to as homologs. For example, any naturally occurring protein described herein can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide homologous to the protein encoded by the original nucleic acid. Homology is generally inferred from the sequence identity between two or more nucleic acids or proteins (or their sequences). The exact percentage of identity between sequences useful for establishing homology varies depending on the nucleic acid and protein in question, but no more than 25% sequence identity is typically used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining percent sequence identity (eg, BLASTP and BLASTN using default parameters) are described herein and are publicly available.

[0234] The term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0235] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a therapeutic agent effective to "treat" a disease or disorder in a subject or mammal. A therapeutically effective amount of a drug has a therapeutic effect and may prevent the onset of a disease or disorder, delay the onset of a disease or disorder, slow the progression of a disease or disorder, alleviate to some extent one or more of the symptoms associated with a disease or disorder, reduce morbidity and mortality, improve quality of life, or some combination of such effects.

[0236] "Treat" or "treatment" or "treating" or "alleviate" or "alleviating" refers to both (1) therapeutic measures that cure, delay, reduce the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent or delay the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder, those prone to have the disorder, and those in whom the disorder is to be prevented.

[0237] "Pharmaceutically acceptable" generally refers to a composition or component of a composition that is non-toxic, inert, and / or physiologically compatible.

[0238] "Pharmaceutical excipients" or "excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, etc. A "pharmaceutical excipient" is a pharmaceutically acceptable excipient.

[0239] As used herein, the terms "scaffold domain," "scaffolding domain," and "scaffold polypeptide" are used interchangeably.

[0240] In one aspect, described herein are fusion polypeptides containing multiple epitopes or antigens fused to a scaffold protein. Epitopes are short peptides that can be approximately 5-50 amino acids in length. Fusion polypeptides, or fusion polyproteins, or "polybodies," can be generated by combining multiple epitopes into a single molecule. Polybodies are useful for generating epitope-containing therapeutics, for example, for immunotherapy, where the epitope is the active therapeutic component. While epitopes alone may rapidly degrade in biological systems without achieving their expected results, polybody forms can be designed to offer advantages over single epitope forms. In polybodies, the scaffold protein can be designed to stabilize the neoantigen, improve epitope solubility, and increase molecular weight, e.g., above 50 kDa, to enable lymph node targeting and retention, target specific tissues or cells, or introduce tags for protein purification or identification.

[0241] From a manufacturing perspective, polybodies reduce batch production of single peptides. The absence of post-translational modifications allows for inexpensive and rapid recombinant production of scaffolds in E. coli. The scaffolds are relatively small and have no known toxicity. In some embodiments, the scaffold protein is a human protein, thereby ensuring a lack of antigenicity. In some embodiments, functional scaffolds can be incorporated, such as for dendritic cell targeting or activation. Ubiquitin scaffolds can enhance proteasomal degradation and antigen processing. In some embodiments, the scaffold protein can be further modified to include, for example, a cell-targeting moiety.

[0242] Fusion Polypeptides In one aspect, disclosed herein is a fusion polypeptide comprising one or more antigen polypeptides and one or more scaffold polypeptides. Also disclosed herein are nucleic acid molecules encoding the fusion polypeptides and compositions comprising the fusion polypeptides or nucleic acid molecules. In some embodiments, the fusion polypeptide comprises a polypeptide sequence comprising one or more antigen polypeptide sequences and one or more scaffold polypeptide sequences. In some embodiments, the fusion polypeptide comprises two or more scaffold polypeptide sequences. In some embodiments, each of the scaffold polypeptide sequences comprises a human polypeptide sequence, a fragment thereof, or a variant thereof. In some embodiments, the molecular weight of the two or more scaffold polypeptide sequences is greater than 11 kDa. In some embodiments, each of the two or more scaffold polypeptide sequences comprises at least 21 amino acid residues. In some embodiments, the scaffold polypeptide is selected from Stefin A, titin-I27, fragments thereof, and variants thereof. In some embodiments, at least one of the scaffold polypeptide sequences is connected to at least one of the one or more antigen polypeptide sequences via one or more linker sequences. In some embodiments, the fusion polypeptide is configured to facilitate cleavage of the linker, cleavage of at least one of the one or more antigen polypeptides, or presentation of at least one of the one or more antigen polypeptides. In some embodiments, the antigen polypeptide is a cancer antigen.

[0243] In some embodiments, the molecular weight of the fusion polypeptide is at least 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, or 200 kDa. In some embodiments, the molecular weight of the fusion polypeptide is at least 75 kDa. In some embodiments, the molecular weight of the fusion polypeptide is at most 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, 200 kDa, or 500 kDa. In some embodiments, the molecular weight of the fusion polypeptide is about 50-80 kDa. In some embodiments, the molecular weight of the fusion polypeptide is about 70-80 kDa. Fusion polypeptides can be configured in a linear or branched format. In some embodiments, the fusion polypeptide is in a linear format. In some embodiments, the fusion polypeptide has multiple branches.

[0244] One or more scaffold polypeptides can be linked to or between the remaining sequences of the fusion polypeptide in various ways. For example, the scaffold polypeptides can be linked via the N-terminus or C-terminus, or via an insertion between the N-terminus and C-terminus. In some embodiments, at least one of the scaffold polypeptide sequences is linked to the remaining sequences of the fusion polypeptide via its N-terminus. In some embodiments, at least one of the scaffold polypeptide sequences is linked to the remaining sequences of the fusion polypeptide via its C-terminus. In some embodiments, each of the one or more scaffold polypeptides is linked to the remaining sequences of the fusion polypeptide via its N-terminus, C-terminus, or both. In some embodiments, at least two of the scaffold polypeptide sequences are not interrupted by an antigen sequence or a linker sequence. In some embodiments, the scaffold polypeptides are not linked by an insertion between their respective N-terminus and C-terminus.

[0245] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (Ia): A n -S m , or S m -A n Formula (Ia), wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, m is an integer greater than or equal to 1, and n is an integer greater than or equal to 1. In some embodiments, each of m and n is an integer independently selected from 1-10. In some embodiments, each of m and n is an integer independently selected from 1-5. In some embodiments, each of m and n is an integer independently selected from 1-3. In some embodiments, each of m and n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 1 and n is 2. In some embodiments, n is 1 and m is 2. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal direction, the structure AAS, AAAS, AAAAS, AAAAAS, AAAAAAS, or AAAAAAAS. In some embodiments, a fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal, the structure SAA, SAAA, SAAAA, SAAAAAA, SAAAAAA, or SAAAAAAA. In some embodiments, a fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal, the structure AS, ASS, ASSS, ASSSS, ASSSSS, ASSSSS, or ASSSSSS. In some embodiments, a fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal, the structure SA, SSA, SSSA, SSSSA, SSSSSA, or SSSSSSA.

[0246] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (Ib): S O -A n -S m Formula (Ib) wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, and each of o, n, and m independently is an integer greater than or equal to 1. In some embodiments, each of o, n, and m independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each of o, m, and n is an integer independently selected from 1-5. In some embodiments, o is 1 or 2, m is 1 or 2, and n is an integer selected from 1-5. In some embodiments, o and m are equal to 1, and n is an integer between 1 and 5. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal direction, the structure SAS, ASAS, SASA, SASAS, SASASAS, SASASASAS, or SASASASASAS. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal direction, the structure SAAS, SAAAS, SAAAAS, SAAAAAS, SAAAAAAS, or SAAAAAAAS.

[0247] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (IIa): (LAL) n -S m , or S m -(LAL)n Formula (IIa),

[0248] wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, each L independently represents a linker sequence or is absent, m is an integer greater than or equal to 1, and n is an integer greater than or equal to 1. In some embodiments of Formula (IIa), m is an integer greater than or equal to 2. In some embodiments of Formula (IIa), n is an integer selected from 1 to 5, and m is 2. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal, the structure ALS, ALALS, ALALALS, SLA, SLALA, or SLALALA.

[0249] In some embodiments, the fusion polypeptide comprises, from N-terminal to C-terminal, a polypeptide sequence having the structure of Formula (IIb): S o -(LAL) n -S m Formula (IIb),

[0250] wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, each L independently represents a linker sequence or is absent, and each of o, n, and m independently is an integer greater than or equal to 1. In some embodiments, o is 1 or 2, m is 1 or 2, and n is an integer selected from 1 to 5. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal direction, the structure SLALS, SALSLALS, SLALSLA, SLALSLALS, SLALSLALSLALS, SLALSLALSLALSLAS, or SLALSLALSLALSLASLALS.

[0251] In some embodiments, the scaffold polypeptide and the antigen polypeptide are positioned in an alternating manner within the fusion polypeptide, with or without a linker sequence between them. For example, in some embodiments, the fusion polypeptide comprises a polypeptide sequence having, from N-terminal to C-terminal, the structure SALSLALS, ALSLALSLA, ASLALSLALS, SLALSLALSLALSA, ASASLALS, or SLALSLALSASA. In some embodiments, each of the scaffold polypeptide sequences is connected to one or two of the antigen polypeptide sequences via at least one of the linker sequences.

[0252] The described fusion polypeptides can result in improved lymph node accumulation or retention compared to corresponding polypeptides lacking a scaffold domain. In some embodiments, the accumulation of the fusion polypeptide in lymph nodes after administration to a subject is at least 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-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the accumulation of the corresponding polypeptide lacking the scaffold polypeptide, as measured by mean radiant efficiency. In some embodiments, the accumulation of the fusion polypeptide in lymph nodes after administration to a subject is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the accumulation of the corresponding polypeptide lacking the scaffold polypeptide, as measured by mean radiant efficiency. For example, lymph node accumulation can be measured according to the method described in Example 3.1.

[0253] The described fusion polypeptides can have improved solubility in aqueous solvents compared to corresponding polypeptides lacking a scaffold domain. In some embodiments, the solubility of a fusion polypeptide in an aqueous solvent is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the solubility of its corresponding polypeptide lacking a scaffold polypeptide, measured in the same solvent. In some embodiments, the solubility of a fusion polypeptide in an aqueous formulation is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the solubility of its corresponding polypeptide lacking a scaffold polypeptide. The described fusion polypeptides can have improved stability, e.g., serum stability, compared to corresponding polypeptides lacking a scaffold domain. In some embodiments, the serum half-life of the fusion polypeptide is at least 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-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer than the serum half-life of its corresponding polypeptide lacking the scaffold polypeptide. In some embodiments, the serum half-life of the fusion polypeptide is at least 1.1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer than the serum half-life of its corresponding polypeptide lacking the scaffold polypeptide. The described fusion polypeptides can have improved solubility compared to the corresponding polypeptide lacking the scaffold domain. In some embodiments, the serum solubility of the fusion polypeptide is at least 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-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the serum solubility of its corresponding polypeptide lacking the scaffold polypeptide.

[0254] The described fusion polypeptides can induce an enhanced immune response compared to the corresponding polypeptide lacking the scaffold domain. In some embodiments, the antigen-specific T cell response of the fusion polypeptide after administration to a subject is at least 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-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or 300-fold higher than the antigen-specific T cell response of the corresponding polypeptide lacking the scaffold polypeptide, as measured by an increase in splenocyte proliferation. For example, antigen-specific T cell responses can be measured according to the method of Example 3.2.

[0255] Scaffold polypeptides or scaffold domains The fusion polypeptide can comprise one or more scaffold polypeptide sequences. In some embodiments, the one or more scaffold polypeptides are the same. In some embodiments, the one or more scaffold polypeptides comprise at least two different scaffold polypeptide sequences. In certain embodiments, the one or more scaffold polypeptides comprise two, three, four, five, six, seven, eight, nine, ten, or more different scaffold polypeptide sequences.

[0256] In some embodiments, one or more scaffold polypeptides comprise a recombinant human polypeptide. In some embodiments, one or more scaffold polypeptides comprise a sequence derived from a human protein. In some embodiments, one or more scaffold polypeptides comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a wild-type mammalian protein, such as a human protein. In some embodiments, one or more scaffold polypeptides are not configured to have target binding properties. In other embodiments, at least one of the scaffold polypeptides is configured to bind to a target molecule. In some embodiments, the scaffold polypeptide lacks post-translational modifications. In some embodiments, the scaffold polypeptide lacks intrapeptide disulfide bonds. In some embodiments, one or more scaffold polypeptides are non-immunogenic. In other embodiments, at least one of the scaffold polypeptides is immunogenic. In some embodiments, the scaffold polypeptides are not configured to have enzymatic activity. In some embodiments, one or more scaffold polypeptides lack a signal sequence.

[0257] In some embodiments, a fusion polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 scaffold polypeptides. In some embodiments, a fusion polypeptide comprises at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 scaffold polypeptides. In some embodiments, a fusion polypeptide comprises 2 to 6 scaffold polypeptides. In some embodiments, a fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 scaffold polypeptides. In certain embodiments, a fusion polypeptide comprises 6 scaffold polypeptides.

[0258] In some embodiments, the molecular weight of the scaffold polypeptide sequence in the fusion polypeptide is at least 11 kDa, 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, or 200 kDa. In some embodiments, the molecular weight of the scaffold polypeptide sequence in the fusion polypeptide is at least 75 kDa. In some embodiments, the molecular weight of the scaffold polypeptide sequence in the fusion polypeptide is at most 15 kDa, 20 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 150 kDa, 200 kDa, or 500 kDa. In some embodiments, the molecular weight of each of the scaffold polypeptide sequences is at least 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, or 100 kDa. In some embodiments, the molecular weight of each of the scaffold polypeptide sequences is at most 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, or 250 kDa. In some embodiments, at least one of the scaffold polypeptides has a molecular weight of 8-12 kDa, or 10-12 kDa. In some embodiments, at least one of the scaffold polypeptides has a molecular weight of about 11 kDa.

[0259] In some embodiments, each of the scaffold polypeptide sequences comprises at least 21, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 amino acid residues. In some embodiments, each of the scaffold polypeptide sequences comprises at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75, at most 80, at most 85, at most 90, at most 100, at most 125, at most 150, at most 175, or at most 200 amino acid residues. In some embodiments, at least one of the scaffold polypeptides has at least 80, at least 85, at least 90, or at least 95 amino acid residues. In some embodiments, at least one of the scaffold polypeptides has a sequence of about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 110, or 120 amino acids.

[0260] In some embodiments, the scaffold protein is a mammalian protein, e.g., a human protein that can be suitably expressed in a bacterial expression system, such as an E. coli expression system, and the protein is small, stable, and either secreted or cytoplasmic. In some embodiments, the scaffold protein is a human protein. Mammalian cells contain an estimated 1 billion protein molecules. The majority of these proteins can be found to function as scaffold proteins, helping to enhance and support biomolecular interactions within the body. These interactions facilitate aspects of cellular behavior, such as signal transduction, intracellular transport of biomolecules, enzymatic activity, or structural rearrangements that promote cell motility. In this disclosure, the inventors provide an efficient and simple method for converting short epitope peptides into a means for generating an active immune response using human scaffold proteins. In some embodiments, the scaffold protein is selected to confer stability to the epitope peptide. In some embodiments, the scaffold protein is selected to confer solubility and increased serum half-life to the epitope peptide. In some embodiments, the scaffold protein is selected to confer a structural advantage in conferring immunogenicity, for example, by increasing the availability for association with cognate MHC molecules. The scaffold protein may be selected so that it is non-toxic. The scaffold protein may be any protein with a scaffolding function or ability that meets any or all of these purposes.

[0261] For example, known cellular scaffolding proteins support other functional proteins to reduce the entropy of a reaction. In some embodiments, scaffolding proteins tether one or more proteins, enzymes, peptides, or other biomolecules, increasing their local concentration or temporarily immobilizing them for a functional outcome. For example, the Cullin scaffolding protein tethers E2 ubiquitin, and the proteins linker for activation of T cells (LAT) and 76 kD SH2 domain-containing leukocyte protein (SLP-76) help organize TCR signaling.

[0262] In some embodiments, at least one of the one or more scaffold polypeptides is a member of the cystatin superfamily, a variant thereof, or a fragment thereof. In some embodiments, the member of the cystatin superfamily is cystatin type 1, cystatin type 2, or cystatin type 3. In some embodiments, at least one of the one or more scaffold polypeptides is Stefin A, such as human Stefin A. In some embodiments, all of the scaffold polypeptides are Stefin A. In some embodiments, the one or more scaffold polypeptides are human Stefin A. A protein sequence includes sequences having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the protein sequence.

[0263] In some embodiments, one or more scaffold polypeptides comprise the sequence of a mammalian titin protein, a variant thereof, or a fragment thereof. The titin protein sequence can be from any domain of the titin protein, such as the Ig-like domain, the FnIII-like domain, and the pseudokinase domain. In some embodiments, the titin protein sequence is an I-band sequence. In some embodiments, one or more scaffold polypeptides comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a titin Ig-like domain sequence. In some embodiments, the sequence from the titin protein is the I27 domain sequence, the I1 domain sequence, the Z1 domain sequence, the Z2 domain sequence, or the M5 domain sequence. In some embodiments, the sequence from the titin protein is the I27 domain sequence. In some embodiments, one or more scaffold polypeptides comprise titin I27, a variant thereof, or a fragment thereof.

[0264] In some embodiments, one or more scaffold polypeptides comprise a sequence of a glycoprotein, such as fibronectin, a variant thereof, or a fragment thereof. In some embodiments, the glycoprotein is fibronectin. The fibronectin sequence can be from any of its subunits, such as a fibronectin type I, type II, or type II domain. In some embodiments, one or more scaffold polypeptides comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the sequence of FnIII, such as 1FNIII, 9FNIII, 10FNIII, or 11FNIII. In some embodiments, the FNIII sequence is a 10FN-III domain sequence.

[0265] In some embodiments, the one or more scaffold polypeptides comprise a polypeptide sequence selected from thioredoxin, 10FNIII, lipocalin, adeno-associated virus capsid polypeptide, alpha-amylase inhibitor, Stefin A, ubiquitin, Ig-L filamin A, tenascin, inactivated staphylococcal nuclease, green fluorescent protein, isolated protein folds such as the Z domain of staphylococcal protein A, ankyrin repeats, bilin-binding proteins, fragments thereof, and variants thereof. In some embodiments, the one or more scaffold polypeptides are each independently selected from titin I27, ubiquitin, Stefin A, 10FN-III, Ig-L filamin A, tenascin, fragments thereof, and variants thereof. In some embodiments, the scaffold polypeptides are each independently selected from Stefin A, titin I27, fragments thereof, and variants thereof. In some embodiments, each of the scaffold polypeptides is Stefin A, a fragment thereof, or a variant thereof.

[0266] The scaffold protein can be a full-length protein or a fragment, or a specific domain. In some embodiments, the scaffold can be a protein A domain, a Src homology domain, a PDZ domain, a WW domain, a zinc finger domain, or a derivative thereof.

[0267] In some embodiments, the scaffold protein can be modified to suit requirements. In some embodiments, the scaffold is mutated at one, two, three, or more residues to reduce toxicity, reduce biological interactions, or reduce immunogenicity. In some embodiments, the scaffold is mutated at one, two, three, or more residues to include a binding site or specificity determinant moiety, e.g., a targeting moiety, such as a dendritic cell (DC) incorporation targeting the CLEC9A moiety.

[0268] antigenic polypeptide The fusion polypeptides described herein can comprise one or more antigen polypeptides. In some embodiments, the fusion polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 antigen polypeptides. In some embodiments, the fusion polypeptide comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 antigen polypeptides. In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5, or 6 antigen polypeptides. In some embodiments, the fusion polypeptide comprises 5 or 6 antigen polypeptides.

[0269] In some embodiments, the fusion polypeptide comprises multiple antigen polypeptides. In some embodiments, each of the antigen polypeptide sequences is different from the other antigen polypeptide sequences on the fusion polypeptide. In some embodiments, all of the antigen polypeptides of the fusion polypeptide have the same sequence. In some embodiments, at least one of the antigen polypeptides is a synthetic polypeptide.

[0270] The one or more antigen polypeptides can include any antigen polypeptide that induces an immune response, for example, an exogenous antigen, an endogenous antigen, an autoantigen, a neoantigen, or a combination thereof. In some embodiments, at least one antigen binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, at least one antigen binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, at least one antigen binds to a CD8 + In some embodiments, at least one antigen activates CD4 T cells. + activates T cells. In some embodiments, the one or more antigenic polypeptides comprise a first antigenic polypeptide and a second antigenic polypeptide. In some embodiments, the first antigenic polypeptide binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second antigenic polypeptide binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second antigenic polypeptide binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first antigenic polypeptide binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the single polypeptide comprises a first antigenic polypeptide and a second antigenic polypeptide. In some embodiments, the first antigenic polypeptide binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the first antigen polypeptide may comprise a CD8 HLA-binding epitope and a class II HLA-binding epitope. + In some embodiments, the second antigenic polypeptide activates CD4 T cells. +activates T cells. In some embodiments, a linker sequence is directly connected to a scaffold polypeptide sequence and an antigen polypeptide sequence. In some embodiments, a linker sequence is directly connected to a scaffold polypeptide sequence and another linker sequence. In some embodiments, a linker sequence is directly connected to an antigen polypeptide sequence and another linker sequence. In some embodiments, a linker sequence is directly connected to two scaffold polypeptide sequences. In some embodiments, a linker sequence is directly connected to two antigen polypeptide sequences. In some embodiments, a linker sequence is directly connected to two other linker sequences. In some embodiments, a linker sequence may be directly connected to three or more of the following: a scaffold polypeptide sequence(s), an antigen polypeptide sequence(s), a linker sequence(s), or any combination thereof.

[0271] In some embodiments, one or more linker sequences are the same. In some embodiments, each of the one or more linker sequences is unique. In some embodiments, the fusion polypeptide comprises at least two different linker sequences. In some embodiments, each of the linker sequences comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 amino acid residues. In some embodiments, each of the linker sequences comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or 100 amino acid residues. In some embodiments, one or more linker sequences comprise between 5 and 20 amino acid residues. In some embodiments, the linker sequence is non-immunogenic. In some embodiments, the linker sequence is not configured to form a disulfide bond. In some embodiments, the linker comprises 1 to 100 amino acid residues, 2 to 50 amino acid residues, or 2 to 25 amino acid residues. In some embodiments, the linker is flexible. In some embodiments, at least one of the linkers is flexible. In some embodiments, the flexible linker comprises small nonpolar amino acids, such as GIy, Ser, and Thr. In some embodiments, the flexible linker comprises a stretch of GIy and Ser residues, such as GIy-GIy-GIy-GIy-Ser. In some embodiments, the flexible linker comprises the sequence KESGSVSSEQLAQFRSLD. In some embodiments, the flexible linker comprises the sequence EGKSSGSGSESKST. In some embodiments, the flexible linker comprises the sequence (GIy)z, where GIy is an integer between 5 and 10. In some embodiments, the flexible linker comprises the sequence (GIy)8. In some embodiments, the flexible linker comprises the sequence GSAGSAAGSGEF. In some embodiments, the linker is rigid. In some embodiments, at least one of the linkers is rigid. In some embodiments, the rigid linker is capable of forming an alpha helix.In some embodiments, the rigid linker comprises the sequence EAAAK. In some embodiments, the rigid linker comprises the sequence Glu-Pro. In some embodiments, the rigid linker comprises the sequence Lys-Pro. Exemplary linker sequences may include those disclosed in Chen, et al., Adv Drug Deliv Rev. 2013 Oct 15;65(10):1357-1369, "Fusion Protein Linkers: Property, Design and Functionality."

[0272] In some embodiments, the fusion polypeptide is configured to facilitate cleavage of the linker. In some embodiments, the fusion polypeptide is configured to facilitate cleavage of at least one of the one or more antigen polypeptides. In some embodiments, the linker is cleavable. In some embodiments, at least one of the linkers is cleavable. The cleavable linker may comprise one or more cleavable sites. In some embodiments, at least one of the linker sequences comprises a cleavage site. In some embodiments, the cleavage site is cleavable by a cellular reducing agent. For example, the cleavable site may comprise a disulfide bond, e.g., a disulfide bond formed between two cysteine ​​residues on the linker. In some embodiments, the cleavage site is cleavable by a peptidase or protease. In some embodiments, the cleavage site is cleavable by a protease, e.g., Kex1, Ste13, and Kex2.

[0273] In some embodiments, the fusion polypeptide is configured to promote presentation of at least one of the one or more antigenic polypeptides. In some embodiments, the linker comprises a context sequence capable of promoting presentation of the antigenic polypeptide. In some embodiments, at least one of the linker sequences comprises a lysine residue, an arginine residue, a serine residue, an asparagine residue, a histidine residue, an alanine residue, a glutamine residue, an aspartic acid residue, a methionine residue, a tyrosine residue, or any combination thereof. In some embodiments, at least one of the linker sequences comprises a lysine residue, an arginine residue, or an alanine residue directly connected to the N-terminus of at least one of the antigenic polypeptides. In some embodiments, at least one of the linker sequences comprises a serine residue, a lysine residue, an arginine residue, or an alanine residue directly connected to the C-terminus of at least one of the antigenic polypeptides. In some embodiments, at least one of the linker sequences comprises an aspartic acid residue, a methionine residue, a leucine residue, a valine residue, an isoleucine residue, a tyrosine residue, a phenylalanine residue, a tryptophan residue, or any combination thereof. In some embodiments, at least one of the linker sequences comprises an aspartic acid, methionine, leucine, tyrosine, or phenylalanine residue directly connected to the N-terminus of at least one of the antigenic polypeptides, hi some embodiments, at least one of the linker sequences comprises a methionine, leucine, valine, isoleucine, tyrosine, phenylalanine, or tryptophan residue directly connected to the C-terminus of at least one of the antigenic polypeptides.

[0274] In some embodiments, at least one of the linkers is functional. For example, the linker can be configured to improve the expression level of the fusion polypeptide, improve the biological activity of the fusion polypeptide, or enable the fusion polypeptide to target a specific site in vivo. In another example, the linker can be configured to affect the PK and PD properties of the fusion polypeptide.

[0275] Targeting capabilities In some embodiments, the fusion polypeptide is configured to target a specific site, such as an in vivo target site. For example, the fusion polypeptide can have one or more binding moieties that have high affinity or bind to the target site. Exemplary target sites can include, but are not limited to, drugs, drugs, proteins or polypeptides, and cells, such as antigen-presenting cells. In some embodiments, the fusion polypeptide comprises one or more binding moieties configured to bind to antigen-presenting cells, adjuvants, or reagents. In some embodiments, the antigen-presenting cells are dendritic cells (DCs), macrophages, Langerhans cells, or B cells.

[0276] In some embodiments, the one or more binding moieties are configured to bind to one or more receptors expressed on a cell, such as a dendritic cell.

[0277] In some embodiments, the one or more receptors comprise a calcium-dependent (C-type) lectin receptor, a scavenger receptor, an F4 / 80 receptor, a DC-specific transmembrane protein (DC-STAMP), an Fc receptor, or any combination thereof.

[0278] In some embodiments, the C-type lectin receptor is mannose receptor, dendritic cell-specific intercellular adhesion molecule-3-linked nonintegrin (DC-SIGN) receptor, L-SIGN or DC-SIGNR receptor, liver and lymph node sinusoidal cell-type lectin (LSECtin) receptor, C-type lectin immunoreceptor (CIRE), Langerin receptor, human macrophage galactose- and N-acetylgalactosamine-specific C-type lectin receptor, Dectin-1 or β-glucan receptor, NK lectin family receptor-1, myeloid inhibitory C-type lectin receptor, C-type lectin-like receptor 2 (CLEC2), CLEC12B receptor, lectin-like receptor for oxidized density lipoprotein-1, DC immunoreceptor subfamily receptor, DC immunoreceptor, Dectin-2 receptor, or blood DC antigen.

[0279] In some embodiments, the one or more receptors include Clec9a.

[0280] In some embodiments, the one or more receptors comprise a chemokine receptor.

[0281] In some embodiments, the one or more receptors include the XCR1 receptor.

[0282] In some embodiments, at least one of the binding moieties is comprised in a scaffold polypeptide.

[0283] In some embodiments, at least one of the binding moieties is attached directly to the N-terminus or C-terminus of one of the scaffold polypeptides.

[0284] In some embodiments, at least one of the binding moieties is directly attached to the N-terminus or C-terminus of one of the antigenic polypeptides.

[0285] In some embodiments, in the N-terminal to C-terminal direction, at least one of the binding moieties is terminally linked to the first or last one of the scaffold polypeptides.

[0286] In some embodiments, the nucleic acid molecule encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 different fusion polypeptides.

[0287] In some embodiments, the nucleic acid molecule encodes at most 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 different fusion polypeptides.

[0288] In some embodiments, the nucleic acid molecule is RNA or DNA.

[0289] In one aspect, disclosed herein are fusion polypeptides encoded by the described nucleic acid molecules.

[0290] In one aspect, disclosed herein is a composition comprising the fusion polypeptide described. In some embodiments, the composition comprises one or more binding moieties that can be conjugated to the fusion polypeptide. In some embodiments, one or more binding moieties are conjugated to the fusion polypeptide.

[0291] In one aspect, disclosed herein is a nucleic acid molecule encoding two or more scaffold polypeptides spaced by one or more linkers and one or more restriction sites located in at least one of the linkers, wherein (i) the two or more scaffold polypeptide sequences have a molecular weight of greater than 11 kDa, or (ii) each of the two or more scaffold polypeptide sequences comprises at least 21 amino acid residues.

[0292] In one aspect, disclosed herein is a plurality of nucleic acid molecules comprising: a first nucleic acid molecule comprising a nucleic acid sequence encoding a first antigenic polypeptide and a nucleic acid sequence encoding a first scaffold polypeptide; and a second nucleic acid molecule comprising a nucleic acid sequence comprising a sequence complementary to the nucleic acid sequence encoding the first antigenic polypeptide and a nucleic acid sequence encoding a second scaffold polypeptide.

[0293] In another aspect, disclosed herein is a plurality of nucleic acid molecules comprising: a first nucleic acid molecule comprising a nucleic acid sequence encoding a first antigenic polypeptide, a nucleic acid sequence encoding a first scaffold polypeptide, and a nucleic acid sequence encoding a second antigenic polypeptide; and a second nucleic acid molecule comprising a nucleic acid sequence comprising a sequence complementary to the nucleic acid sequence encoding the second antigenic polypeptide, and a nucleic acid sequence encoding the second scaffold polypeptide.

[0294] In yet another aspect, disclosed herein is a plurality of nucleic acid molecules comprising: a first nucleic acid molecule comprising a nucleic acid sequence encoding a first antigenic polypeptide, a nucleic acid sequence encoding a first scaffold polypeptide, and a nucleic acid sequence encoding a second antigenic polypeptide; and a second nucleic acid molecule comprising a nucleic acid sequence comprising a sequence complementary to the nucleic acid sequence encoding the second antigenic polypeptide, a nucleic acid sequence encoding the second scaffold polypeptide, and a nucleic acid sequence encoding a third antigenic polypeptide.

[0295] In some embodiments, the first scaffold sequence and the second scaffold sequence are the same.

[0296] In some embodiments, the first antigenic polypeptide and the second antigenic polypeptide are different.

[0297] In some embodiments, the first, second, and third antigenic polypeptides are different.

[0298] In some embodiments, the nucleic acid molecule is RNA or DNA.

[0299] In one aspect, disclosed herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent and a described composition or a described fusion polypeptide.

[0300] In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0301] In some embodiments, the adjuvant is PolyIC:LC.

[0302] In some embodiments, the pharmaceutical composition comprises a pH adjuster.

[0303] In some embodiments, the pharmaceutical composition comprises a second therapeutic agent, such as an immunomodulatory agent, cytokine, chemokine, or checkpoint inhibitor.

[0304] In one aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising expressing a described nucleic acid molecule in a genetically modified cell, thereby producing the immunogenic fusion polypeptide.

[0305] In another aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising providing a nucleic acid molecule described herein; inserting one or more nucleic acid molecules encoding one or more antigenic polypeptides into at least one of the restriction sites, thereby producing a new nucleic acid molecule; and expressing the new nucleic acid molecule in a genetically modified cell, thereby producing the immunogenic fusion polypeptide.

[0306] In some embodiments, one or more nucleic acid molecules encoding one or more antigenic polypeptides are inserted through an isothermal reaction or by restriction enzyme-based cloning.

[0307] In one aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising providing a plurality of nucleic acid molecules described herein, joining the nucleic acid molecules by hybridization, and expressing the joined nucleic acid molecules in a genetically modified cell, thereby producing the immunogenic fusion polypeptide.

[0308] In some embodiments, the fusion polypeptide is expressed in a bacterial expression system.

[0309] In some embodiments, the bacterial expression system is an Escherichia coli expression system.

[0310] In one aspect, disclosed herein is a method of treating or preventing cancer in a human subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0311] In some embodiments, the pharmaceutical composition comprises multiple neoantigenic peptides.

[0312] In some embodiments, the pharmaceutical composition comprises multiple fusion polypeptides.

[0313] In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously.

[0314] In some embodiments, the dose of the fusion polypeptide is divided into at least two, at least three, at least four, or at least five subdoses.

[0315] In some embodiments, each sub-dose of fusion polypeptide comprises 1, 2, 3, 4, 5, or more fusion polypeptides.

[0316] In some embodiments, each fusion polypeptide is administered at a dose of 0.01 to 100 μg.

[0317] In some embodiments, each fusion polypeptide is administered at a dose of 100 μg to 10 mg.

[0318] In some embodiments, the total dose of fusion polypeptide administered is between 0.01 and 100 mg.

[0319] Neoantigens and uses thereof One of the key barriers to developing curative and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic alterations in malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific class of antigens. Neoantigens are rarely used in cancer vaccines or immunogenic compositions due to the technical difficulties involved in identifying them, selecting optimized antigens, and producing neoantigens for use in vaccines or immunogenic compositions. These problems can be addressed by identifying mutations in the neoplasm / tumor that are present at the DNA level in the tumor but not in matched germline cell samples from a large proportion of subjects with cancer, analyzing the identified mutations using one or more peptide-MHC binding prediction algorithms to generate multiple neoantigenic T cell epitopes that are expressed in the neoplasm / tumor and bind to a large proportion of patient HLA alleles, and synthesizing multiple neoantigenic peptides selected from the set of all neoantigenic peptides and predicted binding peptides for use in a cancer vaccine or immunogenic composition suitable for treating a large proportion of subjects with cancer.

[0320] For example, converting peptide sequencing information into a therapeutic vaccine may involve predicting mutant peptides that will bind to HLA molecules in a large proportion of individuals. Effectively selecting which specific mutations to utilize as immunogens requires the ability to predict which mutant peptides will bind efficiently to a large proportion of patients' HLA alleles. Recently, neural network-based learning approaches that validate binding and non-binding peptides have improved the accuracy of prediction algorithms for major HLA-A and -B alleles. However, even with the use of sophisticated neural network-based algorithms to encode HLA-peptide binding rules, several factors limit the ability to predict peptides presented on HLA alleles.

[0321] Another example of converting peptide sequencing information into a therapeutic vaccine may involve formulating a drug as a long peptide multi-epitope vaccine. Targeting as many variant epitopes as practically possible harnesses the vast capabilities of the immune system, prevents the chance of immune evasion due to downregulation of immune target gene products, and compensates for the known inaccuracies of epitope prediction approaches. Provided herein are polypeptides containing multiple epitopes for generating therapeutic products or vaccines, where the polypeptides are expressed from a polynucleotide in a host cell or by in vitro translation. In some embodiments, the polypeptides can be synthetically produced. As described, polypeptides containing multiple epitopes can further include one or more scaffold proteins. In some embodiments, provided herein are synthetic polybodies containing multiple epitopes, scaffold proteins, and linkers for use as therapeutic vaccines.

[0322] Yet another example of converting peptide sequencing information into a therapeutic vaccine may involve combining it with a potent vaccine adjuvant. An effective vaccine may require a potent adjuvant to initiate an immune response. For example, poly-ICLC, an agonist of TLR3 and the RNA helicase domains of MDA5 and RIG3, has demonstrated several desirable properties for a vaccine adjuvant. These properties include inducing local and systemic activation of immune cells in vivo, producing stimulatory chemokines and cytokines, and stimulating antigen presentation by DCs. Furthermore, poly-ICLC induces durable CD4 activation in humans. + and CD8 +Importantly, striking similarities in the upregulation of transcriptional and signaling pathways were observed in subjects vaccinated with poly-ICLC and in volunteers who received a highly effective, replication-competent yellow fever vaccine. Furthermore, over 90% of ovarian cancer patients immunized with poly-ICLC in combination with the NYESO-1 peptide vaccine (in addition to Montanide) demonstrated not only antibody responses to the peptides but also CD4 + and CD8 + Poly-ICLC has been extensively tested in over 25 clinical trials to date and has demonstrated a relatively benign toxicity profile.

[0323] In some aspects, provided herein are compositions comprising a first peptide comprising a first neo-epitope of a protein and a second peptide comprising a second neo-epitope of the same protein, a polynucleotide encoding the first peptide and the second peptide, one or more APCs comprising the first peptide and the second peptide, or a first T cell receptor (TCR) specific for the first neo-epitope in a complex with an HLA protein, and a second TCR specific for the second neo-epitope in a complex with an HLA protein, wherein the first peptide is different from the second peptide, the first neo-epitope comprises a mutation, and the second neo-epitope comprises the same mutation.

[0324] In some aspects, provided herein are compositions comprising a first peptide comprising a first neo-epitope of a region of a protein and a second peptide comprising a second neo-epitope of the same region of the protein, wherein the first neo-epitope and the second neo-epitope comprise at least one amino acid of the region that is the same, a polynucleotide encoding the first peptide and the second peptide, one or more APCs comprising the first peptide and the second peptide, or a first T cell receptor (TCR) specific for the first neo-epitope in a complex with an HLA protein, and a second TCR specific for the second neo-epitope in a complex with an HLA protein, wherein the first peptide is different from the second peptide, the first neo-epitope comprises a mutation, and the second neo-epitope comprises the same mutation.

[0325] In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the first peptide and the second peptide are different molecules. In some embodiments, the first neo-epitope comprises a first neo-epitope of a region of the same protein, and the second neo-epitope comprises a second neo-epitope of a region of the same protein. In some embodiments, the first neo-epitope and the second neo-epitope comprise at least one amino acid of the region that is the same. In some embodiments, a region of a protein comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 consecutive amino acids of the protein. In some embodiments, the region of the protein comprises at most 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 consecutive amino acids of the protein. In some embodiments, the first neo-epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second neo-epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neo-epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neo-epitope binds to a class II HLA protein to form a class II HLA-peptide complex.In some embodiments, the first neo-epitope is a first neo-epitope peptide processed from the first peptide, and / or the second neo-epitope is a second neo-epitope peptide processed from the second peptide. In some embodiments, the first neo-epitope is shorter in length than the first peptide, and / or the second neo-epitope is shorter in length than the second peptide. In some embodiments, the first neo-epitope peptide is processed by an antigen-presenting cell (APC) containing the first peptide, and / or the second neo-epitope peptide is processed by an APC containing the second peptide. In some embodiments, the first neo-epitope is CD8. + In some embodiments, the second neo-epitope activates CD4 T cells. + In some embodiments, the second neo-epitope activates CD8 T cells. + In some embodiments, the first neo-epitope activates CD4 T cells. + In some embodiments, CD4 + The TCR of the T cell binds to a class II HLA-peptide complex containing the first or second peptide. In some embodiments, the CD8 + The TCR of the T cell binds to a class I HLA-peptide complex containing the first or second peptide. In some embodiments, CD4 + The TCR of the T cell binds to a class I HLA-peptide complex containing the first or second peptide. In some embodiments, the CD8 + The TCR of the T cell binds to a class II HLA-peptide complex containing the first or second peptide. In some embodiments, the one or more APCs include a first APC containing the first peptide and a second APC containing the second peptide. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a read-through mutation, a gene fusion mutation, and any combination thereof.

[0326] In some embodiments, a single polypeptide comprises a first peptide and a second peptide, or a single polynucleotide encodes a first peptide and a second peptide. In some embodiments, the first peptide and the second peptide are encoded by sequences transcribed from the same transcription start site. In some embodiments, the first peptide is encoded by a sequence transcribed from a first transcription start site, and the second peptide is encoded by a sequence transcribed from a second transcription start site. In some embodiments, a single polypeptide has a length of at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the polypeptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the first corresponding wild-type sequence. and a second sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding second wild-type sequence.In some embodiments, the polypeptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of at least 8 or 9 contiguous amino acids to the corresponding first wild-type sequence. The present invention relates to a method for preparing a peptide comprising: a first sequence; and a second sequence of at least 16 or 17 consecutive amino acids having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding second wild-type sequence. In some embodiments, the second peptide is longer than the first peptide. In some embodiments, the first peptide is longer than the second peptide. In some embodiments, the first peptide is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 25 , 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids in length.In some embodiments, the second peptide has a length of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the first peptide comprises a sequence of at least 9 contiguous amino acids that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding wild-type sequence. In some embodiments, the second peptide comprises a sequence of at least 17 contiguous amino acids that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding wild-type sequence. In some embodiments, the second neo-epitope is longer than the first neo-epitope. In some embodiments, the first neo-epitope is at least 8 amino acids in length. In some embodiments, the first neo-epitope is 8-12 amino acids in length. In some embodiments, the first neo-epitope comprises a sequence of at least 8 consecutive amino acids, wherein at least two of the 8 consecutive amino acids differ from the corresponding positions in the wild-type sequence. In some embodiments, the second neo-epitope is at least 16 amino acids in length.In some embodiments, the second neo-epitope has a length of 16 to 25 amino acids, hi some embodiments, the second neo-epitope comprises a sequence of at least 16 contiguous amino acids, wherein at least two of the 16 contiguous amino acids differ from the corresponding positions in the wild-type sequence.

[0327] In some embodiments, the first peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the first neo-epitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the first neo-epitope. In some embodiments, the second peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the second neo-epitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the second neo-epitope. In some embodiments, the first peptide, the second peptide, or both, comprise at least one flanking sequence, wherein the at least one flanking sequence is upstream or downstream of the neo-epitope. In some embodiments, at least one flanking sequence has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, at least one flanking sequence comprises a non-wild-type sequence. In some embodiments, at least one flanking sequence is an N-terminal flanking sequence. In some embodiments, at least one flanking sequence is a C-terminal flanking sequence. In some embodiments, at least one flanking sequence of the first peptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one flanking sequence of the second peptide.In some embodiments, at least one flanking region of the first peptide differs from at least one flanking region of the second peptide, hi some embodiments, at least one flanking residue comprises a mutation.

[0328] In some embodiments, the composition comprises one or more additional peptides, wherein the one or more additional peptides comprise a third neo-epitope. In some embodiments, the first and / or second neo-epitope binds to an HLA protein with higher affinity than the corresponding wild-type sequence. In some embodiments, the first and / or second neo-epitope has a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second neo-epitope binds to an HLA protein with a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second neo-epitope binds to an HLA class I protein with a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second neo-epitopes bind to proteins encoded by HLA alleles expressed by the subject. In some embodiments, the mutations are not present in non-cancer cells of the subject. In some embodiments, the first and / or second neo-epitopes are encoded by genes or expressed genes in cancer cells of the subject.

[0329] In some embodiments, a composition comprising a polybody having multiple epitopes can activate a first T cell comprising a first TCR. In some embodiments, a composition having multiple epitopes can activate a second T cell comprising a second TCR. In some embodiments, the first and / or second T cell is a cytotoxic T cell. In some embodiments, the first and / or second T cell is a gamma delta T cell. In some embodiments, the first and / or second T cell is a helper T cell. In some embodiments, the first T cell is a T cell stimulated, expanded, or induced with a first neoepitope, and / or the second T cell is a T cell stimulated, expanded, or induced with a second neoepitope.

[0330] In some embodiments, the first and / or second TCR has a K of less than 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. D or IC 50 In some embodiments, the vector binds to an HLA-peptide complex at a specific site. In some aspects, provided herein is a vector comprising a polynucleotide encoding the first and second peptides described herein. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the vector is a self-amplifying RNA replicon, a plasmid, a phage, a transposon, a cosmid, a virus, or a virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpesvirus, a poxvirus, an alphavirus, a vaccinia virus, a hepatitis B virus, a human papillomavirus, or a pseudotype thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoparticle, a cationic lipid, a cationic polymer, a metal nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere.

[0331] In some aspects, provided herein is a pharmaceutical composition comprising a composition described herein, or a vector described herein, and a pharmaceutically acceptable excipient.

[0332] In some embodiments, the plurality of cells are autologous cells. In some embodiments, the plurality of APC cells are autologous cells. In some embodiments, the plurality of T cells are autologous cells. In some embodiments, the pharmaceutical composition further comprises an immunomodulatory agent or adjuvant. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the adjuvant is Hiltonol.

[0333] In some aspects, provided herein are methods of treating cancer, the method comprising administering to a subject in need of cancer treatment a pharmaceutical composition described herein.

[0334] In some aspects, provided herein are methods of preventing resistance to cancer therapy, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0335] In some aspects, provided herein are methods of inducing an immune response, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0336] In some embodiments, the immune response is a humoral response.

[0337] In some embodiments, provided herein are polypeptides comprising one or more epitope peptides and a scaffold protein that together form a polybody. In some embodiments, the polybody is a homopolymer. In some embodiments, the polybody comprises a plurality of first peptides. In some embodiments, the polybody comprises a plurality of second peptides. In some embodiments, the polybody comprises a plurality of third peptides. In some embodiments, the first peptide and the second peptide are administered simultaneously, separately, or sequentially. In some embodiments, the first peptide is administered sequentially after the second peptide. In some embodiments, the second peptide is administered sequentially after the first peptide. In some embodiments, the first peptide is administered sequentially after a period sufficient for the second peptide to activate T cells. In some embodiments, the second peptide is administered sequentially after a period sufficient for the first peptide to activate T cells. In some embodiments, the first peptide is administered sequentially after the second peptide has restimulated T cells. In some embodiments, the second peptide is administered sequentially after the first peptide has restimulated T cells. In some embodiments, a first peptide is administered to stimulate T cells and a second peptide is administered after the first peptide has restimulated the T cells. In some embodiments, a second peptide is administered to stimulate T cells and a first peptide is administered after the second peptide has restimulated the T cells.

[0338] In some embodiments, provided herein are polypeptides comprising one or more epitope peptides and a scaffold protein that together form a polybody. In some embodiments, the polybody is a heteropolymer. In some embodiments, the polybody comprises a plurality of first peptides and a plurality of second peptides, and / or third peptides, and / or fourth peptides, and / or fifth peptides, and / or sixth peptides, and / or more than six peptides. In some embodiments, the polybody comprises a plurality of third peptides. In some embodiments, the subject has cancer, and the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, and chronic lymphocytic leukemia (CLL). In some embodiments, the subject has breast cancer that is resistant to anti-estrogen therapy. In some embodiments, the breast cancer expresses a mutated estrogen receptor. In some embodiments, the subject has CLL that is resistant to ibrutinib therapy. In some embodiments, the CLL expresses a Bruton's tyrosine kinase with a mutation, such as a C481S mutation. In some embodiments, the subject has lung cancer that is resistant to a tyrosine kinase inhibitor. In some embodiments, the lung cancer expresses an epidermal growth factor receptor (EGFR) with a mutation, such as a T790M, L792F, or C797S mutation. In some embodiments, the APC cells containing the first peptide and the APC cells containing the second peptide are administered simultaneously, separately, or sequentially. In some embodiments, the method further comprises administering at least one additional therapeutic agent or modality. In some embodiments, the at least one additional therapeutic agent or modality is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and an APC, a polynucleotide, an oncolytic virus, or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and an anti-PD-L1 agent, an anti-CTLA-4 agent, or an anti-CD40 agent.In some embodiments, the additional therapeutic agent is administered before, simultaneously with, or after administering the pharmaceutical composition described herein. In some embodiments, the additional treatment is a chemokine or cytokine. Exemplary cytokines are interleukins such as IL-1, IL-12, or combinations of more than two cytokines, more than three cytokines, or more than four cytokines, etc.

[0339] peptide Polypeptides or peptides, in either their neutral (uncharged) or salt form, can be of various lengths, free of modifications such as glycosylation, side chain oxidation, or phosphorylation, or containing these modifications, provided that the modifications do not destroy the biological activity of the polypeptides described herein.

[0340] In some embodiments, sequencing methods are used to identify tumor-specific mutations. Any suitable sequencing method, such as next-generation sequencing (NGS) technology, can be used in accordance with the present disclosure. Third-generation sequencing methods may replace NGS technology in the future to speed up the sequencing step of the method. For clarity, the term "next-generation sequencing" or "NGS" in the context of this disclosure refers to any novel high-throughput sequencing technology that randomly reads nucleic acid templates in parallel across the entire genome by breaking the entire genome into small fragments, in contrast to the "traditional" sequencing methodology known as Sanger chemistry. Such NGS technologies (also known as massively parallel sequencing technologies) can deliver nucleic acid sequence information for the whole genome, exome, transcriptome (all transcribed sequences in the genome), or methylome (all methylated sequences in the genome) in a very short period of time, e.g., within 1-2 weeks, e.g., within 1-7 days, or even within 24 hours, enabling a single-cell sequencing approach. Several NGS platforms that are commercially available or mentioned in the literature, such as those described in detail in WO 2012 / 159643, can be used in the context of the present disclosure.

[0341] In certain embodiments, the peptides described herein are selected from the group consisting of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, In certain embodiments, the neo-antigenic peptide molecule is 100 or less amino acids in length, including, but not limited to, about 49, 50, 60, 70, 80, 90, 100, 110, 120, 150, 200, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, 10,000 or more amino acids, and any range derivable therein.

[0342] In some embodiments, the peptides can be about 8 to about 50 amino acid residues in length, or about 8 to about 30, about 8 to about 20, about 8 to about 18, about 8 to about 15, or about 8 to about 12 amino acid residues in length. In some embodiments, the peptides can be about 8 to about 500 amino acid residues in length, or about 8 to about 450, about 8 to about 400, about 8 to about 350, about 8 to about 300, about 8 to about 250, about 8 to about 200, about 8 to about 150, about 8 to about 100, about 8 to about 50, or about 8 to about 30 amino acid residues in length.

[0343] In some embodiments, peptides can be at least 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 acid residues in length. In some embodiments, the peptides are at least 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, It can be 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues. In some embodiments, peptides can be at most 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 fewer amino acid residues in length. In some embodiments, the peptides are at most 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, It can be 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or fewer amino acid residues.

[0344] In some embodiments, the peptides have an overall length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0345] In some embodiments, the peptides have an overall length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.

[0346] Longer peptides can be designed in several ways. In some embodiments, if HLA-binding peptides are predicted or known, the longer peptide comprises (1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-termini of each corresponding gene product, or (2) a concatenation of some or all of the binding peptides with their respective extension sequences. In other embodiments, if sequencing reveals long (more than 10 residues) neo-epitope sequences present in the tumor (e.g., due to frameshifts, readthrough, or intron inclusion resulting in novel peptide sequences), the longer peptide can consist of the entire stretch of novel tumor-specific amino acids, either as a single longer peptide or several overlapping longer peptides. In some embodiments, the use of longer peptides is presumed to allow endogenous processing by patient cells, resulting in more effective antigen presentation and induction of T cell responses. In some embodiments, two or more peptides can be used, overlapping and displayed side-by-side on a long neo-antigenic peptide.

[0347] In some embodiments, the peptides can have a pI value of about 0.5 to about 12, about 2 to about 10, or about 4 to about 8. In some embodiments, the peptides can have a pI value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the peptides can have a pI value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.

[0348] In some embodiments, the peptides described herein may be in solution, lyophilized, or in crystalline form. In some embodiments, the peptides described herein may be synthetically prepared by recombinant DNA technology or chemical synthesis, or isolated from natural sources such as native tumors or pathogenic organisms. Neoepitopes may be synthesized individually or directly or indirectly conjugated to the peptide. The peptides described herein may be substantially free of other naturally occurring host cell proteins and fragments thereof, although in some embodiments, the peptides may be synthetically conjugated to natural fragments or particles.

[0349] In some embodiments, the peptides described herein can be prepared in a variety of ways. In some embodiments, the peptides can be expressed in host cells, such as bacteria. In some embodiments, the peptides can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols. See, for example, Stewart & Young, Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co., 1984. Furthermore, individual peptides can be joined using chemical ligation to generate larger peptides that are still within the scope of the present disclosure.

[0350] Alternatively, recombinant DNA technology can be utilized, involving a nucleotide sequence encoding the peptide inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression. These procedures are generally known in the art, as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). Thus, recombinant peptides, including one or more neoantigenic peptides described herein, can be used to present appropriate T cell epitopes.

[0351] In some embodiments, the peptide is encoded by a gene with a point mutation that results in an amino acid substitution of the native peptide. In some embodiments, the peptide is encoded by a gene with a point mutation that results in a frameshift mutation. A frameshift occurs when a mutation disrupts the normal phase of a gene's codon periodicity (also known as the "reading frame"), resulting in the translation of a non-native protein sequence. Different mutations in a gene can achieve the same altered reading frame. In some embodiments, the peptide is encoded by a gene with mutations that result in fusion polypeptides, in-frame deletions, insertions, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of the polypeptide. In some embodiments, the peptide is encoded by a fusion of a first gene with a second gene. In some embodiments, the peptide is encoded by an in-frame fusion of a first gene with a second gene. In some embodiments, the peptide is encoded by a fusion of a first gene with an exon of a splice variant of the first gene. In some embodiments, the peptide is encoded by a fusion of a first gene with a cryptic exon of the first gene. In some embodiments, the peptide is encoded by a fusion of a first gene and a second gene, and the peptide comprises an amino acid sequence encoded by an out-of-frame sequence resulting from the fusion.

[0352] In some aspects, the present disclosure provides compositions comprising at least two or more peptides. In some embodiments, the compositions described herein contain at least two different peptides. In some embodiments, the compositions described herein contain a first peptide comprising a first neoepitope and a second peptide comprising a second neoepitope. In some embodiments, the first and second peptides are derived from the same protein. The at least two different peptides can vary in length, amino acid sequence, or both. The peptides can be derived from any protein known or found to contain tumor-specific mutations. In some embodiments, the compositions described herein include a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein, where the first peptide is different from the second peptide, the first neoepitope comprises a mutation, and the second neoepitope comprises the same mutation. In some embodiments, the compositions described herein comprise a first peptide comprising a first neoepitope of a first region of a protein and a second peptide comprising a second neoepitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide differs from the second peptide, the first neoepitope comprises a first mutation, and the second neoepitope comprises a second mutation. In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a readthrough mutation, a gene fusion mutation, and any combination thereof.

[0353] In some embodiments, the peptides can be derived from proteins with substitution mutations, such as KRAS G12C, G12D, G12V, Q61H, or Q61L mutations, or NRAS Q61K or Q61R mutations. The substitution can be located anywhere along the length of the peptide. For example, it can be located in the N-terminal third of the peptide, the central third of the peptide, or the C-terminal third of the peptide. In another embodiment, the substituted residue is located 2-5 residues away from the N-terminus or 2-5 residues away from the C-terminus. Peptides can also be derived from tumor-specific insertion mutations, where the peptide includes one or more or all of the inserted residues. In some embodiments, the first neo-epitope and / or the second neo-epitope bind to an HLA protein with higher affinity than the corresponding neo-epitope without the substitution. In some embodiments, the first neo-epitope and / or the second neo-epitope bind to an HLA protein with higher affinity than the corresponding wild-type sequence without the substitution.

[0354] In some embodiments, the first peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the first neo-epitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the first neo-epitope. In some embodiments, the second peptide comprises at least one additional mutation. In some embodiments, one or more of the at least one additional mutation is not a mutation in the second neo-epitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the second neo-epitope.

[0355] In some aspects, the present disclosure provides compositions comprising a single polypeptide comprising a first peptide and a second peptide, or a single polynucleotide encoding the first peptide and the second peptide. In some embodiments, the compositions provided herein comprise one or more additional peptides, wherein the one or more additional peptides comprise a third neoepitope. In some embodiments, the first peptide and the second peptide are encoded by sequences transcribed from the same transcription start site. In some embodiments, the first peptide is encoded by a sequence transcribed from a first transcription start site, and the second peptide is encoded by a sequence transcribed from a second transcription start site. In some embodiments, the polypeptide has a length of at least 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the polypeptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding wild-type sequence. and a second sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding wild-type sequence.In some embodiments, the polypeptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of at least 8 or 9 contiguous amino acids to the corresponding wild-type sequence. a first sequence and a second sequence of at least 16 or 17 contiguous amino acids having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding wild-type sequence.

[0356] In some embodiments, the second peptide is longer than the first peptide. In some embodiments, the first peptide is longer than the second peptide. In some embodiments, the first peptide is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000, 1200, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 240 , 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids in length. In some embodiments, the second peptide has a length of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, or 10,000 amino acids. In some embodiments, the first peptide comprises a sequence of at least 9 contiguous amino acids that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding wild-type sequence.In some embodiments, the second peptide comprises a sequence of at least 17 contiguous amino acids having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the corresponding wild-type sequence.

[0357] In some embodiments, the first peptide, the second peptide, or both, comprise at least one flanking sequence, and the at least one flanking sequence is upstream or downstream of the neo-epitope. In some embodiments, the at least one flanking sequence has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, at least one flanking sequence comprises a non-wild-type sequence. In some embodiments, at least one flanking sequence is an N-terminal flanking sequence. In some embodiments, at least one flanking sequence is a C-terminal flanking sequence. In some embodiments, at least one flanking sequence of a first peptide has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one flanking sequence of a second peptide. In some embodiments, at least one flanking region of the first peptide differs from at least one flanking region of the second peptide, hi some embodiments, at least one flanking residue comprises a mutation.

[0358] In some embodiments, the peptide comprises a neo-epitope sequence comprising at least one mutated amino acid, hi some embodiments, the peptide comprises a neo-epitope sequence comprising at least 1, 2, 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, or more mutated amino acids. In some embodiments, the peptide comprises a neo-epitope sequence derived from a protein that includes at least one mutated amino acid and at least 1, 2, 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, or more non-mutated amino acids. In some embodiments, the peptide comprises a neo-epitope sequence derived from a protein that includes at least one mutated amino acid and at least 1, 2, 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, or more non-mutated amino acids upstream of the at least one mutated amino acid. In some embodiments, the peptide comprises a neo-epitope sequence derived from a protein that includes at least one mutated amino acid and at least 1, 2, 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, or more non-mutated amino acids downstream of the at least one mutated amino acid.In some embodiments, the peptide comprises at least one mutated amino acid and at least 1, 2, 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, or more non-mutated amino acids upstream of the at least one mutated amino acid. and at least 1, 2, 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, or more non-mutated amino acids downstream of the at least one mutated amino acid.

[0359] In some embodiments, the peptide comprises a neo-epitope sequence derived from a protein comprising at least one mutant amino acid and a sequence upstream of the at least one mutant amino acid that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. In some embodiments, the peptide comprises a neo-epitope sequence derived from a protein comprising at least one mutant amino acid and a sequence downstream of the at least one mutant amino acid that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence.In some embodiments, the peptide comprises at least one mutant amino acid and at least one mutant amino acid that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence. and a sequence downstream of at least one mutant amino acid that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding wild-type sequence.

[0360] In some embodiments, the peptide comprises at least one mutant amino acid and a sequence similar to or different from the corresponding wild-type sequence by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. and a sequence upstream of the at least one variant amino acid comprising at least 1, 2, 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 or more consecutive amino acids that have 99%, 99%, or 100% sequence identity to the protein. In some embodiments, the peptide comprises at least one mutant amino acid and a sequence similar to or different from the corresponding wild-type sequence by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. and a sequence downstream of the at least one mutant amino acid comprising at least 1, 2, 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 or more consecutive amino acids with 99%, 99%, or 100% sequence identity to the protein.In some embodiments, the peptide comprises at least one mutant amino acid and a sequence similar to or different from the corresponding wild-type sequence by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100% sequence identity upstream of at least one variant amino acid, including at least 1, 2, 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, or more consecutive amino acids. sequences and at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity relative to the corresponding wild-type sequence. and a sequence downstream of the at least one mutant amino acid comprising at least 1, 2, 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, or more consecutive amino acids having sequence identity with the protein.

[0361] Peptide Modification In some embodiments, the present disclosure includes modified peptides. Modifications can include covalent chemical modifications that do not alter the primary amino acid sequence of the antigen peptide itself. Modifications can produce peptides with desirable properties, such as increased in vivo half-life, increased stability, reduced clearance, altered immunogenicity or allergenicity, increased specific antibody production, cellular targeting, antigen uptake, antigen processing, HLA affinity, HLA stability, or antigen presentation. In some embodiments, the peptides can include one or more sequences that enhance epitope processing and presentation by APCs, for example, for generating an immune response.

[0362] In some embodiments, peptides can be modified to provide desired attributes. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a T helper cell response. In some embodiments, the immunogenic peptide / T helper conjugate is linked by a spacer molecule. In some embodiments, the spacer comprises a relatively small, neutral molecule, such as an amino acid or amino acid mimetic, that is substantially uncharged under physiological conditions. The spacer can be selected, for example, from Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that, optionally, any spacers present need not be composed of the same residues and can therefore be hetero- or homo-oligomers. The neo-antigenic peptide can be linked to the T helper peptide at either the amino or carboxy terminus of the peptide, either directly or via a spacer. The amino terminus of either the neo-antigenic peptide or the T helper peptide can be acylated. Examples of T helper peptides include tetanus toxoid residues 830-843, influenza residues 307-319, and malaria sporozoite circumferential residues 382-398 and residues 378-389.

[0363] The peptide sequences of the present disclosure may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the peptide at preselected bases to generate codons that translate into desired amino acids.

[0364] Peptides may also be modified by extending or reducing the amino acid sequence of the compound, for example, by adding or deleting amino acids. Peptides or analogs may also be modified by changing the order or composition of specific residues. Those skilled in the art will understand that certain amino acid residues that are essential for biological activity, such as amino acid residues at critical contact sites or conserved residues, generally cannot be altered without adversely affecting biological activity.

[0365] In some embodiments, peptides may be modified using a series of peptides with single amino acid substitutions to determine the effect of electrostatic charge, hydrophobicity, and the like on HLA binding. For example, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions may be made along the length of the peptide, revealing distinct patterns of sensitivity to various HLA molecules and T cell receptors. Additionally, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be utilized. Substitutions may be homo- or hetero-oligomeric. The number and type of residues substituted or added depend on the required spacing between essential contact points and the particular functional attributes sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for HLA molecules or T cell receptors may also be achieved by such substitutions compared to the affinity of the parent peptide. In any case, such substitutions should utilize amino acid residues or other molecular fragments selected to avoid, for example, steric and charge interferences that may interfere with binding. Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at the final peptide.

[0366] In some embodiments, the peptides described herein are terminally acylated, e.g., alkanoyl (C1-C 20 ) or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for linking to supports or other molecules. In some embodiments, the peptides described herein may contain modifications such as, but not limited to, glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of surfactants, e.g., lipids, or may be chemically modified, e.g., by acetylation. Furthermore, bonds in peptides may be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0367] In some embodiments, the peptides described herein can include a carrier, such as those known in the art, e.g., thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acid residues such as poly-L-lysine and poly-L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.

[0368] Peptides may be further modified to contain additional chemical moieties not normally part of proteins. These derivatized moieties can improve solubility, biological half-life, protein absorption, or binding affinity. The moieties can also reduce or eliminate any desired side effects of the peptide. A summary of these moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000). For example, neoantigenic peptides with desired activity can be modified as needed to provide certain desired attributes, e.g., improved pharmacological properties, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind to the desired HLA molecule and activate appropriate T cells. For example, peptides can be subjected to various changes, such as conservative or non-conservative substitutions, which may confer certain advantages in their use, such as improved HLA binding. Such conservative substitutions can involve replacing an amino acid residue with another that is biologically and / or chemically similar, e.g., one hydrophobic residue for another amino acid, or one polar residue for another amino acid. The effect of single amino acid substitution can also be examined using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, for example, as described in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (NY, Academic Press), pp.1-284 (1979), and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2nd Ed. (1984).

[0369] In some embodiments, the peptides described herein may be conjugated to large, slowly metabolized macromolecules such as proteins, polysaccharides such as sepharose, agarose, cellulose, cellulose beads, polymeric amino acids such as polyglutamic acid, polylysine, amino acid copolymers, inactivated virus particles, inactivated bacterial toxins such as toxoids derived from diphtheria, tetanus, cholera, and leukotoxin molecules, inactivated bacteria, and dendritic cells.

[0370] Alterations to the peptide can include, but are not limited to, conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylated HESylation, recombinant PEG mimics, Fc fusions, albumin fusions, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusions, iron fusions, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of surfactants, addition of amino acid mimetics, or addition of unnatural amino acids.

[0371] Additional suitable moieties and molecules for conjugation include, for example, molecules for targeting the lymphatic system, thyroglobulin, albumins such as human serum albumin (HAS), tetanus toxoid, diphtheria toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid), VP6 polypeptide of rotavirus, influenza virus hemagglutinin, influenza virus nucleoprotein, keyhole limpet hemocyanin (KLH), and hepatitis B virus core protein and surface antigen, or any combination of the foregoing.

[0372] Another type of modification is the conjugation (e.g., linking) of one or more additional components or molecules to the N-terminus and / or C-terminus of the polypeptide sequence, such as another protein (e.g., a protein having an amino acid sequence heterologous to the protein of interest) or a carrier molecule. Thus, exemplary polypeptide sequences can be provided as conjugates with other components or molecules. In some embodiments, fusion of albumin to a peptide or protein of the present disclosure can be achieved by genetic engineering, for example, by joining DNA encoding HSA, or a fragment thereof, to DNA encoding one or more polypeptide sequences. A suitable host can then be transformed or transfected with the fusion nucleotide sequence, for example, in the form of a suitable plasmid, to express the fusion polypeptide. Expression can be carried out in vitro, for example, from prokaryotic or eukaryotic cells, or in vivo, for example, from transgenic organisms. In some embodiments of the present disclosure, expression of the fusion protein is carried out in mammalian cell lines, for example, CHO cell lines. Furthermore, albumin itself can be modified to extend its circulating half-life. The fusion of modified albumin to one or more polypeptides can be achieved by the above-mentioned genetic engineering techniques or by chemical conjugation, and the resulting fusion molecule has a half-life that exceeds that of fusion with unmodified albumin (see, for example, WO 2011 / 051489).As an alternative to direct fusion, several albumin binding strategies have been developed, including albumin binding via conjugated fatty acid chains (acylation).Since serum albumin is a transport protein for fatty acids, these natural ligands with albumin binding activity have been used to extend the half-life of small protein therapeutics.

[0373] Further candidate components and molecules for conjugation include those suitable for isolation or purification.Non-limiting examples include binding molecules such as biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, lectins, or molecules containing solid supports, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.Purification methods such as cation exchange chromatography can be used to separate conjugates based on charge differences, thereby effectively separating conjugates into their various molecular weights.The contents of the fractions obtained by cation exchange chromatography can be identified by molecular weight using conventional methods, such as mass spectrometry, SDS-PAGE, or other known methods for separating molecular entities based on molecular weight.

[0374] In some embodiments, the amino or carboxyl terminus of a peptide or protein sequence of the present disclosure can be fused to an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule). Fc fusion conjugates have been shown to increase the systemic half-life of biopharmaceuticals, thus allowing them to be administered less frequently. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels, protecting the Fc fusion molecule from degradation and allowing it to be re-released into the circulation, prolonging its life in the circulation. This Fc binding is thought to be the mechanism by which endogenous IgG maintains its long plasma half-life. More recent Fc fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of biopharmaceuticals compared to traditional Fc fusion conjugates.

[0375] The present disclosure contemplates the use of other modifications of peptides, now known or developed in the future, to improve one or more properties, such as extending the circulating half-life of the peptide, increasing stability, reducing clearance, or altering immunogenicity or allergenicity.

[0376] Peptide stability can be assayed in several ways. For example, stability is tested using peptidases and various biological media, such as human plasma and serum. See, for example, Verhoef, et al., Eur. J. Drug Metab. Pharmacokinetics 11:291 (1986). The half-life of the peptides described herein is conveniently determined using a 25% human serum (v / v) assay. The protocol is as follows: pooled human serum (type AB, non-heat-inactivated) is disrupted by centrifugation before use. The serum is then diluted to 25% with RPMI-1640 or another suitable tissue culture medium. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid (TCA) or ethanol. The cloudy reaction sample is cooled for 15 minutes (4°C) and then spun to pellet precipitated serum proteins. The presence of the peptide is then determined by reverse-phase HPLC using stability-specific chromatography conditions.

[0377] Problems associated with short plasma half-lives or susceptibility to protease degradation can be overcome by various modifications, including conjugating or linking the peptide or protein sequence to any of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes (see, e.g., typically via a linking moiety, e.g., PEG, covalently attached to both the protein and the nonproteinaceous polymer). Such PEG-conjugated biomolecules have been shown to have clinically useful properties, including good physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulatory half-lives and reduced clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

[0378] PEG suitable for conjugation to polypeptide or protein sequences is generally soluble in water at room temperature and has the general formula R—(O—CH—CH) nThe formula has the formula -OR, where R is hydrogen or a protecting group, e.g., an alkyl or alkanol group, and n is an integer between 1 and 1000. When R is a protecting group, it generally has 1 to 8 carbon atoms. PEG conjugated to a polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-arm PEGs are contemplated by the present disclosure. The present disclosure also contemplates compositions of conjugates in which PEGs have different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain mixtures of conjugates in which n=1, 2, 3, and 4. In some compositions, the percentage of conjugates in which n=1 is 18-25%, the percentage of conjugates in which n=2 is 50-66%, the percentage of conjugates in which n=3 is 12-16%, and the percentage of conjugates in which n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. For example, cation exchange chromatography may be used to separate the conjugates, and then purified fractions are identified that contain, for example, conjugates with a desired number of PEGs attached, and are free of unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0379] PEG can be attached to the peptides or proteins of the present disclosure via a terminal reactive group ("spacer"). The spacer is, for example, a terminal reactive group that mediates the bond between the polypeptide sequence and one or more free amino or carboxyl groups of PEG. PEGs having a spacer that can be attached to a free amino group include N-hydroxysuccinimide PEG, which can be prepared by activating the succinic acid ester of PEG with N-hydroxysuccinimide. Another activated PEG that can be attached to a free amino group is 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, which can be prepared by reacting PEG monomethyl ether with cyanyl chloride. Activated PEGs that are attached to free carboxyl groups include polyoxyethylenediamine.

[0380] Conjugation of one or more of the peptide or protein sequences of the present disclosure to PEG having a spacer can be carried out by a variety of conventional methods. For example, the conjugation reaction can be carried out in solution using a molar ratio of reagent to peptide / protein of 4:1 to 30:1, for 30 minutes to 20 hours, at temperatures between 4°C and room temperature, and at a pH between 5 and 10. Reaction conditions can be selected to drive the reaction to produce primarily the desired degree of substitution. Generally, low temperatures, low pH (e.g., pH = 5), and short reaction times tend to decrease the number of attached PEGs, while high temperatures, neutral to high pH (e.g., pH > 7), and long reaction times tend to increase the number of attached PEGs. Various means known in the art can be used to terminate the reaction. In some embodiments, the reaction is terminated by acidifying the reaction mixture and freezing, for example, at -20°C.

[0381] The present disclosure also contemplates the use of PEG mimetics. Recombinant PEG mimetics have been developed to retain the attributes of PEG (e.g., enhanced serum half-life) while imparting several additional advantageous properties. For example, simple polypeptide chains (e.g., containing Ala, Glu, Gly, Pro, Ser, and Thr) capable of forming an extended conformation similar to PEG can be produced already recombinantly fused to a peptide or protein drug of interest (e.g., Amunix XTEN technology, Mountain View, CA). This eliminates the need for an additional conjugation step during the manufacturing process. Furthermore, established molecular biology techniques allow for control of the side chain composition of the polypeptide chain, enabling optimization of immunogenicity and manufacturing properties.

[0382] New epitopes Neoepitopes include neoantigenic peptides or neoantigenic determinant portions of neoantigenic polypeptides that are recognized by the immune system. Neoepitopes refer to epitopes that are not present in reference non-diseased cells, e.g., non-cancer cells or germline cells, but are found in diseased cells, e.g., cancer cells. This includes situations where the corresponding epitope is found in normal non-diseased cells or germline cells, but one or more mutations in the diseased cells, e.g., cancer cells, change the sequence of the epitope to create a neoepitope. The term "neoepitope" is used interchangeably with "tumor-specific neoepitope" herein and typically designates a series of residues, typically L-amino acids, connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Neoepitopes can be of various lengths, in either their neutral (uncharged) or salt form, and free of or containing modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptides described herein.

[0383] In some embodiments, the neo-epitopes described herein for HLA class I are 13 residues or less in length, typically consisting of about 8 to about 12 residues, particularly 9 or 10 residues. In some embodiments, the neo-epitopes described herein for HLA class II are 25 residues or less in length, typically consisting of about 16 to about 25 residues.

[0384] In some embodiments, the compositions described herein comprise a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein, wherein the first peptide is different from the second peptide, the first neoepitope comprises a mutation, and the second neoepitope comprises the same mutation. In some embodiments, the compositions described herein comprise a first peptide comprising a first neoepitope of a first region of a protein and a second peptide comprising a second neoepitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide is different from the second peptide, the first neoepitope comprises a first mutation, and the second neoepitope comprises a second mutation. In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a readthrough mutation, a gene fusion mutation, and any combination thereof.

[0385] In some embodiments, the first neo-epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second neo-epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neo-epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neo-epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the first neo-epitope binds to a CD8 + In some embodiments, the first neo-epitope activates CD4 T cells. + In some embodiments, the second neo-epitope activates CD4 T cells. + In some embodiments, the second neo-epitope activates CD8 T cells. + In some embodiments, CD4 +The TCR of the T cell binds to a class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to a class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to a class I HLA-peptide complex. In some embodiments, CD4 + The TCR of the T cell binds to the class I HLA-peptide complex.

[0386] In some embodiments, the second neo-epitope is longer than the first neo-epitope. In some embodiments, the first neo-epitope is at least 8 amino acids in length. In some embodiments, the first neo-epitope is 8-12 amino acids in length. In some embodiments, the first neo-epitope comprises a sequence of at least 8 contiguous amino acids, wherein at least one of the 8 contiguous amino acids differs from the corresponding position in the wild-type sequence. In some embodiments, the first neo-epitope comprises a sequence of at least 8 contiguous amino acids, wherein at least two of the 8 contiguous amino acids differ from the corresponding position in the wild-type sequence. In some embodiments, the second neo-epitope is at least 16 amino acids in length. In some embodiments, the second neo-epitope is 16-25 amino acids in length. In some embodiments, the second neo-epitope comprises a sequence of at least 16 contiguous amino acids, wherein at least two of the 16 contiguous amino acids differ from the corresponding position in the wild-type sequence. In some embodiments, the second neo-epitope comprises a sequence of at least 16 contiguous amino acids, wherein at least two of the 16 contiguous amino acids differ from the corresponding positions in the wild-type sequence.

[0387] In some embodiments, the neo-epitope binds to an HLA protein (e.g., HLA class I or HLA class II). In some embodiments, the neo-epitope binds to an HLA protein with higher affinity than the corresponding wild-type peptide. In some embodiments, the neo-epitope has an IC of less than 5,000 nM, less than 1,000 nM, less than 500 nM, less than 100 nM, less than 50 nM, or less than that. 50 It has.

[0388] In some embodiments, the neo-epitopes can have an HLA binding affinity of about 1 pM to about 1 mM, about 100 pM to about 500 μM, about 500 pM to about 10 μM, about 1 nM to about 1 μM, or about 10 nM to about 1 μM. In some embodiments, the neo-epitopes can have an HLA binding affinity of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, or 1,000 nM or more. In some embodiments, the neo-epitope can have an HLA binding affinity of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, or 1,000 nM.

[0389] In some embodiments, the first and / or second neo-epitopes bind to an HLA protein with higher affinity than the corresponding wild-type neo-epitope. In some embodiments, the first and / or second neo-epitopes bind to an HLA protein with a K of less than 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50In some embodiments, the first and / or second neo-epitope binds to an HLA protein with a K of less than 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second neo-epitope binds to an HLA class I protein with a K of less than 2,000 nM, 1,500 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to HLA class II proteins.

[0390] In one embodiment, the first and / or second neo-epitopes bind to proteins encoded by HLA alleles expressed by the subject. In another embodiment, the mutation is not present in non-cancer cells of the subject. In yet another embodiment, the first and / or second neo-epitopes are encoded by genes or expressed genes in cancer cells of the subject.

[0391] Polynucleotides Alternatively, nucleic acids (e.g., polynucleotides) encoding the peptides of the present disclosure can be used to produce polypeptides using host cells or in vitro, e.g., by in vitro translation. In some embodiments, in vitro translation is used to produce the peptides. Polynucleotides encoding polypeptides that can be used as therapeutics, e.g., polybodies comprising neoantigenic peptides, are also contemplated. The polynucleotides may be, for example, DNA, cDNA, or RNA, and may be either single-stranded and / or double-stranded.

[0392] Provided herein are neoantigenic polynucleotides encoding each of the neoantigenic peptides described in this disclosure. The terms "polynucleotide," "nucleotide," or "nucleic acid" are used interchangeably in this disclosure with "mutant polynucleotide," "mutant nucleotide," "mutant nucleic acid," "neoantigenic polynucleotide," "neoantigenic nucleotide," or "neoantigenic mutant nucleic acid." Various nucleic acid sequences can encode the same peptide due to redundancy in the genetic code. Each of these nucleic acids is within the scope of this disclosure. The nucleic acid encoding the peptide can be DNA or RNA, e.g., mRNA, or a combination of DNA and RNA. In some embodiments, the nucleic acid encoding the peptide is a self-amplifying mRNA (Brito et al., Adv. Genet. 2015;89:179-233). Any suitable polynucleotide encoding the peptides described herein is within the scope of this disclosure.

[0393] The term "RNA" includes "mRNA" and, in some embodiments, relates to "mRNA." The term "mRNA" refers to "messenger RNA" and refers to a "transcript" generated using a DNA template and encoding a peptide or polypeptide. Typically, mRNA includes a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In some embodiments, mRNA is self-amplifying. In the context of the present disclosure, mRNA can be generated by in vitro transcription from a DNA template. In vitro transcription methodologies are known to those skilled in the art. For example, various in vitro transcription kits are commercially available.

[0394] The stability and translation efficiency of RNA can be modified as needed. For example, RNA can be stabilized, and its translation can be increased by one or more modifications that have a stabilizing effect and / or increase the translation efficiency of RNA. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of the RNA used in accordance with the present disclosure, it can be modified in the coding region, i.e., the sequence encoding the expressed peptide or protein, without changing the sequence of the expressed peptide or protein, to increase mRNA stability by increasing GC content, and to perform codon optimization, thereby improving translation in cells.

[0395] As used in this disclosure, the term "modified" in the context of RNA includes any modification of RNA that does not naturally occur in the RNA. In some embodiments, the RNA does not have an uncapped 5'-triphosphate. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase. In other embodiments, the RNA may have modified ribonucleotides to increase its stability and / or reduce cytotoxicity. In some embodiments, 5-methylcytidine may be partially or completely substituted within the RNA, for example, for cytidine. Alternatively, pseudouridine may be partially or completely substituted for, for example, uridine.

[0396] In some embodiments, the term "modified" refers to providing RNA with a 5' cap or a 5' cap analog. The term "5' cap" refers to the cap structure found on the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide attached to the mRNA via a rare 5' to 5' triphosphate linkage. In some embodiments, this guanosine is methylated at the 7 position. The term "conventional 5' cap" refers to the naturally occurring RNA 5' cap relative to the 7-methylguanosine cap (m G). In the context of the present disclosure, the term "5' cap" includes 5' cap analogs that resemble RNA cap structures and are modified to have the ability to stabilize RNA and / or enhance translation of RNA when attached thereto in vivo and / or in cells.

[0397] In certain embodiments, mRNA encoding the new antigenic peptide of the present disclosure is administered to a subject in need thereof.In some embodiments, the present disclosure provides RNA, oligoribonucleotide, and polyribonucleotide molecules that contain modified nucleosides, gene therapy vectors that contain them, and gene therapy methods and gene transcription silencing methods that contain them.In some embodiments, the mRNA that is administered contains at least one modified nucleoside.

[0398] Polynucleotides encoding the peptides described herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Polynucleotides encoding peptides that comprise or consist of analogs can be made simply by substituting the appropriate desired nucleobase(s) for those encoding the native epitope.

[0399] The polynucleotides described herein may contain one or more synthetic or naturally occurring introns in the transcribed region. The inclusion of mRNA stabilizing sequences and sequences for replication in mammalian cells may also be considered to increase polynucleotide expression. Furthermore, the polynucleotides described herein may contain immunostimulatory sequences (ISS or CpG). These sequences may be included in the vector outside the polynucleotide coding sequence to enhance immunogenicity.

[0400] In some embodiments, a polynucleotide can include a coding sequence for a peptide or protein fused in the same reading frame to a polynucleotide that aids in the expression and / or secretion of the peptide or protein from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide from the cell). A polypeptide with a leader sequence can be a preprotein, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0401] In some embodiments, a polynucleotide can include a coding sequence for a peptide or protein fused in the same reading frame to a marker sequence that allows for purification of the encoded peptide, which can then be incorporated into a personalized disease vaccine or immunogenic composition. For example, the marker sequence can be a hexa-histidine tag provided by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in bacterial hosts, or a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when using mammalian hosts (e.g., COS-7 cells). Additional tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Sof tag1, Sof tag3, V5 tag, Xpress tag, Isopep tag, SpyTag, biotin carboxyl carrier protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose-binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, etc. Polynucleotides encoding the neoantigenic peptides described herein can also include sequences encoding ubiquitination signal sequences and / or targeting sequences, such as reticular (ER) signal sequences, to facilitate translocation of the resulting peptide into the endoplasmic reticulum.

[0402] In some embodiments, the polynucleotide can comprise the coding sequences of one or more presently described peptides or proteins fused in the same reading frame to create a single concatemerized neo-antigenic peptide construct capable of producing multiple neo-antigenic peptides.

[0403] In some embodiments, DNA sequences are constructed using recombinant techniques by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-directed mutagenesis to obtain functional analogs thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585. In some embodiments, DNA sequences encoding a peptide or protein of interest are constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired peptide, and codons preferred in the host cell in which the recombinant polypeptide of interest will be produced can be selected. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. Additionally, DNA oligomers containing nucleotide sequences encoding a particular isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0404] Once assembled (e.g., by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into an expression vector and optionally operably linked to an expression control sequence suitable for protein expression in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene can be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0405] Accordingly, the present disclosure is also directed to vectors and expression vectors useful for producing and administering the neo-antigenic peptides and neo-epitopes described herein, as well as host cells containing such vectors.

[0406] vector In some embodiments, an expression vector capable of expressing the peptide or protein described herein can also be prepared.Expression vectors for different cell types are well known in the art and can be selected without undue experimentation.Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate orientation and correct reading frame for expression.If necessary, DNA can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequence recognized by the desired host (e.g., bacteria), and such control is generally available in expression vectors.The vector is then introduced into host bacteria for cloning using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0407] Numerous vectors and host systems suitable for producing and administering the neoantigenic peptides described herein are known to those of skill in the art and are commercially available. The following vectors are provided by way of example: Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pCR (Invitrogen). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia); p75.6 (Valentis); pCEP (Invitrogen); pCEI (Epimmune). However, any other plasmid or vector can be used as long as it is replicable and viable in the host.

[0408] For expression of the neoantigenic peptides described herein, the coding sequence is provided with an operably linked initiation and termination codon, a promoter and terminator region, and, in some embodiments, a replication system to provide an expression vector for expression in a desired cellular host. For example, a promoter sequence compatible with a bacterial host is provided in a plasmid containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vector is transformed into a suitable bacterial host.

[0409] Mammalian expression vectors contain a replication origin, a suitable promoter and enhancer, and also any necessary ribosome binding site, polyadenylation site, splice donor and acceptor sites, transcription termination sequence, and 5' flanking non-transcribed sequence. Such promoters can also be derived from viral sources, such as human cytomegalovirus (CMV-IE promoter) or herpes simplex virus type 1 (HSV TK promoter). Nucleic acid sequences derived from SV40 splice and polyadenylation sites can be used to provide the necessary non-transcribed genetic elements.

[0410] Recombinant expression vectors can be used to amplify and express DNA encoding the peptides or proteins described herein. A recombinant expression vector is a replicable DNA construct containing a synthetic or cDNA-derived DNA fragment encoding a peptide or bioequivalent analog operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally contains (1) one or more genetic elements that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) an assembly of appropriate transcriptional and translational initiation and termination sequences, as described in detail herein. Such regulatory elements may include an operator sequence to control transcription. Additional components, typically conferred by an origin of replication, include the ability to replicate in a host and a selection gene to facilitate recognition of transformants. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue, which can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0411] Generally, recombinant expression vectors contain an origin of replication and a selectable marker, e.g., the E. coli and S. cerevisiae TRP1 gene, that enable transformation of host cells, as well as a promoter derived from a highly expressed gene to direct transcription of downstream structural sequences. Such promoters may be derived from operons encoding glycolytic enzymes, such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences and, in some embodiments, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein containing an N-terminal identification peptide that confers desirable characteristics, such as stabilization or simplified purification of the expressed recombinant product.

[0412] In some embodiments, the neoantigenic peptides described herein can also be expressed by bacterial vectors. Examples of bacterial expression vectors include the most commonly used E. coli expression system. Escherichia coli (E. coli) is one of the most widely used hosts for the production of heterologous proteins, and its genes are far better characterized than those of any other microorganism. Fusion proteins can be expressed in E. coli expression vectors and grown in a variety of host strains. E. coli strains DH5α, JM101, RRI, DH5c, S17-1λ / pir, K12, Top10, and BL21(DE3) are exemplary strains that can be used as heterologous gene expression hosts. In many embodiments, suitable bacteria contain one or more mutations or other genetic modifications that increase expression levels.

[0413] Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for constructing recombinant nucleic acids, such as expression vectors, are well known to those of skill in the art. Examples of these techniques and explanations sufficient to guide one of skill through many cloning practices can be found in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, ed. 4, Cold Spring Harbor Laboratory Press; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); and Current Protocols in Molecular Biology, F.M.A. Subel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley Sons, Inc., (1994 Supplement) (Ausubel). Examples of protocols sufficient to guide one of skill in the art through in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qp-replicase amplification, and other RNA polymerase-mediated techniques, can be found in Berger, Sambrook, and Ausubel, and in Mullis et al. (1987) U.S. Pat. No. 4,683,202.

[0414] Briefly, protein expression methods in E. coli systems include the following steps: A nucleic acid encoding a protein or polypeptide of interest is cloned into a desired plasmid. The plasmid is then introduced into a bacterial host compatible with plasmid propagation (e.g., by transfection, electroporation, or any other suitable method known to those skilled in the art). The bacteria are cultured and grown in a culture medium, while the protein product encoded by the nucleic acid within the bacteria is amplified (or, if the encoded protein is a secreted protein, is secreted into the medium). The protein is then harvested from the grown bacteria (for endogenous proteins) or the culture supernatant (for secreted proteins). High-copy-number expression plasmids are typically selected for this purpose. The basic elements of any plasmid for bacterial cloning include, among others, an ORI (origin of replication) site, control elements such as a promoter and enhancer at the 5' end, one or more expression stabilizers at the 3' end, and a selectable marker gene such as antibiotic resistance. Most E. coli strains can be used to propagate the plasmid. Expression plasmids often contain tags for protein purification. When a nucleic acid encoding a protein of interest is cloned into a designated cloning site of a commercially available vector with a suitable tag, the expressed protein is a fusion protein containing the tag. For example, the plasmid pDEST-HisMBP contains MBP and a hexa-histidine tag. In this case, the MBP protein and His tag are used to isolate and purify the protein from E. coli cells. A variety of such plasmids and expression systems are commercially available and known to those skilled in the art. Construction of an expression vector involves the use of P lac , P trp , P tac , λP L , P T7 , and P BAD Many promoters are commonly used, including lacUV5, tac, and P T7The lacUV5-based system is widely used because expression can be easily regulated by varying the concentration of the inducer, isopropyl-β-D-thiogalactopyranolate (IPTG). Other promoters, called λPL and λPR, are generally induced by temperature shift. To achieve high-level expression of cloned genes, expression cassettes can include other sequences, such as ribosome binding sites for translation initiation and transcription / translation termination sequences. High-level expression of desired peptides or polypeptides can be achieved by using bacterial expression vectors containing dual promoters. Cells can be grown in shake flasks or other vessels, but for large-scale production, polypeptide growth in fermenters is preferred. To achieve maximum levels of expression, galactose is added to the nutrient medium at the appropriate time during the growth cycle to induce increased expression of the desired polypeptide. For example, growth of host cells can be initiated in medium containing fructose (0.25% final concentration) as a carbon source, which causes an increase in intracellular cAMP (adenosine 3',5'-cyclic monophosphate) concentrations; other sugars (glycerol, acetate) can also be used as carbon sources.

[0415] To enable the selection of cells containing the construct, one or more selectable marker genes, such as antibiotic resistance genes, are preferably included in the expression vector. These genes encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells that are not transformed with a vector containing a selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, or tetracycline.

[0416] Alternatively, the selectable marker may encode a protein that complements an auxotrophic deficiency or may supply a critical nutrient unavailable from complex media, such as the gene encoding D-alanine racemase for Bacillus. Several selectable markers are known to those skilled in the art and are described, for example, in Sambrook et al., supra. A preferred selectable marker for use when using a dual tac-lac promoter to express a desired polypeptide is the kanamycin resistance marker (Vieira and Messing, Gene 19:259 (1982)). The use of kanamycin selection is advantageous over, for example, ampicillin selection, because ampicillin is rapidly degraded by p-lactamase in the medium, removing selection pressure and allowing the culture to overgrow cells that do not contain the vector.

[0417] Construction of suitable vectors containing one or more of the components listed above can utilize standard ligation techniques, such as those described in the above-mentioned references. The vector may also contain other sequences that allow the vector to be cloned in a prokaryotic host. Those skilled in the art will recognize that each of these vector components can be modified without substantially affecting their function.

[0418] The isolated plasmid or DNA fragment is then cleaved, tailored, and religated in the desired configuration to generate the desired plasmid. To confirm the correct sequence in the constructed plasmid, the plasmid is analyzed by standard techniques, such as restriction endonuclease digestion and / or sequencing, according to known methods.

[0419] Affinity tags and other types of genetically engineered fusion partners are widely used as tools in molecular biology. Affinity tags are very useful for purifying expressed proteins. The basic steps of purification include protein dissolution, protein binding to a matrix, washing, and elution. As an example, a polyhistidine (His6) tag is commonly placed at the N- or C-terminus of a protein during cloning. Each protein of interest is expressed with the tag. Finally, the His can be captured by a metal because the nitrogen of the imidazole moiety of the polyhistidine interacts with the metal. Typically, the metal is immobilized to support the capture. Washing and elution are performed using a suitable buffer. The tag can be designed to be cleaved from the expressed protein after purification. Similarly, various other tags, such as GST tags, Halo tags, and HA tags, are well known to those skilled in the art for this purpose. The tag portion is typically small and does not interfere with the expressed protein structure. While these were originally developed to facilitate the detection and purification of recombinant proteins, in recent years it has become clear that certain tags can also improve yield, enhance solubility, and even promote proper folding of their fusion partners. Recombinant protein insolubility can be a particular problem in Escherichia coli during the production of bioactive substances for structural and functional studies; however, the use of solubility-enhancing tags to avoid the formation of insoluble protein aggregates is rapidly increasing in popularity. Many proteins that are highly soluble when overproduced in E. coli have been reported to possess solubility-enhancing properties as fusion partners, although in most cases there is little evidence to support these claims. Among known solubility-enhancing fusion partners, MBP is unique in that it is also a natural affinity tag. MBP fusion proteins can be purified using amylose affinity chromatography. Further examples include His tags, FLAG tags, HA tags, and others.

[0420] Examples of expression vectors that can infect mammalian cells include attenuated viral hosts such as vaccinia or fowlpox. In one example of this approach, vaccinia virus is used as a vector to express the nucleotide sequences encoding the neoantigenic peptides described herein. Vaccinia vectors and methods useful for immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991).

[0421] A wide variety of other vectors useful for therapeutic administration or immunization with the neoantigenic polypeptides described herein will be apparent to those skilled in the art from the description herein, such as adenovirus and adeno-associated virus vectors, retrovirus vectors, Salmonella Typhimurium vectors, detoxified anthrax toxin vectors, Sendai virus vectors, poxvirus vectors, canarypox vectors, and fowlpox vectors. In some embodiments, the vector is Modified Vaccinia Ankara (VA) (e.g., Bavarian Noridic (MVA-BN)).

[0422] Among the vectors that can be used in the practice of this disclosure, integration into the cellular host genome is possible through retroviral gene transfer methods, often resulting in long-term expression of the inserted transgene. In some embodiments, the retrovirus is a lentivirus. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues. Retroviral tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Retroviruses can also be engineered to enable conditional expression of the inserted transgene, so that only specific cell types are infected by the lentivirus. Cell-type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and therefore, both lentiviral and retroviral vectors can be used in the practice of this disclosure). Furthermore, lentiviral vectors can transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats, capable of packaging up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which then integrates into target cells with the desired nucleic acid to provide persistent expression. Widely used retroviral vectors that can be used in the practice of the present disclosure include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., Buchscher et al., (1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfelt et al., (1990) Virol. 176:58-59; Wilson et al. al., (1998) J. Virol. 63:2374-2378; Miller et al., (1991) J. Virol. 65:2220-2224; see PCT / US94 / 05700).

[0423] Also useful in implementing the present disclosure are minimal non-primate lentiviral vectors, such as lentiviral vectors based on equine infectious anemia virus (EIAV). The vector may have a cytomegalovirus (CMV) promoter driving the expression of a target gene. Thus, the present disclosure contemplates viral vectors, including retroviral and lentiviral vectors, among the vector(s) useful in implementing the present disclosure.

[0424] Adenovirus vectors are also useful in the implementation of the present disclosure.One advantage is that recombinant adenovirus can efficiently transcribe and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, resulting in high expression of transcribed nucleic acid.In addition, the ability to productively infect quiescent cells expands the usefulness of recombinant adenovirus vectors.Furthermore, high expression level ensures that nucleic acid products are expressed to a sufficient level to generate immune response (see, for example, U.S. Patent No. 7,029,848, which is incorporated herein by reference).

[0425] With regard to adenovirus vectors useful in carrying out the present disclosure, reference is made to U.S. Patent No. 6,955,808. The adenovirus vector used may be selected from the group consisting of Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; the contents of which are incorporated herein by reference). Ad35 vectors are described in U.S. Patent Nos. 6,974,695, 6,913,922, and 6,869,794. Ad11 vectors are described in U.S. Patent No. 6,913,922. C6 adenoviral vectors are described in U.S. Patent Nos. 6,780,407, 6,537,594, 6,309,647, 6,265,189, 6,156,567, 6,090,393, 5,942,235, and 5,833,975. C7 vectors are described in U.S. Patent No. 6,277,558. E1-deleted or deleted, E3-deleted or deleted, and / or E4-deleted or deleted adenoviral vectors can also be used. Certain adenoviruses with mutations in the E1 region offer an improved safety margin, as E1-deleted adenoviral mutants are replication-deficient or at least highly attenuated in nonpermissive cells. Adenovirus with mutation in E3 region can enhance immunogenicity by destroying the mechanism that adenovirus downregulates MHC class I molecules.Adenovirus with E4 mutation can reduce the immunogenicity of adenoviral vector due to the suppression of late gene expression.This vector can be particularly useful when repeated revaccination using the same vector is desired.The adenoviral vector that is deleted or mutated in E1, E3, E4; E1 and E3; and E1 and E4 can be used according to the present disclosure.

[0426] Furthermore, "gutless" adenoviral vectors, in which all viral genes have been deleted, can also be used in accordance with the present disclosure. Such vectors require a helper virus for their replication and require a specialized human 293 cell line that expresses both E1a and Cre, a condition not present in the natural environment. Such "gutless" vectors are non-immunogenic, and therefore, the vectors can be administered multiple times for revaccination. "Gutless" adenoviral vectors can be used to insert heterologous inserts / genes, such as the transgenes of the present disclosure, and can even be used to simultaneously deliver multiple heterologous inserts / genes.

[0427] In some embodiments, delivery is via adenovirus, and the adenovirus may be delivered in a single booster dose. In some embodiments, the adenovirus is delivered via multiple doses. For in vivo delivery, AAV is advantageous over other viral vectors due to its low toxicity and low likelihood of causing insertional mutagenesis because it does not integrate into the host genome. AAV has a packaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb significantly reduce virus production. There are many promoters that can be used to drive nucleic acid molecule expression. AAV ITRs can serve as promoters, advantageously eliminating the need for additional promoter elements.

[0428] For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc. For brain expression, the following promoters can be used: synapsin I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis can include Pol III promoters such as U6 or H1. The use of Pol II promoters and intron cassettes can be used to express guide RNAs (gRNAs). For the AAV vector useful in carrying out the present disclosure, reference is made to United States Patent Nos. 5,658,785, 7,115,391, 7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926, 6,537,540, 6,475,769 and 6,258,595 and the documents cited therein.For AAV, AAV can be AAV1, AAV2, AAV5 or any combination thereof. AAV can be selected based on the cell type to be targeted, for example, AAV serotype 1, 2, 5, or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected to target brain or neural cells, and AAV4 can be selected to target cardiac tissue. AAV8 is useful for delivery to the liver. In some embodiments, delivery is via AAV. Dosage can be adjusted to balance the therapeutic benefit against any side effects.

[0429] In some embodiments, poxviruses are used in the compositions described herein. These include orthopoxviruses, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see, for example, Verardiet et al., Hum. Vaccin. Immunother. 2012 July;8(7):961-70, and Moss, Vaccine. 2013;31(39):4220-4222). Poxvirus expression vectors were described in 1982 and quickly became widely used in vaccine development and research in many fields. The advantages of this vector include simple construction, the ability to accommodate large amounts of foreign DNA, and high expression levels. Information regarding poxviruses that can be used in practicing the present disclosure, such as Chordopoxvirinae Subfamily Poxviruses (vertebrate poxviruses), e.g., orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth strain, WR strain (e.g., ATCC® VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 strain, ALVAC), fowlpox virus (e.g., FP9 strain, Webster strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, among others, synthetic or non-naturally occurring recombinants thereof, their uses, and methods for making and using such recombinants, can be found in the scientific and patent literature.

[0430] In some embodiments, vaccinia viruses are used in disease vaccines or immunogenic compositions to express antigens (Rolph et al., Recombinant viruses as vaccines and immunological tools. Curr. Opin. Immunol. 9:517-524, 1997). Recombinant vaccinia viruses can replicate in the cytoplasm of infected host cells, thus allowing the polypeptide of interest to induce an immune response. Furthermore, poxviruses have been widely used as vaccine or immunogenic composition vectors due to their ability to target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells, particularly antigen-presenting cells, but also due to their ability to self-adjuvant.

[0431] In some embodiments, ALVAC is used as a vector in disease vaccines or immunogenic compositions. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W,et al.Phase I clinical trial of a recombinant canarypoxvirus(ALVAC)vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule.Cancer Immunol.Immunother.2000;49:504-14, von Mehren M,Arlen P,Tsang KY,et al.Pilot study of a dual gene recombinant avipox vaccine containing both carcinoembryonic antigen(CEA)and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas.Clin.Cancer.Res.2000;6:2219-28, Musey L,Ding Y,Elizaga M,et al.HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals.J.Immunol.2003;171:1094-101, Paoletti E. Applications of pox virus vectors to vaccination: an update.Proc.Natl.Acad.Sci.USA 1996;93:11349-53, U.S. Patent No. 7,255,862). In a phase I clinical trial, ALVAC virus expressing the tumor antigen CEA demonstrated a favorable safety profile and induced increased CEA-specific T cell responses in selected patients, but no objective clinical responses were observed (Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant vaccine that expresses human carcinoembryonic antigen. J. Clin. Oncol. 1999;17:332-7).

[0432] In some embodiments, modified vaccinia Ankara (MVA) virus can be used as a viral vector for antigen vaccines or immunogenic compositions. MVA is a member of the orthopoxvirus family and has been generated by approximately 570 serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (see, e.g., Mayr, A., et al., Infection 3, 6-14, 1975). As a result of these passages, the resulting MVA virus contains 31 kilobases less genomic information than CVA and is highly host-cell restricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extreme attenuation, i.e., reduced virulence or infectivity, yet still possesses excellent immunogenicity. When tested in various animal models, MVA has proven nonpathogenic, even in immunosuppressed individuals. Additionally, MVA-BN®-HER2 is a candidate immunotherapy designed for the treatment of HER-2-positive breast cancer and is currently undergoing clinical trials. (Mandl et al., Cancer Immunol. Immunother. Jan 2012;61(1):19-29) Methods for making and using recombinant MVA have been described (see, e.g., U.S. Patent Nos. 8,309,098 and 5,185,146, which are incorporated herein in their entireties).

[0433] Suitable host cells for expressing polypeptides include prokaryotes, yeast, insects, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0434] Various mammalian or insect cell culture systems can also be advantageously utilized to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be performed to ensure that such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, as well as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0435] Host cells are genetically engineered (transduced or transformed or transfected) with a vector, which may be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as necessary for activating promoters, selecting transformants, or amplifying the polynucleotide. Culture conditions, such as temperature, pH, and the like, will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0436] Representative examples of suitable hosts include bacterial cells such as E. coli, Bacillus subtilis, Salmonella typhimurium, and various species of the genera Pseudomonas, Streptomyces, and Staphylococcus; fungal cells such as yeast; insect cells such as Drosophila and Sf9; animal cells such as the COS-7 strain of monkey kidney fibroblasts described by Gluzman, Cell 23:175 (1981), as well as other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0437] Yeast, insect, or mammalian cell hosts can also be used, utilizing appropriate vectors and control sequences. Examples of mammalian expression systems include the COS-7 strain of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), as well as other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines.

[0438] The polynucleotides described herein can be administered and expressed in human cells (e.g., immune cells, including dendritic cells). A human codon usage table can be used to guide the codon selection for each amino acid. Such polynucleotides can include spacer amino acid residues between epitopes and / or analogs, such as those described above, or naturally occurring flanking sequences (and / or CTL (e.g., CD8)) flanking the epitopes and / or analogs. + ), Th (e.g., CD4 + ), and B-cell epitopes).

[0439] Standard regulatory sequences well known to those skilled in the art can be included in the vector to ensure expression in human target cells. Several vector elements are desirable: a promoter with a cloning site downstream of the polynucleotide, such as a minigene insert; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). Many promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. For other suitable promoter sequences, see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466. In some embodiments, the promoter is a CMV-IE promoter.

[0440] Useful expression vectors for eukaryotic hosts, particularly mammalian or human, include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as those derived from Escherichia coli, including pCR1, pBR322, pMB9, and their derivatives, and broader host range plasmids such as M13 and filamentous single-stranded DNA phages.

[0441] Vectors can be introduced into animal tissues by several different methods. The two most common approaches are injection of DNA in saline using a standard hypodermic needle and gene gun delivery. An overview of the construction of DNA vaccine plasmids and their subsequent delivery to the host by these two methods is illustrated in Scientific American (Weiner et al., (1999) Scientific American 281(1):34-41). Injection in saline is usually performed intramuscularly (IM) or intradermally (ID) in skeletal muscle, with DNA being delivered to the extracellular space. This can be assisted by electroporation, which involves temporarily damaging muscle fibers with myotoxins such as bupivacaine, or by using hypertonic solutions of saline or sucrose (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410). The immune response to this delivery method can be affected by many factors, including needle type, needle alignment, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the injected animal (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410).

[0442] Another commonly used delivery method, gene gun delivery, uses compressed helium as an accelerator to ballistically accelerate plasmid DNA (pDNA) adsorbed to gold or tungsten microparticles into target cells (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88).

[0443] Alternative delivery methods include aerosol instillation of naked DNA onto mucosal surfaces such as the nasal and pulmonary mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88), and topical administration of pDNA to the ocular and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54:129-88). Mucosal surface delivery can also be achieved using cationic liposomal DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors, and recombinant adenovirus vectors for oral administration to the intestinal mucosa. DNA or RNA can also be delivered to cells after mild mechanical disruption of the cell membrane, allowing cells to temporarily permeate. Such mild mechanical disruption of the membrane can be achieved by gently passing the cells through a small opening (Sharei et al., Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, PLOS ONE (2015)).

[0444] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-10 (1991)). However, compositions that have structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may assume a micellar structure or may exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0445] The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist as a bilayer structure, micelles, or in a "collapsed" structure. They may also simply be interspersed in solution, or in some cases, form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include the naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0446] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol and is therefore used as the sole solvent. In some embodiments, lipids are essential for intracellular delivery of nucleic acids encoding proteins of interest. Nucleic acid constructs encoding the fusion polypeptides described herein can be delivered via lipid compositions, e.g., liposomes, into cells for expression of the fusion polypeptides. In some embodiments, the liposomes comprise one or more cationic lipids, hi some embodiments, the liposomes comprise at least a cationic lipid and at least a non-cationic lipid.

[0447] In some embodiments, the vector comprises a polynucleotide encoding a first peptide comprising a first neoepitope and a second peptide comprising a second neoepitope. In some embodiments, the first and second peptides are derived from the same protein. The at least two different peptides can vary in length, amino acid sequence, or both. The peptides are derived from any protein known or found to contain a tumor-specific mutation. In some embodiments, the vector comprises a first peptide comprising a first neoepitope of a protein and a second peptide comprising a second neoepitope of the same protein, wherein the first peptide is different from the second peptide, the first neoepitope comprises a mutation, and the second neoepitope comprises the same mutation. In some embodiments, the vector comprises a first peptide comprising a first neo-epitope of a first region of a protein and a second peptide comprising a second neo-epitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide differs from the second peptide, the first neo-epitope comprises a first mutation, and the second neo-epitope comprises a second mutation. In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the mutation is selected from the group consisting of a point mutation, a splice site mutation, a frameshift mutation, a read-through mutation, a gene fusion mutation, and any combination thereof.

[0448] In some embodiments, the vector comprises a polynucleotide operably linked to a promoter. In some embodiments, the vector is a self-amplifying RNA replicon, a plasmid, a phage, a transposon, a cosmid, a virus, or a virion. In some embodiments, the vector is derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpesvirus, a poxvirus, an alphavirus, a vaccinia virus, a hepatitis B virus, a human papillomavirus, or a pseudotype thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a nanoparticle, a cationic lipid, a cationic polymer, a metal nanopolymer, a nanorod, a liposome, a micelle, a microbubble, a cell-penetrating peptide, or a liposphere.

[0449] T cells and T cell receptors T cells are T lymphocytes, immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells can be naive, i.e., not exposed to antigen, and show increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO, compared to more mature T cells with antigen exposure and memory, and are classified as memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). M are a subset of central memory T cells (TC M , increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA, compared with naive T cells), and effector memory T cells (T EM , naive T cells or TCs M They can be further divided into effector T cells (T E ) is a TC MThese T cells refer to antigen-experienced CD8+ cytotoxic T lymphocytes that have reduced expression of CD62L, CCR7, and CD28, and are positive for granzymes and perforin, compared to other T cells. Other exemplary T cells include CD4+CD25+ (Foxp3+) regulatory T cells and Tregl7 cells, as well as regulatory T cells such as Trl, Th3, CD8+CD28-, and Qa-1-restricted T cells.

[0450] In one aspect, the present disclosure provides a mechanism for targeting neoantigens to expose naive T cells. Lymph nodes harbor naive T cells. Targeting antigenic peptides to lymph nodes promotes exposure of resident naive T cells to the antigen, thus generating new primed T cells. Polybodies are prepared with neoantigen-enriched peptides, and subsequently primed T cells in lymph nodes are primed against neoantigens and become immunogenic against tumors or cancers.

[0451] In another embodiment, the present disclosure provides a composition comprising a first peptide comprising a first neo-epitope of a first region of a protein and a second peptide comprising a second neo-epitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide is different from the second peptide, the first neo-epitope comprises a first mutation, and the second neo-epitope comprises a second mutation. In some embodiments, the first neo-epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neo-epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neo-epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neo-epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neo-epitope forms an HLA-peptide complex. +In some embodiments, the first neo-epitope activates CD4 T cells. + In some embodiments, the second neo-epitope activates CD4 T cells. + In some embodiments, the second neo-epitope activates CD8 T cells. + In some embodiments, CD4 + The TCR of the T cell binds to a class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to a class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to a class I HLA-peptide complex. In some embodiments, CD4 + The TCR of the T cell binds to the class I HLA-peptide complex.

[0452] In some embodiments, the first TCR is a first chimeric antigen receptor specific for a first neoepitope, and the second TCR is a second chimeric antigen receptor specific for a second neoepitope. In some embodiments, the first T cell is a cytotoxic T cell. In some embodiments, the first T cell is a gamma delta T cell. In some embodiments, the second T cell is a helper T cell. In some embodiments, the first and / or second TCR has a K of less than 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 In some embodiments, the first and / or second TCR binds to an HLA-peptide complex with a K of less than 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50In some embodiments, the first and / or second TCR binds to an HLA class I-peptide complex with a K of less than 2,000, 1,500, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM, or 10 nM. D or IC 50 It binds to HLA class II-peptide complexes.

[0453] antigen presenting cells The neo-antigenic peptides or proteins may be provided as antigen-presenting cells (e.g., dendritic cells) containing such peptides, proteins, or polynucleotides described herein. In other embodiments, such antigen-presenting cells are used to stimulate T cells for use in a patient. Accordingly, one embodiment of the present disclosure is a composition containing at least one antigen-presenting cell (e.g., dendritic cell) pulsed or loaded with one or more neo-antigenic peptides or polynucleotides described herein. In some embodiments, such APCs are autologous (e.g., autologous dendritic cells). Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with the neo-antigenic peptides or polynucleotides ex vivo. In related embodiments, such APCs or PBMCs are infused back into the patient. In some embodiments, the antigen-presenting cells are dendritic cells. In related embodiments, the dendritic cells are autologous dendritic cells pulsed with the neo-antigenic peptide or nucleic acid. The neo-antigenic peptide can be any suitable peptide that generates an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides derived from tumor-associated antigens is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278. In some embodiments, the T cells are CTLs (e.g., CD8 + In some embodiments, the T cells are helper T lymphocytes (Th (e.g., CD4 + )).

[0454] In some embodiments, the present disclosure provides compositions, including cell-based immunogenic pharmaceutical compositions, which can be administered to a subject. For example, antigen-presenting cell (APC)-based immunogenic pharmaceutical compositions can be formulated using any of the well-known techniques, carriers, and excipients suitable and understood in the art. APCs include monocytes, monocyte-derived cells, macrophages, and dendritic cells. In some cases, the APC-based immunogenic pharmaceutical composition may be a dendritic cell-based immunogenic pharmaceutical composition.

[0455] Dendritic cell-based immunogenic pharmaceutical compositions can be prepared by any method known in the art. In some cases, dendritic cell-based immunogenic pharmaceutical compositions can be prepared through ex vivo or in vivo methods. Ex vivo methods can include using autologous DCs pulsed ex vivo with a polypeptide described herein to activate or load the DCs before administration to a patient. In vivo methods can include targeting specific DC receptors using an antibody coupled to a polypeptide described herein. DC-based immunogenic pharmaceutical compositions can further include DC activators such as TLR3, TLR-7-8, and CD40 agonists. DC-based immunogenic pharmaceutical compositions can further include an adjuvant and a pharmaceutically acceptable carrier.

[0456] Antigen-presenting cells (APCs) can be prepared from a variety of sources, including humans and non-human primates, other mammals, and vertebrates. In certain embodiments, APCs can be prepared from the blood of humans or non-human vertebrates. APCs can also be isolated from enriched populations of leukocytes. The leukocyte population can be prepared by methods known to those skilled in the art. Such methods typically include heparinized blood collection, apheresis or leukopheresis, buffy coat preparation, rosette formation, centrifugation, density gradient centrifugation (e.g., using Ficoll, colloidal silica particles, and sucrose), differential lysis of non-leukocyte cells, and filtration. Leukocyte populations can also be prepared by collecting blood from a subject, defibrinating to remove platelets, and lysing red blood cells. The leukocyte population can optionally be enriched for monocytic dendritic cell precursors.

[0457] Blood cell population can be obtained from various subjects according to the desired use of enriched leukocyte population.The subject can be a healthy subject.Alternatively, blood cells can be obtained from subjects that require immune stimulation, such as cancer patients or other patients who benefit from immune stimulation.Similarly, blood cells can be obtained from subjects that require immunosuppression, such as patients with autoimmune disorders (e.g., rheumatoid arthritis, diabetes, lupus, multiple sclerosis, etc.).Leukocyte population can also be obtained from HLA-matched healthy individuals.

[0458] When blood is used as a source of APCs, blood leukocytes can be obtained using conventional methods that maintain their viability. According to one embodiment of the present disclosure, blood can be diluted in a medium that may or may not contain heparin or other suitable anticoagulants. The blood to medium volume ratio can be approximately 1:1. Cells can be concentrated by centrifugation of the blood in the medium at approximately 1,000 rpm (150 g) at 4°C. Platelets and red blood cells can be depleted by resuspending the cells in any number of solutions known in the art that lyse red blood cells, such as ammonium chloride. For example, the mixture can be approximately 1:1 volume of medium and ammonium chloride. The cells can be concentrated by centrifugation and washed in the desired solution until a population of white blood cells substantially free of platelets and red blood cells is obtained. Any isotonic solution commonly used in tissue culture can be used as a medium for separating blood leukocytes from platelets and red blood cells. Examples of such isotonic solutions include phosphate-buffered saline, Hank's balanced salt solution, and complete growth medium. APCs and / or APC precursor cells can also be purified by elutriation.

[0459] In some embodiments, APC can be non-nominal APC under inflammatory or other activated conditions.For example, non-nominal APC can include epithelial cells stimulated with interferon-gamma, T cells, B cells and / or monocytes activated by factors or conditions that induce APC activity.Such non-nominal APC can be prepared according to methods known in the art.

[0460] APCs can be cultured, expanded, differentiated, and / or matured according to the type of APC, as desired. APCs can be cultured in any suitable culture vessel, such as, for example, culture plates, flasks, culture bags, and bioreactors.

[0461] In certain embodiments, APCs can be cultured in a suitable culture or growth medium to maintain and / or expand the number of APCs in the preparation. The culture medium can be selected according to the type of isolated APC. For example, mature APCs, such as mature dendritic cells, can be cultured in a suitable growth medium for their maintenance and expansion. The culture medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. Additionally, cytokines, growth factors, and / or hormones can be included in the growth medium. For example, cytokines, such as granulocyte / macrophage colony-stimulating factor (GM-CSF) and / or interleukin 4 (IL-4), can be added to maintain and / or expand mature dendritic cells. In other embodiments, immature APCs can be cultured and / or expanded. Immature dendritic cells can retain the ability to take up target mRNA and process new antigens. In some embodiments, immature dendritic cells can be cultured in a suitable medium for their maintenance and culture. The culture medium can be supplemented with amino acids, vitamins, antibiotics, divalent cations, etc. Additionally, cytokines, growth factors and / or hormones may be included in the growth medium.

[0462] Other immature APCs can be similarly cultured or expanded. Preparations of immature APCs can be matured to form mature APCs. APC maturation can occur during or after exposure to neoantigenic peptides. In certain embodiments, preparations of immature dendritic cells can be matured. Suitable maturation factors include, for example, the cytokine TNF-α, bacterial products (e.g., BCG), etc. In another aspect, isolated APC precursors can be used to prepare preparations of immature APCs. The APC precursors can be cultured, differentiated, and / or matured. In certain embodiments, monocytic dendritic cell precursors can be cultured in the presence of a suitable culture medium supplemented with amino acids, vitamins, cytokines, and / or divalent cations to promote the differentiation of the monocytic dendritic cell precursors into immature dendritic cells. In some embodiments, APC precursors are isolated from PBMCs. PBMCs can be obtained from a donor, e.g., a human donor, and can be used fresh or frozen for future use. In some embodiments, APCs are prepared from one or more APC preparations. In some embodiments, the APC comprises an APC loaded with a first and a second neo-antigenic peptide comprising the first and second neo-epitopes, or a polynucleotide encoding the first and the second neo-antigenic peptide comprising the first and the second neo-epitopes, hi some embodiments, the APC is an autologous APC, an allogeneic APC, or an artificial APC.

[0463] In one embodiment, the disclosure provides a composition comprising an APC comprising a first peptide comprising a first neoepitope and a second peptide comprising a second neoepitope, wherein the first peptide is different from the second peptide, the first neoepitope comprises a mutation, and the second neoepitope comprises the same mutation. In some embodiments, the first and second peptides are derived from the same protein. In another embodiment, the disclosure provides a composition comprising an APC comprising a first peptide comprising a first neoepitope of a first region of a protein and a second peptide comprising a second neoepitope of a second region of the same protein, wherein the first region comprises at least one amino acid of the second region, the first peptide is different from the second peptide, the first neoepitope comprises a first mutation, and the second neoepitope comprises a second mutation. In some embodiments, the first mutation and the second mutation are the same.

[0464] Adjuvants Adjuvants can be used to enhance the immune response (humoral and / or cellular) elicited in patients receiving the compositions provided herein. The active ingredient in the composition includes an antigenic peptide. The incorporation of an adjuvant can further enhance the antigen response of the composition. In some cases, the adjuvant can induce a Th1-type response. In other cases, the adjuvant can induce a Th2-type response. A Th1-type response is In contrast to Th2-type responses, which may be characterized by the production of cytokines such as IL-4, IL-5, and IL-10, IFN-γ may be produced.

[0465] In some embodiments, lipid-based adjuvants such as MPLA and MDP can be used with the immunogenic pharmaceutical compositions disclosed herein. For example, monophosphoryl lipid A (MPLA) is an adjuvant that causes increased presentation of liposomal antigens to specific T lymphocytes. In addition, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in conjunction with the immunogenic pharmaceutical formulations described herein.

[0466] Suitable adjuvants are known in the art (see WO 2015 / 095811) and include poly(I:C), poly-ICLC, Hiltonol, STING agonist, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide Adjuvants include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel® Vector System, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants also include incomplete Freund's or GM-CSF. Several immunological adjuvants specific for dendritic cells and their preparations (e.g., MF59) have been previously described (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27, Allison AC; Dev. Biol. Stand. 1998; 92:3-11) (Mosca et al. Frontiers in Bioscience, 2007; 12:4050-4060) (Gamvrellis et al. Immunol & Cell Biol. 2004; 82:506-516). Cytokines can also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-1b, IL-4, IL-6, and CD40L) (U.S. Pat. No. 5,849,589, incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J. Immunother. Emphasis Tumor Immunol. 1996(6):414-418).

[0467] Adjuvants may also include stimulatory molecules such as cytokines. Non-limiting examples of cytokines include CCL20, α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ (lymphotoxin alpha (LTα)), GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1, IL-8, L-selectin, P-selectin, E-selectin, CD34, GI yCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor , Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2 , DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP2.

[0468] Additional adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, Fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR 5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IκB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFκB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and their functional fragments.

[0469] In some embodiments, the adjuvant may be a toll-like receptor modulator. Examples of toll-like receptor modulators include TLR-9 agonists, but are not limited to small molecule toll-like receptor modulators such as imiquimod. Other examples of adjuvants used in combination with the immunogenic pharmaceutical compositions described herein may include, but are not limited to, saponins, CpG ODNs, and the like. In some cases, the adjuvant is selected from bacterial toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or combinations thereof. In some cases, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion may include at least one oil and at least one surfactant, and the oil(s) and surfactant(s) are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion can be less than 5 μm in diameter, or even submicron in diameter; these small sizes are achieved using a microfluidizer to provide a stable emulsion. Droplets with a size less than 220 nm can be subjected to sterilization by filtration.

[0470] Methods for generating polybodies bearing epitope peptides Polybodies can be generated by cloning. In one approach, a parent scaffold assembly containing the antigenic peptide is first generated. The parent scaffold can be digested with a restriction enzyme, and then the epitope-containing peptide or polypeptide is cloned into the restriction site to obtain peptide scaffold units (multiple units), which are then inserted into a linearized expression vector. The peptide can contain flanking linker sequences. Alternatively, the peptide sequence with the linker can be synthesized or generated by PCR. Gibson assembly can be used to easily join a vector with an insert (a peptide or polypeptide to be inserted into the vector). The Gibson cloning method allows any fragment to be joined without specific restriction sites, providing a fast and flexible cloning strategy regardless of sequence. This involves using T5 exonuclease Chewback to generate single-stranded overhangs at the 5' and 3' ends of the DNA fragment (within the insert), while the vector typically has sticky ends. Upon contact, the insert overhangs thus created hybridize with the cohesive ends of the vector, and fill-in ligation is performed using a suitable ligation enzyme. In some cases, larger overhangs can be generated, and the overhang regions of the vector and insert are joined by overhang hybridization and then ligated using a suitable ligation enzyme to form an intact vector. Variations from the above scheme will occur to those skilled in the art and are intended to be within the scope of the present disclosure.

[0471] Artificial miniproteome vaccine Active tumor eradication immunotherapy requires therapeutic products that are effective, can be rapidly generated, and can be widely adopted. Personalized medicines that use a patient's own neoantigen peptides to activate T cells are a new and promising area in immuno-oncology, but the preparation of neoantigen vaccines can take days, weeks, or even months. Furthermore, this method is expensive. A more effective, time- and cost-effective approach is needed. The goal is to quickly identify and develop a rapid, inexpensive vaccine that contains most neoantigens and other potentially useful antigens from the patient. Such a product should be easily integrated with any off-the-shelf combination of approved and developmental immunotherapies, inexpensive to repeat preparation from additional tumor samples after recurrence or to reduce the impact of tumor heterogeneity, incorporate a significantly broader representation of potentially useful epitopes, and, among other things, activate both the subject's CD8+ and CD4+ cells. Provided herein are compositions and methods for vaccine production that fulfill certain unmet needs, as described above.

[0472] In one aspect, provided herein is a highly efficient method for generating subject-specific cancer vaccines that avoids the step of neoantigen prediction. This method relates to the generation of artificial miniproteome vaccines (AmpVax). In this method, personalized cancer vaccines can be generated by preparing a library of whole-exome-captured RNA fragments from tumor cells or fresh-frozen paraffin-embedded (FFPE) tissue slices from a subject that encode all or a selected portion of the proteins expressed by tumor cells, including all or nearly all neoantigens, as well as tumor-associated antigens, and additional regions adjacent to introns and untranslated regions, which may contain neoantigen translation products that cannot be easily determined by nucleic acid sequencing and antigen prediction algorithms. cDNA is prepared from the library of whole-exome-captured RNA fragments. Each of the cDNA products from the RNA fragments can be cloned into a suitable expression vector and expressed in a suitable system, such as a bacterial system (e.g., an E. coli expression system). The expressed peptides can then be isolated and used as vaccines. In some embodiments, isolated peptides expressed from a library of whole exome-captured RNA from a diseased subject are an artificial miniproteome, where the exome-captured RNA is derived from diseased cells of the subject. The expressed peptides can then be purified and used as a vaccine without further modification.

[0473] In some embodiments, polynucleotides encoding peptides expressed from a library of whole-exome-captured RNA fragments (or, in other words, a whole-exome cDNA library) are isolated and subcloned upstream of a protein(s) or protein fragment(s) (e.g., a scaffold protein as described elsewhere herein) to generate peptide fusion constructs such as polybodies, resulting in a peptide fusion library that can be easily expressed in high yields and in rapid biological expression systems such as E. coli systems. Proteins expressed from the peptide fusion library can be isolated, purified, and used in their entirety as an artificial miniproteome (AmpVax) vaccine.

[0474] In one aspect, the subject'...

Claims

[Claim 1] The invention described in this specification.

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