Multi-domain protein vaccine

Abstract

Disclosed herein are protein fusion technologies that allow for the combination of one or more cancer vaccine epitopes with a scaffold domain. Also disclosed herein are polypeptide and polynucleic acid compositions encompassed by the protein fusion technologies and methods of using them.

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 on October 9, 2019, the entire content of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Cancer immunotherapy aims to utilize a patient's immune system to treat cancer. This takes advantage of the fact that cancer cells often have on their surface molecules, known as tumor antigens, which can be detected by the immune system and which are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy instructs the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing anti - tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Active immunotherapy aims to induce a new immune response in the patient. Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands) that act together to induce antigen - specific cytotoxic T lymphocytes (CTLs) that recognize and lyse tumor cells. Some of the important barriers to developing curative and tumor - specific immunotherapies are to efficiently identify and select highly specific and restricted tumor antigens and deliver the antigens to subjects in a way that maximizes the activation of the subject's T cells for the generation of high anti - tumor immunogenicity.

[0003] Tumor neoantigens resulting from genetic changes (e.g., inversion, translocation, deletion, missense mutation, splice site mutation, etc.) within malignant cells 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 the tumor. They also avoid central tolerance and are therefore likely to be immunogenic. Thus, 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 in identifying them, selecting optimized antigens, and generating 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 incorporated herein by reference 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 Problems

[0005] In some aspects, provided herein is a composition 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, and each of the two or more scaffold polypeptide sequences comprises a human polypeptide sequence, a fragment thereof, or a variant thereof, and (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 embodiments, provided herein is a composition 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, and 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. 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.

[0008] In some embodiments, provided herein is a composition 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, 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 tenascin 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, tenascin 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 6 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 exceeds 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 exceeds 75 kDa.

[0029] In some embodiments, the molecular weight of the fusion polypeptide is 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 or less.

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

[0031] In some embodiments, each of the one or more antigen 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 to the remainder of the fusion polypeptide via its N-terminus.

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

[0034] In some embodiments, at least two of the scaffold polypeptide sequences are not interrupted by an antigen sequence or a linker sequence.

[0035] In some embodiments, the fusion polypeptide comprises a polypeptide sequence having the structure of formula (Ia) in the direction from the N-terminus to the C-terminus, 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 of 2 or more, and n is an integer of 1 or more. In some embodiments, n is an integer selected from 1 to 5, and m is 2.

[0036] In some embodiments, the fusion polypeptide comprises a polypeptide sequence having the structure of formula (Ib) in the direction from the N-terminus to the C-terminus, S O -A n -S m Formula (Ib) In the formula, each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, and each of o, n, and m is independently an integer of 1 or more. 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 a structure of S-A-S, A-S-A-S, S-A-S-A, S-A-S-A-S, S-A-S-A-S-A-S, S-A-S-A-S-A-S-A-S, or S-A-S-A-S-A-S-A-S-A-S.

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

[0038] In some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of formula (IIa) in the direction from the N-terminus to the C-terminus, (L-A-L) n -S m or S m -(L-A-L) 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 of 2 or more, and n is an integer of 1 or more. In some embodiments, n is an integer selected from 1 to 5 and m is 2.

[0039] In some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of formula (IIb) in the direction from the N-terminus to the C-terminus, S o --(L-A-L) n -S m Formula (IIb), In the formula, 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 is independently an integer of 1 or more. 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 a structure of S-L-A-L-S, S-A-L-S-L-A-L-S, S-L-A-L-S-L-A, S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S-L-A-S, or S-L-A-L-S-L-A-L-S-L-A-L-S-L-A-S-L-A-L-S.

[0040] In some embodiments, the scaffold polypeptide and the antigen polypeptide are located in the fusion polypeptide in an alternating pattern.

[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 antigen 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 includes 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 facilitate presentation of at least one of the one or more antigen polypeptides.

[0051] In some embodiments, at least one of the linker sequences includes 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 includes a lysine residue, an arginine residue, or an alanine residue directly connected to the N-terminus of at least one of the antigen polypeptides.

[0053] In some embodiments, at least one of the linker sequences includes 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 antigen polypeptides.

[0054] 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 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 residue, an arginine residue, or an 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 residue, a lysine residue, an arginine residue, or an 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 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, one or more antigen polypeptides are HLA class II antigen polypeptides, and 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 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 antigen polypeptides.

[0058] In some embodiments, 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.

[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 the 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 the corresponding polypeptide lacking the scaffold polypeptide, as 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 higher than the solubility of the 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 higher than the serum solubility of the corresponding polypeptide lacking the scaffold polypeptide.

[0066] 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 the corresponding polypeptide lacking the scaffold polypeptide. In some embodiments, the serum half-life of the fusion polypeptide is at least 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 5000-fold, or 10000-fold higher than the serum solubility of the corresponding polypeptide lacking the scaffold polypeptide.

[0067] 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, 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 higher than the accumulation of the corresponding polypeptide lacking the scaffold polypeptide, as measured by average radiation 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 higher 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 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), macrophage, Langerhans cell, or B cell.

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

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

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

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

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

[0077] In some embodiments, at least one of the linking moieties is directly connected to the N-terminus or C-terminus of one of the antigen polypeptides.

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

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

[0081] In some embodiments, the antigen polypeptide is a neoantigen 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 at least one expressed gene of the subject's cancer cells, and at least one of the cancer neoantigen peptides or portions thereof comprises one or more mutations that are not present in the subject's normal tissues.

[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 the protein encoded by the HLA allele expressed by the subject with an IC50 of less than 500 nM and with a 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 neoantigen peptide of the plurality binds to the protein encoded by a first HLA allele expressed by the subject, a second cancer neoantigen peptide of the plurality binds to the protein encoded by a second HLA allele expressed by the subject, and 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 the protein encoded by the 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 is a fusion polypeptide encoded by the described nucleic acid molecule.

[0091] In one aspect, disclosed herein is a composition comprising the described fusion polypeptide. In some embodiments, the composition comprises one or more binding moieties attached to the fusion polypeptide. In some embodiments, the 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 apart by one or more linkers and one or more restriction sites located in at least one of the linkers, 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. In some embodiments, the scaffold polypeptide is 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 antigen polypeptide and a nucleic acid sequence encoding a first scaffold polypeptide, and a second nucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the first antigen 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 antigen polypeptide, a nucleic acid sequence encoding a first scaffold polypeptide, and a nucleic acid sequence encoding a second antigen polypeptide, and a second nucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the second antigen polypeptide and a nucleic acid sequence encoding a second scaffold polypeptide.

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

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

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

[0098] In some embodiments, the first, second, and third antigen 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 the described composition or the described fusion polypeptide.

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

[0102] In some embodiments, the adjuvant is poly IC: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 immunomodulatory agent, a cytokine or chemokine, or a checkpoint inhibitor. In some embodiments, the second therapeutic is administered to a subject in need thereof prior to administering a pharmaceutical composition comprising the 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 the 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 the 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 the nucleic acid molecule described in a genetically modified cell, thereby producing an immunogenic fusion polypeptide.

[0106] In another aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising providing the nucleic acid molecule described herein; inserting one or more nucleic acid molecules encoding one or more antigen 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 an immunogenic fusion polypeptide.

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

[0108] In one aspect, disclosed herein is a method for producing an immunogenic fusion polypeptide, the method comprising providing a plurality of nucleic acid molecules as described herein, joining the nucleic acid molecules by hybridization, and expressing the joined nucleic acid molecules in a genetically modified cell, thereby producing an 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 for producing an immunogenic fusion polypeptide, the method comprising: (a) providing a nucleic acid molecule as described in the above paragraph, or specifically any one of paragraphs 89-96; (b) inserting one or more nucleic acid molecules encoding one or more antigen 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 is a method for producing an immunogenic fusion polypeptide, the method comprising: (a) providing a plurality of nucleic acid molecules as described in the above paragraph, or specifically 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 an 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, the method comprising administering to the subject in need thereof a pharmaceutical composition as described herein.

[0115] In some embodiments, the pharmaceutical composition comprises a plurality of neoantigen peptides.

[0116] In some embodiments, the pharmaceutical composition comprises a plurality of 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 sub-doses.

[0119] In some embodiments, each sub-dose of the fusion polypeptide comprises one, two, three, four, five, or more than five fusion polypeptides.

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

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

[0122] In some embodiments, the total dose of the fusion polypeptide administered is 0.01 - 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, wherein each of the plurality of expression constructs comprises (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, wherein the first polynucleotide sequence comprises different template polynucleotide sequences, and different templates from the polynucleotide sequences are derived from a sample comprising diseased cells from a subject having a disease, and encode a peptide sequence of a protein encoded by the diseased cells from the subject, and (iii) a second polynucleotide sequence downstream of the first polynucleotide sequence, wherein the second polynucleotide sequence comprises (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, and a sequence encoding a fusion polypeptide. In some embodiments, if the different template polynucleotide sequences or copies thereof are out of frame with the sequence encoding the affinity tag, the frame check sequence operates to terminate translation of the fusion polypeptide. In some embodiments, the frame check sequence is N1-N2-N3-N4-N5-N6-N7-N8-N9 having the formula, wherein each N is, independently, a nucleic acid selected from the group consisting of A, T, U, C, and G, each of N1-N2-N3 and 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 links a first polynucleotide sequence to a sequence encoding an 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 crystallizable fragment (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-enhanced polypeptide and / or (ii) each expression construct of the library comprises a sequence encoding a size-enhanced polypeptide. In some embodiments, the sequence encoding the size-enhanced polypeptide is downstream of the first polynucleotide sequence and / or at least one of the sequences encoding the size-enhanced polypeptide is upstream of a different template polynucleotide sequence. In some embodiments, the affinity tag is an epitope of the size-enhanced polypeptide. In some embodiments, the sequence encoding the size-enhanced polypeptide encodes at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more size-enhanced polypeptides. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the plurality of size-enhanced polypeptides are the same. In some embodiments, the sequence encoding the size-enhanced polypeptide encodes one or more linkers between two or more of the size-enhanced polypeptides. In some embodiments, the molecular weight of the plurality of size-enhanced polypeptides is at least 15 kDa. In some embodiments, the molecular weight of the plurality of size-enhanced polypeptides is 200 kDa or less. In some embodiments, the molecular weight of the plurality of size-enhanced 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. In 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 includes a biopsy, a blood sample, or a peripheral blood mononuclear cell (PBMC) sample, and the sample contains cells having the disease. In some embodiments, each of the plurality of expression constructs includes 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 has a length of at least 24 bps, at least 45 bps, at most 450 bps, at most 250 bps, a length of 24 - 300 bps, a length of 45 - 450 bps, or any combination thereof.

[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 includes a plurality of different template polynucleotides representing at least 5% to a maximum of 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 the 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 the proteome. In some embodiments, the exosome or proteome is the exosome or proteome of diseased cells. 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 polypeptides that are attached to an affinity tag. In some embodiments, the library comprises at least 100, 1000, 10000, 100000, 1×10 6 individuals, 1×10 7 individuals, 1×10 8 individuals, 1×10 9 individuals or up to 1×10 10 individuals of different template polynucleotide sequences. In some embodiments, the different template polynucleotides encode peptide sequences of at least 10, 50, 100, 500, 1000, 10000 or 100000 different proteins.

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

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

[0137] In some embodiments, the first polynucleotide sequence further comprises an upstream adapter sequence immediately upstream of a different template polynucleotide sequence and / or a downstream adapter sequence immediately downstream of a 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, or an f1 phage vector, or an fd phage vector.

[0141] In some embodiments, the polypeptides of the library are expressed as part of virus-like particles (VLPs). In some embodiments, the VLP-like particles can be expressed in a host cell, such as a bacterial cell, such as an Escherichia coli cell. In some embodiments, the VLPs self-assemble 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 concentrated at least via 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 comprise 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, and each of the polypeptide sequences encoded by the plurality of at least 10 different template polynucleotides, in the N-to-C direction, (i) a polypeptide sequence encoded by a different template polynucleotide of the plurality of 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 comprising a sequence encoding an affinity tag, and / or a size enhancing polypeptide sequence of at least 40 kDa, an individualized recombinant proteome library.

[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 comprise 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, an individualized recombinant proteome library.

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

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

[0149] In one aspect, provided herein is a pharmaceutical composition comprising the above polypeptide library or individualized 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 above pharmaceutical composition.

[0151] In one aspect, provided herein is a cell population comprising the polypeptide library or individualized 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 an expression vector, 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; (e) expressing the polypeptides encoded by the library of recombinant expression constructs; and concentrating the expressed polypeptides.

[0153] In some embodiments, the method comprises contacting a plurality of different polynucleotides with a library of exosome capture oligonucleotides, the capture oligonucleotides comprising a target sequence.

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

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

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

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

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

[0159] In some embodiments, selectively enriching comprises contacting the double-stranded polynucleotides with an agent that specifically binds to double-stranded polynucleotides containing 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 derived from bacterial species including, but not limited to, E coli, Salmonella sp., Haemophilus sp., Azotobater sp., Acinetobacter sp., Bacillus sp., Borrelia sp., Chlamydia sp., Helicobacter sp., Neisseria sp., Deinococcus radiodurans, and Streptococcus sp.

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

[0163] In some embodiments, the method further includes (i) combining a selectively enriched double-stranded polynucleotide containing a mismatch with (ii) a plurality of different template polynucleotides from a sample containing diseased cells from a subject having a non-selectively enriched disease.

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

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

[0166] In some embodiments, the plurality of different target polynucleotides include sequences derived from the whole genome.

[0167] In some embodiments, the plurality of different target polynucleotides include sequences derived from a set of whole genome sequences enriched with a library of exon 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 a subject.

[0170] In some embodiments, the library of exome capture oligonucleotides comprises high-frequency human polymorphic sequences.

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

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

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

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

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

[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 an affinity tag containing an expressed polypeptide.

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

[0180] In some embodiments, the method further comprises shearing the 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 to a subject a concentrated expressed polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0183] The features of the present disclosure are set forth in detail 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 which sets forth exemplary embodiments in which the principles of the disclosure are utilized, and the accompanying drawings.

[0184]

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[0185] The present disclosure focuses on important aspects of therapeutic agent development involving peptides or nucleic acids encoding therapeutic peptides, and includes modifications of peptides for stability and target delivery within the recipient of the therapeutic agent. In some aspects, the peptide is modified due to increased immunogenicity. In some aspects, the peptide is a neoantigen peptide. In some aspects, the peptide is a neoantigen for treating a disease in a subject, and the disease is an immune disease. In some aspects, the disease is cancer. In some aspects, the modification includes a peptide fusion. Provided herein are methods and pharmaceutical compositions related to the delivery of neoantigens in a fusion form. Furthermore, the neoantigen peptide can be efficiently delivered to lymph nodes in a fusion form, thereby exposing and priming a greater number of naive T lymphocyte populations to the neoantigen peptide.

[0186] In one aspect, described herein is a highly modular protein fusion technology named polybody that enables efficient delivery, tissue uptake, and functionality of neoantigen peptides. In some embodiments, the polybody protects the neoantigen peptide from enzymatic digestion in serum after subcutaneous injection. In some embodiments, the polybody helps target the neoantigen peptide to lymph nodes. In some embodiments, the polybody helps retain the neoantigen peptide in lymph nodes. In some embodiments, the polybody helps in the effective priming and activation of T lymphocytes.

[0187] In some embodiments, the modular protein fusion technology enables 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 scaffold domains. In some embodiments, these scaffold domains keep the antigen peptide more soluble and prevent premature degradation caused by the patient's circulating proteases and peptidases. In some embodiments, these scaffold domains are functionalized to add desired functions to the polybody to further increase the immunogenicity and anti-tumor efficacy of the vaccine.

[0188] Described herein are novel immunotherapeutic agents and their use based on the discovery of neoantigens arising from mutation events specific to an individual's tumor. Accordingly, the present disclosure described herein can be used, for example, to stimulate an immune response against tumor-associated antigens or neoepitopes and to produce immunogenic compositions or cancer vaccines for use in the treatment of diseases, by providing peptides, polynucleotides encoding the peptides, and peptide-binding agents.

[0189] The following description and examples illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and is thus modifiable. Those skilled in the art will recognize that numerous variations and modifications of the present disclosure exist within the scope of the present disclosure.

[0190] All terms are intended to be understood as would be 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 purposes of organization only and should not be construed as limiting the subject matter described.

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

[0193] The various technical terms used in this specification are for the sole purpose of describing specific instances and are not intended to be limiting. In this application, unless otherwise specified, the use of the singular form includes the plural form. As used in this specification, the singular forms "a", "an", and "the" are intended to include the plural as well, unless the context clearly indicates otherwise. As understood in this specification, "or" means "and / or" unless otherwise specified. The terms "and / or" and "any combination thereof", as well as their grammatical equivalents used in this specification, may be used interchangeably. These terms can convey that any combination is specifically contemplated. Merely for illustrative purposes, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "individually A, individually B, individually C, A and B, B and C, A and C, as well as A, B, and C". The term "or" can be used conjunctively or disjunctively, except when the context specifically indicates a 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 one standard deviation or more than one standard deviation according to the conventions in the relevant technical field. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, this term can mean within one order of magnitude, within fivefold, more preferably within twofold of the value. When a specific value is recited in this application and the claims, the term "about", meaning within an acceptable error range for the specific value, should be assumed unless otherwise stated.

[0195] As used in this specification and the claims (if any), the words "comprising" (and any form of comprising, 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. Any embodiment discussed herein is contemplated to be implemented with respect to any method or composition of the present disclosure, and vice versa. Further, the methods of the present disclosure can be achieved using the compositions of the present disclosure.

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

[0197] The major histocompatibility complex or "MHC" is a cluster of genes that plays a role in the control of cell interactions that underlie the physiological immune response. 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 rdSee Ed., Raven Press, New York (1993). "Major histocompatibility complex (MHC) proteins or molecules", "MHC molecules", "MHC proteins" or "HLA proteins" are to be understood as meaning proteins that can represent potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes) that bind to peptides resulting from the proteolytic cleavage of protein antigens, transport them to the cell surface, and present them there to specific cells, in particular cytotoxic T lymphocytes, T helper cells, or B cells. The major histocompatibility complex in the genome contains a gene region where gene products expressed on the cell surface are important for the binding and presentation of endogenous and / or exogenous antigens and are thus important for regulating immunological processes. The major histocompatibility complex is classified into two groups of genes encoding different proteins, namely, MHC class I molecules and MHC class II molecules. The cell biology and expression patterns of the two MHC classes are adapted to these different roles.

[0198] Human leukocyte antigen or "HLA" is a human class I or class II major histocompatibility complex (MHC) protein (e.g., Stites, et al., Immunology, 8 th See Ed., Lange Publishing, Los Altos, Calif. (1994).

[0199] As used herein, "polypeptide," "peptide," and their grammatical equivalents refer to polymers of amino acid residues. A polymer of amino acid residues is typically a series of amino acid residues connected to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acid residues. The polypeptides and proteins (including functional portions and functional variants thereof) disclosed herein can include 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 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 the expression of the polypeptides described herein in engineered cells can 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, acetylation and acylation including formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylization, 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 a peptide that contains an allele-specific motif such that the peptide binds to an HLA molecule and induces a cell-mediated or humoral response, such as a cytotoxic T lymphocyte (CTL (e.g., CD8 + )), helper T lymphocyte (Th (e.g., CD4 + )) and / or B lymphocyte response. Thus, the immunogenic peptides described herein can bind to an appropriate HLA molecule and then induce a CTL (cytotoxic) response, or an HTL (and humoral) response to the peptide.

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

[0202] The terms "peptide" and "polypeptide" are used interchangeably and typically refer to a series of residues of L-amino acids, typically connected to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. In some embodiments, a polypeptide comprises a single translation product that includes multiple distinct epitopes. In some embodiments, a polypeptide comprises a single translation product that includes a single distinct peptide, such as an epitope or a neoepitope, 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 can be a branched multipeptide structure. A polypeptide or peptide can be of any length, either in their neutral (uncharged) form or as salts, and can contain no modifications, such as glycosylation, side chain oxidation, or phosphorylation, or can contain these modifications 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 neoantigenic peptide that is not part of a neoepitope. The terms "variant polypeptide", "neoantigenic polypeptide", "neoantigenic polypeptide", "variant peptide", "neoantigenic peptide" and "neoantigenic peptide" are used interchangeably and refer to a peptide or polypeptide that contains a mutation.

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

[0204] "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 can include at least one "anchor residue" and at least one "anchor residue adjacent region". A neoepitope may further include a "separation region". The term "anchor residue" refers to an amino acid residue that binds to a specific pocket on HLA and confers specificity of interaction with HLA. In some cases, the anchor residue can be in a standard anchor position. In other cases, the anchor residue may be in a non-standard anchor position. A neoepitope can bind to an HLA molecule through primary and secondary anchor residues that protrude into pockets within the peptide-binding groove. In the peptide-binding groove, specific amino acids form pockets that accommodate the corresponding side chains of the anchor residues of the presented neoepitope. There is a preference for peptide binding between different alleles of both HLA I and HLA II molecules. HLA class I molecules bind to short neoepitopes, and their N- and C-termini are fixed in pockets located at the ends of the neoepitope-binding groove. Most HLA class I-binding neoepitopes are about 9 amino acids in length, but longer neoepitopes can be accommodated by bulges in their central portions, resulting in binding neoepitopes of about 8 - 12 amino acids. Neoepitopes that bind to HLA class II proteins are not size-restricted and can vary in length up to about 16 - 25 amino acids. The neoepitope-binding groove within HLA class II molecules is open at both ends, allowing peptide binding over a relatively long length. A core segment of about 9 amino acid residues contributes most to the recognition of the neoepitope, but anchor residues adjacent to this region are also important for the specificity of the peptide for HLA class II alleles. In some cases, the anchor residue adjacent to the core region contributing to peptide specificity is present at the N-terminus of the core region residues. In other cases, the anchor residue adjacent to the core region is present at the C-terminus of the core region residues.In yet another case, the anchor residues adjacent to the core region that contribute to the peptide specificity for HLA are at both the N-terminus and C-terminus of the core region residues.

[0205] "Reference" can be used to correlate and compare the results obtained by the methods of the present disclosure from tumor specimens. Typically, the "reference" can be based on one or more normal specimens, especially specimens not affected by cancer disease, obtained from a patient or one or more different individuals, such as healthy individuals, especially individuals of the same species. The "reference" can be determined experimentally by testing a sufficiently large number of normal specimens.

[0206] An "epitope" is the collective features of molecules such as primary, secondary, and tertiary peptide structures, as well as charge, which together form the site recognized by, for example, immunoglobulins, T cell receptors, HLA molecules, or chimeric antigen receptors. Alternatively, an epitope can be defined as a set of amino acid residues involved in recognition by a specific immunoglobulin, or in the context of T cells, as a set of amino acid residues necessary for recognition by a T cell receptor protein, a chimeric antigen receptor, and / or a major histocompatibility complex (MHC) receptor. A "T cell epitope" means a peptide sequence that can be bound by a class I or II MHC molecule in the form of an MHC molecule or MHC complex presenting the peptide, and then, in this form, can be recognized and bound by a T cell such as a T lymphocyte or a T helper cell. 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 mimics" 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, in some cases, be referred to as peptides or peptide epitopes. It should be understood that the epitopes or analogs described herein, and proteins or peptides containing additional amino acid(s), are still within the scope of this disclosure. In certain embodiments, the peptide contains a fragment of an antigen. In certain embodiments, there is a limitation on the length of the peptides of this disclosure. Embodiments where the length is limited occur when a protein or peptide containing an epitope described herein contains a region (i.e., a continuous series of amino acid residues) having 100% identity to a native sequence.

[0207] The nomenclature used to describe peptides or proteins follows the conventional practice in which the amino group is presented on the left side (amino terminus or N-terminus) of each amino acid residue and the carboxyl group is presented on the right side (carboxyl terminus or C-terminus) of each amino acid residue. 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 a peptide or protein that can be part of it. In the formulas representing certain selected embodiments of the present disclosure, although not specifically shown, the amino-terminal group and the carboxyl-terminal group are in the form assumed at physiological pH values unless otherwise specified. In amino acid structural formulas, each residue is generally represented by the standard three-letter or one-letter notation, but when using three-letter symbols or full names without capital letters, they can refer to L-amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or "G". The amino acid sequences of the peptides shown herein are generally specified using the standard single-letter symbols. (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) as compared to a reference. A somatic mutation is a genetic change acquired by a cell that can be passed on to the progeny of the mutant cell during the process of cell division. Somatic mutations are distinct from germline mutations, which are genetic changes that occur 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 nucleotide substitution that results in an amino acid change, such as an amino acid substitution in a 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 an unnatural protein sequence. Different mutations in a gene can achieve the same altered reading frame.

[0209] A conservative amino acid substitution is one in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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), nonpolar 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, such as between an HLA-binding peptide and class I or II HLA. K Dis the dissociation constant and has the unit of molar concentration. The affinity constant is the reciprocal of the dissociation constant. The affinity constant may be used as a general term to describe this chemical substance. It is a direct measure of the energy of binding. Affinity can be determined experimentally, for example, by surface plasmon resonance (SPR) using commercially available Biacore SPR units. Affinity can also be expressed as the inhibitory concentration 50 (IC 50 ), at which concentration 50% of the peptide is displaced. Similarly, ln(IC 50 ) refers to the natural logarithm of IC 50 . K off refers to the off-rate constant, for example, for the dissociation of HLA-binding peptides and class I or II HLA. Throughout this disclosure, the results of "binding data" can be expressed from the perspective of "IC 50 ". IC 50 is the concentration of the tested peptide in a binding assay at which 50% inhibition of the binding of the labeled reference peptide is observed. Considering the conditions under which the assay is performed (i.e., limiting the HLA protein and labeled reference peptide concentrations), these values approximate the K D values. Assays for determining binding are well known in the art and are described in detail in other publications such as PCT Publication WO 94 / 20127 and WO 94 / 03205, as well 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 as compared to the binding by a reference standard peptide. For example, compared to the IC 50 of the reference standard peptide, its IC 50It can be based on. Binding can also be determined using other assay systems including those that use: 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., 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-throughput 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., J. Immunol. 149:1896 (1992)). "Cross-reactive binding" is evident in some peptides, i.e., the peptide shows promiscuous binding to two or more HLA molecules.

[0211] When used to discuss epitopes, the term "derived" and its grammatical equivalents are synonyms for "prepared" and its grammatical equivalents. A derived epitope can be isolated from a natural source or it can be synthesized according to standard protocols in the art. Synthetic epitopes can include "amino acid mimics", which are artificial amino acid residues such as D-isomers of naturally occurring L-amino acid residues or non-natural amino acid residues such as cyclohexylalanine. A derived or prepared epitope can be an analog of a natural epitope.

[0212] "Diluent" includes water and sterile liquids such as oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is also a diluent for pharmaceutical compositions. Physiological saline as well as aqueous solutions of dextrose and glycerol can also be used as diluents, for example, in injection solutions.

[0213] A "natural" or "wild-type" sequence refers to a sequence found in nature. Such a sequence can, in essence, include longer sequences.

[0214] "Receptor" should be understood to mean a biomolecule or group of molecules that can bind a ligand. A receptor can play a role in transmitting information in a cell, cell formation or organism. A receptor includes, for example, at least one receptor unit, where each receptor unit can consist of a protein molecule. A receptor has a structure that complements the structure of the ligand and can complex the ligand as a binding partner. 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, a receptor should be understood to mean a specific protein of MHC class I and II that can form a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of a suitable length.

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

[0216] In some embodiments, "receptor / ligand complex" should also be understood to mean "receptor / peptide complex" or "receptor / peptide fragment complex" that includes 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, such as an artificial peptide. Such peptides can be produced using methods such as chemical synthesis or recombinant DNA technology. "Synthetic peptide" includes "fusion protein".

[0218] The term "motif" refers to a pattern of residues of a peptide of defined length, for example, for a class I HLA motif, less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, such as about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13), and for a class II HLA motif, 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), which 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 patterns of primary and secondary anchor residues. In some embodiments, the MHC class I motif specifies a peptide that is 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 a living organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in the laboratory is naturally occurring.

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

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

[0222] "Antigen processing" or "processing", and its grammatical equivalents, refer to the breakdown of a polypeptide or antigen into process products that are fragments of the polypeptide or antigen (e.g., the breakdown of a polypeptide into peptides), and the association of one or more of these fragments (e.g., by binding) with MHC molecules for presentation by cells, such as antigen-presenting cells, 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 surfaces. Some APCs can activate antigen-specific T cells. Professional antigen-presenting cells are very efficient in internalizing antigens either by phagocytosis or receptor-mediated endocytosis and then displaying fragments of the antigens 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. Subsequently, additional co-stimulatory signals are generated by the antigen-presenting cell, leading to the activation of T cells. The expression of co-stimulatory molecules is a defining feature 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 the MHC class II and I antigen presentation pathways. It is well known that dendritic cells are powerful inducers of immune responses, and the activation of these cells is an important step for the induction of anti-tumor immunity. Dendritic cells are conveniently classified as "immature" and "mature" cells, which can be used as a simple way to distinguish 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 high capacity for antigen uptake and processing, correlated with high expression of Fc receptors (FcR) and mannose receptors. The mature phenotype typically features lower expression of these markers but high expression of cell surface molecules involved in T cell activation, such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB).

[0224] In the context of two nucleic acid or amino acid sequences of a polypeptide, as used herein, the terms "identical" and its grammatical equivalents, or "sequence identity", refer to residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. As used herein, an exemplary "comparison window" refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150, with the sequences being compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of aligning sequences for comparison are well known in the art.Optimal alignment of arrays for comparison can be done 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. U.S.A., 85:2444 (1988), 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., U.S.A.), the CLUSTAL program is well 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), as well as Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Also, alignment is often carried out 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 by BLASTP (or CLUSTAL, or any other available alignment software), using, for example, default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, and for example, 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 by BLASTN (or CLUSTAL, or any other available alignment software), using, for example, default parameters. When one molecule is said to have a certain percentage of sequence identity to a larger molecule, it means that when the two molecules are optimally aligned, that percentage of the residues in the smaller molecule find matching residues in the larger molecule, following the order in which the two molecules are optimally aligned.

[0225] "Substantially identical" and its grammatical equivalents as applied to a nucleic acid or amino acid sequence mean that the nucleic acid or amino acid sequence has 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 programs, e.g., BLAST, with standard parameters. For example, the BLASTN program (for nucleotide sequences) by default uses an 11-word length (W), an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program by default uses a 3-word length (W), an expectation value (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, and the portion of the polynucleotide sequence within the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences, obtaining the number of matched positions, dividing the number of matched 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 a sequence that is at least about 50 residues in length, 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 full length of the coding region.

[0226] A vector generally refers to a construct that can deliver one or more genes and / or sequences of interest in a host cell and can usually be expressed. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

[0227] A polypeptide, antibody, polynucleotide, vector, cell, or composition that is "isolated" 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 are purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, the polypeptides, antibodies, polynucleotides, vectors, cells, or compositions that are isolated are substantially pure. In some embodiments, "isolated polynucleotide" includes polynucleotides amplified in a PCR or quantitative PCR reaction, including PCR or quantitative PCR reactions.

[0228] The terms "isolated" and "biologically pure", or their grammatical equivalents, refer to a material that substantially or essentially does not contain the components normally associated with the material as found in its natural state. Thus, an isolated peptide described herein does not contain some or all of the materials that normally associate with the peptide in its in situ environment. An "isolated" epitope refers to an epitope that does not include the entire sequence of the antigen from which the epitope is derived. Typically, an "isolated" epitope is not attached to additional amino acid residues that would result in a sequence having 100% identity over the full length of the native sequence. The native sequence can be a sequence such as a tumor-associated antigen from which the epitope is derived. Thus, the term "isolated" means that the material has been removed from its original environment (e.g., the natural environment if it occurs naturally). An "isolated" nucleic acid is a nucleic acid 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 materials coexisting in the natural system is isolated. Such a polynucleotide can be part of a vector and / or such a polynucleotide or peptide can be part of a composition, and such a vector or composition can still be "isolated" in that it 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 refer to nucleic acid sequences, polypeptides, proteins or other compounds that essentially do not contain polynucleotides, proteins, polypeptides, polypeptides, and other molecules that naturally associate therewith, i.e., contain no more than about 50%, no more than about 70%, no more than about 90%.

[0230] As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e., free of 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 and refer to polymers of nucleotides of any length, including DNA and RNA, such as mRNA. Thus, these terms include double-stranded and single-stranded DNA, triple-stranded DNA, as well as double-stranded and single-stranded RNA. Also included are, for example, modifications by methylation and / or capping, as well as unmodified forms of polynucleotides. This term also means that it includes molecules containing non-natural or synthetic nucleotides, as well as nucleotide analogs. Nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into cells, for example, by transfection, transformation, or transduction. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be mRNA transcribed in vitro. 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 a host cell by using physical or chemical methods. Many transfection techniques are known in the art, including, for example, calcium phosphate DNA coprecipitation (e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)), DEAE-dextran, electroporation, cationic liposome-mediated transfection, tungsten particle-facilitated microparticle 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 a host cell after the growth of infectious particles in suitable packaging cells, and many of them 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 acids and proteins in question, but a sequence identity of only 25% is commonly used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or levels exceeding that can also be used to establish homology. Methods for determining the percentage of sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.

[0234] The term "subject" refers to any animal (e.g., mammal) that should 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 with respect to human subjects.

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

[0236] "Treat" or "treatment" or "treating" or "alleviate" or "alleviating" refers to both (1) therapeutic means to cure, delay, reduce the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventive means to prevent or delay the onset of a targeted pathological condition or disorder. Accordingly, those in need of treatment include those already having a disorder, those having a tendency to have a disorder, and those in whom a disorder should be prevented.

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

[0238] "Pharmaceutical excipient" or "excipient" includes materials such as adjuvants, carriers, pH adjusters and buffers, isotonicity adjusters, 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 is a fusion polypeptide comprising a plurality of epitopes or antigens fused to a scaffold protein. The epitope is a short peptide that can be about 5 to 50 amino acids in length. The fusion polypeptide, or fusion protein, or "polybod" can be generated by combining a plurality of epitopes into one molecule. The polybod is useful, for example, for immunotherapy, to generate a therapeutic agent comprising an epitope, where the epitope is the active therapeutic component. An epitope alone can be rapidly degraded in a biological system without achieving its expected result, while the polybod form can be designed to provide advantages over a single epitope form. In a polybod, the scaffold protein stabilizes the neoantigen, improves the solubility of the epitope, increases the molecular weight to, for example, 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, polybods reduce the batch production of single peptides. The absence of post-translational modifications enables inexpensive and rapid recombinant production of the scaffold in E. coli. The scaffold is of relatively small size and has no known toxicity. In some embodiments, the scaffold protein is a human protein, thereby ensuring the lack of antigenicity. In some embodiments, a functional scaffold can be incorporated, for example, for dendritic cell targeting or activation. A ubiquitin scaffold can enhance proteasomal degradation and antigen processing. In some embodiments, the scaffold protein can be further modified, for example, to include a cell targeting moiety.

[0242] Fusion polypeptide 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 a nucleic acid molecule encoding the fusion polypeptide, and a composition comprising the fusion polypeptide or the nucleic acid molecule. In some embodiments, the fusion polypeptide comprises a polypeptide sequence that includes 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 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 a 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. The fusion polypeptide can be configured to take 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 the remainder of the fusion polypeptide in a variety of ways. For example, the scaffold polypeptide 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 remainder of the fusion polypeptide via its N-terminus. In some embodiments, at least one of the scaffold polypeptide sequences is linked to the remainder of the fusion polypeptide via its C-terminus. In some embodiments, each of the one or more scaffold polypeptides is linked to the remainder of the fusion polypeptide via its N-terminus, C-terminus, or both termini. 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 a polypeptide sequence having the structure of formula (Ia) in the N-terminus to C-terminus direction, A n -S m or S m -A n Formula (Ia), In the formula, each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, m is an integer of 1 or more, and n is an integer of 1 or more. In some embodiments, each of m and n is an integer independently selected from 1 to 10. In some embodiments, each of m and n is an integer independently selected from 1 to 5. In some embodiments, each of m and n is an integer independently selected from 1 to 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 a structure of A-A-S, A-A-A-S, A-A-A-A-S, A-A-A-A-A-S, A-A-A-A-A-A-S, or A-A-A-A-A-A-A-S in the direction from the N-terminus to the C-terminus. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of S-A-A, S-A-A-A, S-A-A-A-A, S-A-A-A-A-A, S-A-A-A-A-A-A, or S-A-A-A-A-A-A-A in the direction from the N-terminus to the C-terminus. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of A-S, A-S-S, A-S-S-S, A-S-S-S-S, A-S-S-S-S-S, or A-S-S-S-S-S-S in the direction from the N-terminus to the C-terminus. In some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of S-A, S-S-A, S-S-S-A, S-S-S-S-A, S-S-S-S-S-A, or S-S-S-S-S-S-A in the direction from the N-terminus to the C-terminus.

[0246] In some embodiments, the fusion polypeptide comprises a polypeptide sequence having the structure of formula (Ib) in the direction from the N-terminus to the C-terminus, 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 is independently an integer of 1 or more. In some embodiments, each of o, n, and m is independently 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 to 5. 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, o and m are equal to 1, and n is an integer from 1 to 5. In some embodiments, the fusion polypeptide has a polypeptide sequence having a structure of S-A-S, A-S-A-S, S-A-S-A, S-A-S-A-S, S-A-S-A-S-A-S, S-A-S-A-S-A-S-A-S, or S-A-S-A-S-A-S-A-S-A-S in the direction from the N-terminus to the C-terminus. In some embodiments, the fusion polypeptide has a polypeptide sequence having a structure of S-A-A-S, S-A-A-A-S, S-A-A-A-A-S, S-A-A-A-A-A-S, S-A-A-A-A-A-A-S, or S-A-A-A-A-A-A-A-S in the direction from the N-terminus to the C-terminus.

[0247] In some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of formula (IIa) in the direction from the N-terminus to the C-terminus, (L-A-L) n -S m or S m -(L-A-L)n formula (IIa),

[0248] In the formula, 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 of 1 or more, and n is an integer of 1 or more. In some embodiments of formula (IIa), m is an integer of 2 or more. 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 has a polypeptide sequence having a structure of A-L-S, A-L-A-L-S, A-L-A-L-A-L-S, S-L-A, S-L-A-L-A, or S-L-A-L-A-L-A in the direction from the N-terminus to the C-terminus.

[0249] In some embodiments, the fusion polypeptide has a polypeptide sequence having a structure of formula (IIb) in the direction from the N-terminus to the C-terminus, S o -(L-A-L) n -S m Formula (IIb),

[0250] In the formula, 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 is independently an integer of 1 or more. 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 has a polypeptide sequence having a structure of S-L-A-L-S, S-A-L-S-L-A-L-S, S-L-A-L-S-L-A, S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S-L-A-S, or S-L-A-L-S-L-A-L-S-L-A-L-S-L-A-S-L-A-L-S in the direction from the N-terminus to the C-terminus.

[0251] In some embodiments, the scaffold polypeptide and the antigen polypeptide are positioned within the fusion polypeptide in an alternating fashion, with or without a linker sequence therebetween. For example, in some embodiments, the fusion polypeptide comprises a polypeptide sequence having a structure of S-A-L-S-L-A-L-S, A-L-S-L-A-L-S-L-A, A-S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S-A, A-S-A-S-L-A-L-S, or S-L-A-L-S-L-A-L-S-A-S-A in the N-terminal to C-terminal direction. 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 polypeptide can result in improved lymph node accumulation or retention compared to the corresponding polypeptide lacking the scaffold domain. In some embodiments, the accumulation of the fusion polypeptide in the 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 radiance efficiency. In some embodiments, the accumulation of the fusion polypeptide in the 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 radiance efficiency. For example, lymph node accumulation can be measured according to the method described in Example 3.1.

[0253] The described fusion polypeptide can have improved solubility in an aqueous solvent as compared to the corresponding polypeptide lacking the scaffold domain. In some embodiments, the solubility of the 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 the corresponding polypeptide lacking the scaffold polypeptide, measured in the same solvent. 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 higher than the solubility of the corresponding polypeptide lacking the scaffold polypeptide. The described fusion polypeptide can have improved stability, e.g., serum stability, as compared to the corresponding polypeptide lacking the 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 the 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 the corresponding polypeptide lacking the scaffold polypeptide. The described fusion polypeptide can have improved solubility as 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 higher than the serum solubility of the corresponding polypeptide lacking the scaffold polypeptide.

[0254] The described fusion polypeptide can induce an enhanced immune response compared to the corresponding polypeptide lacking a 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 its corresponding polypeptide lacking a scaffold polypeptide, as measured by an increase in splenocyte proliferation. For example, the antigen-specific T cell response can be measured according to the method of Example 3.2.

[0255] Scaffold polypeptide or scaffold domain 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 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different scaffold polypeptide sequences.

[0256] In some embodiments, one or more scaffold polypeptides comprise recombinant human polypeptides. In some embodiments, one or more scaffold polypeptides comprise sequences derived from human proteins. In some embodiments, one or more scaffold polypeptides comprise sequences having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to wild-type mammalian proteins such as human proteins. 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 polypeptides lack post-translational modifications. In some embodiments, the scaffold polypeptides lack 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, the 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, the fusion polypeptide comprises at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 scaffold polypeptides. In some embodiments, the fusion polypeptide comprises 2-6 scaffold polypeptides. In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 scaffold polypeptides. In certain embodiments, the 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 an amino acid 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, such as a human protein that can be suitably expressed in a bacterial expression system such as an E. coli expression system, the protein is small, stable, and is either a secreted protein or a cytoplasmic protein. In some embodiments, the scaffold protein is a human protein. Mammalian cells contain an estimated 1 billion protein molecules. Most of these proteins can be found to function as scaffold proteins that help enhance and support biological molecule interactions in the body, and this interaction promotes aspects of cell behavior such as the exertion of signal transduction, intracellular transport of biomolecules, enzyme activity, or structural remodeling that promotes cell motility. In the present disclosure, the inventors provide an efficient and concise method of using a human scaffold protein to convert a short epitope peptide into a means for generating an active immune response. 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 structural advantages in conferring immunogenicity, for example, by increasing the availability for association with cognate MHC molecules. The scaffold protein can be selected such that it is non-toxic. The scaffold protein can be any protein having a scaffolding function or ability that meets any or all of these purposes.

[0261] For example, known cellular scaffold proteins assist other functional proteins in reducing the entropy of the reaction. In some embodiments, the scaffold protein serves to link one or more proteins, enzymes, peptides, or other biomolecules together to increase local concentration or to temporarily immobilize them for functional outcome. For example, the Cullin scaffold protein links E2 ubiquitin together, and proteins such as the linker for activation of T cells (LAT) and the 76 kD SH2 domain-containing leukocyte protein (SLP-76) serve to organize TCR signal transduction.

[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 comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the human Stefin A protein sequence.

[0263] In some embodiments, the one or more scaffold polypeptides comprise a sequence of a mammalian titin protein, a variant thereof, or a fragment thereof. The sequence of the titin protein can be a sequence from any domain of the titin protein, such as an Ig-like domain, an FnIII-like domain, and a pseudo-kinase domain. In some embodiments, the sequence of the titin protein is the sequence of the I-band. In some embodiments, the one or more scaffold polypeptides comprise a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the titin Ig-like domain sequence. In some embodiments, the sequence from the titin protein is an I27 domain sequence, an I1 domain sequence, a Z1 domain sequence, a Z2 domain sequence, or an M5 domain sequence. In some embodiments, the sequence from the titin protein is an I27 domain sequence. In some embodiments, the 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 the sequence of a glycoprotein, such as fibronectin, variants thereof, or fragments thereof. In some embodiments, the glycoprotein is fibronectin. The sequence of fibronectin can be from any of its subunits, such as the sequence from fibronectin type I, type II, or type III domains. 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 sequence of FNIII is the 10FN-III domain sequence.

[0265] In some embodiments, one or more scaffold polypeptides comprise a polypeptide sequence selected from isolated protein folds, such as thioredoxin, 10FNIII, lipocalin, the capsid polypeptide of adeno-associated virus, an α-amylase inhibitor, Stefin A, ubiquitin, Ig-L filamin A, tenascin, inactivated staphylococcal nuclease, green fluorescent protein, the Z domain of staphylococcal protein A, ankyrin repeats, villin-binding proteins, fragments thereof, and variants thereof. In some embodiments, each of the one or more scaffold polypeptides is 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 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 fragment, or a specific domain. In some embodiments, the scaffold can be a Protein A domain, Src homology domain, PDZ domain, WW domain, zinc finger domain, or derivatives thereof.

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

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

[0269] In some embodiments, the fusion polypeptide includes a plurality of 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] One or more antigen polypeptides can include any antigen polypeptide that induces an immune response, such as an exogenous antigen, an endogenous antigen, a self-antigen, 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 activates CD8 + T cells. In some embodiments, at least one antigen activates CD4 + T cells. In some embodiments, one or more antigen polypeptides include a first antigen polypeptide and a second antigen polypeptide. In some embodiments, the first antigen polypeptide binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second antigen polypeptide binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second antigen polypeptide binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first antigen polypeptide binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, a single polypeptide can include a class I HLA-binding epitope and a class II HLA-binding epitope. In some embodiments, the first antigen polypeptide activates CD8 + T cells. In some embodiments, the second antigen polypeptide activates CD4 +Activates T cells. In some embodiments, the linker sequence is directly connected to the scaffold polypeptide sequence and the antigen polypeptide sequence. In some embodiments, the linker sequence is directly connected to the scaffold polypeptide sequence and another linker sequence. In some embodiments, the linker sequence is directly connected to the antigen polypeptide sequence and another linker sequence. In some embodiments, the linker sequence is directly connected to two scaffold polypeptide sequences. In some embodiments, the linker sequence is directly connected to two antigen polypeptide sequences. In some embodiments, the linker sequence is directly connected to two other linker sequences. In some embodiments, the linker sequence can be directly connected to three or more of the following: scaffold polypeptide sequence(s), antigen polypeptide sequence(s), 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 5 to 20 amino acid residues. In some embodiments, the linker sequence is non-immunogenic. In some embodiments, the linker sequence is not configured to form disulfide bonds. 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, a flexible linker comprises small non-polar amino acids such as Gly, Ser, and Thr. In some embodiments, a flexible linker comprises a stretch of Gly and Ser residues such as Gly-Gly-Gly-Gly-Ser. In some embodiments, a flexible linker comprises the sequence KESGSVSSEQLAQFRSLD. In some embodiments, a flexible linker comprises the sequence EGKSSGSGSESKST. In some embodiments, a flexible linker comprises the sequence (Gly)z, where z is an integer from 5 to 10. In some embodiments, a flexible linker comprises the sequence (Gly)8. In some embodiments, a 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, a rigid linker can form 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 can 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. A cleavable linker can include one or more cleavable sites. In some embodiments, at least one of the linker sequences includes a cleavage site. In some embodiments, the cleavage site is cleavable by a cellular reducing agent. For example, the cleavable site can include 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 the presentation of at least one of the one or more antigen polypeptides. In some embodiments, the linker comprises a context sequence capable of promoting the presentation of the antigen 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 antigen 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 antigen 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 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 antigen polypeptides. In some embodiments, 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.

[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, to improve the bioactivity of the fusion polypeptide, or to 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 function 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, agents, drugs, proteins or polypeptides, and cells such as antigen-presenting cells. In some embodiments, the fusion polypeptide includes one or more binding moieties configured to bind to an antigen-presenting cell, an adjuvant, or a reagent. In some embodiments, the antigen-presenting cell is a dendritic cell (DC), a macrophage, a Langerhans cell, or a B cell.

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

[0277] In some embodiments, one or more of the receptors include 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 a mannose receptor, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (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 group receptor-1, myeloid inhibitory C-type lectin receptor, C-type lectin-like receptor 2 (CLEC2), CLEC12B receptor, lectin-like receptor for oxidized low density lipoprotein-1, DC immunoreceptor subfamily receptor, DC immunoreceptor, Dectin-2 receptor, or blood DC antigen.

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

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

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

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

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

[0285] In some embodiments, in the direction from the N-terminus to the C-terminus, at least one of the binding moieties is terminally linked to the first or last scaffold polypeptide 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 is a fusion polypeptide encoded by the described nucleic acid molecule.

[0290] In one aspect, disclosed herein is a composition comprising the described fusion polypeptide. In some embodiments, the composition comprises one or more binding moieties that can conjugate to the fusion polypeptide. In some embodiments, the 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 apart by one or more linkers and one or more restriction sites located in at least one of the linkers, 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.

[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 antigen polypeptide and a nucleic acid sequence encoding a first scaffold polypeptide, and a second nucleic acid molecule comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the first antigen polypeptide and a nucleic acid sequence encoding a second scaffold polypeptide.

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

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

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

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

[0297] In some embodiments, the first, second, and third antigen 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 the described composition or the described fusion polypeptide.

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

[0301] In some embodiments, the adjuvant is poly IC: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, a cytokine, a chemokine, or a checkpoint inhibitor.

[0304] In one aspect, disclosed herein is a method of producing an immunogenic fusion polypeptide, the method comprising expressing the 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 the nucleic acid molecule described herein; inserting one or more nucleic acid molecules encoding one or more antigen 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, the one or more nucleic acid molecules encoding one or more antigen 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, the method comprising administering to the subject in need thereof a pharmaceutical composition described herein.

[0311] In some embodiments, the pharmaceutical composition comprises a plurality of neoantigen peptides.

[0312] In some embodiments, the pharmaceutical composition comprises a plurality of fusion polypeptides.

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

[0314] In some embodiments, the dosage of the fusion polypeptide is divided into at least two, at least three, at least four, or at least five sub-dosages.

[0315] In some embodiments, each sub-dosage of the fusion polypeptide comprises one, two, three, four, five, or more than five fusion polypeptides.

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

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

[0318] In some embodiments, the total dosage of the fusion polypeptide administered is 0.01 - 100 mg.

[0319] Neoantigens and their uses One of the important barriers to developing curative and tumor-specific immunotherapies is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens resulting from genetic changes (e.g., inversions, translocations, deletions, missense mutations, splice-site mutations, etc.) within malignant cells represent the most tumor-specific class of antigens. Neoantigens are rarely used in cancer vaccines or immunogenic compositions due to the technical difficulties in identifying them, selecting optimized antigens, and generating neoantigens for use in vaccines or immunogenic compositions. These problems can be addressed by identifying mutations in neoplasms / tumors that are present at the DNA level in tumors but not in matching 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 neoantigen T cell epitopes that are expressed within the neoplasm / tumor and bind to a large proportion of the patient's HLA alleles, and synthesizing multiple neoantigenic peptides selected from the set of all neoantigen 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 can bind to HLA molecules of a large proportion of individuals. Efficiently selecting which specific mutations to utilize as immunogens requires the ability to predict which mutant peptides will efficiently bind to a large proportion of the patient's HLA alleles. Recently, a neural network-based learning approach with validated binding and non-binding peptides has improved the accuracy of prediction algorithms for major HLA-A and -B alleles. However, even using advanced 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 the drug as a multi-epitope vaccine of a long peptide. Targeting as many variant epitopes as is practically possible takes advantage of the vast capabilities of the immune system, prevents opportunities for immune evasion by downregulating immune target gene products, and compensates for the known inaccuracies of epitope prediction approaches. Provided herein is a polypeptide comprising a plurality of epitopes for generating a therapeutic product, or a vaccine, wherein the polypeptide is expressed from a polynucleotide in a host cell or by in vitro translation. In some embodiments, the polypeptide can be synthetically produced. As described, a polypeptide comprising a plurality of epitopes can further comprise one or more scaffold proteins. In some embodiments, provided herein is a synthetic polybody comprising a plurality of epitopes, a scaffold protein, and a linker for use as a therapeutic vaccine.

[0322] Yet another example of converting peptide sequencing information into a therapeutic vaccine may involve a combination with a potent vaccine adjuvant. An effective vaccine may require a potent adjuvant to initiate an immune response. For example, poly ICLC, which is an agonist of TLR3 and the RNA helicase domains of MDA5 and RIG3, exhibits several desirable properties for a vaccine adjuvant. These properties include induction of local and systemic activation of immune cells in vivo, production of stimulatory chemokines and cytokines, and stimulation of antigen presentation by DCs. Further, poly ICLC elicits durable CD4 + and CD8 +Responses can be induced. Importantly, significant similarities in the upregulation of transcription and signaling pathways were seen in subjects vaccinated with poly-ICLC and volunteers who received a highly effective replicating yellow fever vaccine. Additionally, over 90% of ovarian cancer patients immunized with poly-ICLC in combination with (in addition to Montanide) the NYESO-1 peptide vaccine showed, in a recent Phase I trial, not only an antibody response to the peptide but also CD4 + and CD8 + T cell induction. At the same time, poly-ICLC has been extensively tested in over 25 clinical trials to date and has shown a relatively favorable toxicity profile.

[0323] In some aspects, provided herein is a composition comprising a first peptide comprising a first novel epitope of a protein and a second peptide comprising a second novel 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 novel epitope in a complex with an HLA protein, and a second TCR specific for the second novel epitope in a complex with an HLA protein, wherein the first peptide is different from the second peptide, the first novel epitope comprises a mutation, and the second novel epitope comprises the same mutation.

[0324] In some embodiments, provided herein is a first peptide comprising a first novel epitope of a region of a protein and a second peptide comprising a second novel epitope of the same region of the protein, wherein the first novel epitope and the second novel epitope comprise at least one amino acid of the region that is the same, the first novel epitope and the second novel epitope, 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 novel epitope in a complex with an HLA protein, and a second TCR specific for the second novel epitope in a complex with an HLA protein, a composition, wherein the first peptide is different from the second peptide, the first novel epitope comprises a mutation, and the second novel 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 neoepitope comprises a first neoepitope of a region of the same protein, and the second neoepitope comprises a second neoepitope of a region of the same protein. In some embodiments, the first neoepitope and the second neoepitope comprise at least one amino acid of a region that is the same. In some embodiments, the region of the 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 neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex.In some embodiments, the first neoepitope is a first neoepitope peptide processed from a first peptide, and / or the second neoepitope is a second neoepitope peptide processed from a second peptide. In some embodiments, the first neoepitope is shorter in length than the first peptide, and / or the second neoepitope is shorter in length than the second peptide. In some embodiments, the first neoepitope peptide is processed by an antigen-presenting cell (APC) comprising the first peptide, and / or the second neoepitope peptide is processed by an APC comprising the second peptide. In some embodiments, the first neoepitope is CD8. + activates T cells. In some embodiments, the second neoepitope is CD4 + activates T cells. In some embodiments, the second neoepitope is CD8 + activates T cells. In some embodiments, the first neoepitope is CD4 + activates T cells. In some embodiments, CD4 + The TCR of T cells binds to a class II HLA-peptide complex comprising the first or second peptide. In some embodiments, CD8 + The TCR of T cells binds to a class I HLA-peptide complex comprising the first or second peptide. In some embodiments, CD4 + The TCR of T cells binds to a class I HLA-peptide complex comprising the first or second peptide. In some embodiments, CD8 + The TCR of T cells binds to a class II HLA-peptide complex comprising the first or second peptide. In some embodiments, one or more APCs comprise a first APC comprising the first peptide and a second APC comprising the second peptide. In some embodiments, the mutation is selected from the group consisting of point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, 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 comprises a first 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 first 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 comprises a first sequence of at least 8 or 9 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 first wild-type 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 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 has a length of 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, 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 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 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% 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% identity to the corresponding wild-type sequence. In some embodiments, the second new epitope is longer than the first new epitope. In some embodiments, the first new epitope has a length of at least 8 amino acids. In some embodiments, the first new epitope has a length of 8 to 12 amino acids. In some embodiments, the first new epitope comprises a sequence of at least 8 contiguous amino acids, and at least 2 of the 8 contiguous amino acids are different at the corresponding positions in the wild-type sequence. In some embodiments, the second new epitope has a length of at least 16 amino acids.In some embodiments, the second new epitope has a length of 16 to 25 amino acids. In some embodiments, the second new epitope comprises a sequence of at least 16 contiguous amino acids, and at least 2 of the 16 contiguous amino acids differ at 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 are not mutations in the first new epitope. In some embodiments, one or more of the at least one additional mutation are mutations in the first new 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 are not mutations in the second new epitope. In some embodiments, one or more of the at least one additional mutation are mutations in the second new epitope. 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 new 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, the at least one flanking sequence comprises a non-wild-type sequence. In some embodiments, the at least one flanking sequence is an N-terminal flanking sequence. In some embodiments, the 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 adjacent region of the first peptide is different from at least one adjacent region of the second peptide. In some embodiments, at least one adjacent residue comprises a mutation.

[0328] In some embodiments, the composition comprises one or more additional peptides, and the one or more additional peptides comprise a third neoepitope. In some embodiments, the first and / or second neoepitope binds to an HLA protein with a higher affinity than the corresponding wild-type sequence. In some embodiments, the first and / or second neoepitope has a K D or IC 50 for binding to an HLA protein 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 for binding to an HLA class I protein 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 for binding to an HLA class II protein 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. In some embodiments, the first and / or second neoepitope binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, the mutation is not present in the non-cancerous cells of the subject. In some embodiments, the first and / or second neoepitope is encoded by a gene or an expressed gene of the 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 cells are cytotoxic T cells. In some embodiments, the first and / or second T cells are gamma-delta T cells. In some embodiments, the first and / or second T cells are helper T cells. In some embodiments, the first T cell is a T cell stimulated, expanded, or induced by a first neoepitope, and / or the second T cell is a T cell stimulated, expanded, or induced by a second neoepitope.

[0330] In some embodiments, the first and / or second TCR binds to an HLA peptide complex with a K D or IC 50 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. 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, plasmid, phage, transposon, cosmid, virus, or virion. In some embodiments, the vector is a viral vector. In some embodiments, the vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, alphavirus, vaccinia virus, hepatitis B virus, 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, cationic lipid, cationic polymer, metal nanopolymer, nanorod, liposome, micelle, microbubble, cell-penetrating peptide, or lipospheres.

[0331] In some embodiments, 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 an adjuvant. In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the adjuvant is Hiltonol.

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

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

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

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

[0337] In some embodiments, provided herein is a polypeptide 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, the first peptide is administered to stimulate T cells, and the second peptide is administered after the first peptide has restimulated T cells. In some embodiments, the second peptide is administered to stimulate T cells, and the first peptide is administered after the second peptide has restimulated T cells.

[0338] In some embodiments, provided herein is a polypeptide comprising one or more epitope peptides and a scaffold protein that together form a polypody. In some embodiments, the polypody is a heteropolymer. In some embodiments, the polypody comprises a plurality of first peptides, and a plurality of second peptides, and / or a third peptide, and / or a fourth peptide, and / or a fifth peptide, and / or a sixth peptide, and / or more than six peptides. In some embodiments, the polypody 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 an estrogen receptor having a mutation. In some embodiments, the subject has CLL that is resistant to ibrutinib therapy. In some embodiments, the CLL expresses a Bruton's tyrosine kinase having a mutation such as the C481S mutation. In some embodiments, the subject has lung cancer that is resistant to tyrosine kinase inhibitors. In some embodiments, the lung cancer expresses an epidermal growth factor receptor (EGFR) having a mutation such as the T790M, L792F, or C797S mutation. In some embodiments, the plurality of APC cells comprising the first peptide and the plurality of APC cells comprising 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 administration of the pharmaceutical composition described herein. In some embodiments, the additional therapy is a chemokine or a cytokine. Exemplary cytokines are interleukins such as IL-1, IL-12, or combinations such as two or more cytokines, three or more cytokines, or four or more cytokines.

[0339] Peptide The polypeptide or peptide can be of various lengths, either in their neutral (uncharged) form or in the form of a salt, and can contain modifications such as glycosylation, side-chain oxidation, or phosphorylation, provided that these modifications do not destroy the biological activity of the polypeptides described herein.

[0340] In some embodiments, the sequencing method is 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 potentially replace NGS technology in the future to speed up the sequencing steps of the method. For purposes of clarity, the term "next-generation sequencing" or "NGS" in the context of the present disclosure refers to all new high-throughput sequencing technologies that read nucleic acid templates randomly along the entire genome in parallel by breaking the entire genome into small fragments, as opposed to the "conventional" sequencing methodology known as Sanger chemistry. Such NGS technologies (also known as massively parallel sequencing technologies) can deliver nucleic acid sequence information of the entire genome, exome, transcriptome (all transcribed sequences of the genome), or methylome (all methylated sequences of the genome) in a very short period of time, such as within 1 to 2 weeks, such as within 1 to 7 days or less than 24 hours, and enable single-cell sequencing approaches. A plurality of 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 include, but are not limited to, from 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, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 150, about 200, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 4,000, about 5,000, about 7,500, about 10,000 amino acids, or more amino acid residues, and any range derivable therefrom. In certain embodiments, the neoantigenic peptide molecule is 100 amino acids or less.

[0342] In some embodiments, the peptide can be from about 8 to about 50 amino acid residues in length, or can be from 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 peptide can be from about 8 to about 500 amino acid residues in length, or can be from 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, the peptide 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 peptide 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, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more amino acid residues in length. In some embodiments, the peptide 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 less amino acid residues in length. In some embodiments, the peptide 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, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or less amino acid residues in length.

[0344] In some embodiments, the peptide has a full 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 peptide has a full 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 an HLA-binding peptide is predicted or known, the longer peptide comprises (1) individual binding peptides having an extension of 2-5 amino acids towards the N-terminus and C-terminus of their respective corresponding gene products, or (2) some or all of the ligation of binding peptides having an extension sequence for each. In other embodiments, when sequencing reveals long (more than 10 residues) neoepitope sequences present within a tumor (e.g., due to a frameshift, readthrough, or intron inclusion that results in a novel peptide sequence), 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 for endogenous processing by patient cells, resulting in more effective antigen presentation and induction of a T cell response. In some embodiments, two or more peptides can be used, and these peptides overlap and are displayed side by side on a long neoantigenic peptide.

[0347] In some embodiments, the peptide can have a pI value of from about 0.5 to about 12, from about 2 to about 10, or from about 4 to about 8. In some embodiments, the peptide 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 peptide 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 can be in solution, can be lyophilized, or can be in crystalline form. In some embodiments, the peptides described herein can be synthetically prepared by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources such as natural tumors or pathogenic organisms. New epitopes can be synthesized individually or can be conjugated directly or indirectly to the peptide. The peptides described herein may not substantially contain other naturally occurring host cell proteins and fragments thereof, although in some embodiments the peptides may be synthetically conjugated to be 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 peptide can be expressed in a host cell such as bacteria. In some embodiments, the peptide 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. Further, individual peptides can be joined using chemical ligation to produce larger peptides that are still within the scope of the present disclosure.

[0350] Alternatively, recombinant DNA techniques can be utilized that involve a nucleotide sequence encoding a 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, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus, recombinant peptides containing one or more of the novel antigenic peptides described herein can be used to present appropriate T cell epitopes.

[0351] In some embodiments, the peptide is encoded by a gene having a point mutation that results in an amino acid substitution of the native peptide. In some embodiments, the peptide is encoded by a gene having a point mutation that results in a frameshift mutation. A frameshift occurs when the mutation disrupts the normal phase of the 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 having a mutation that results in a fusion polypeptide, an in-frame deletion, an insertion, the expression of an endogenous retroviral polypeptide, and the tumor-specific overexpression of a polypeptide. In some embodiments, the peptide is encoded by the fusion of a first gene and a second gene. In some embodiments, the peptide is encoded by the in-frame fusion of a first gene and a second gene. In some embodiments, the peptide is encoded by the fusion of a first gene and an exon of a splice variant of the first gene. In some embodiments, the peptide is encoded by the fusion of a first gene and a cryptic exon of the first gene. In some embodiments, the peptide is encoded by the fusion of a first gene and a second gene, and the peptide contains an amino acid sequence encoded by an out-of-frame sequence resulting from the fusion.

[0352] In some embodiments, the disclosure provides a composition 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 by 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, wherein the first peptide is different from the second peptide, the first neoepitope contains a mutation, and the second neoepitope contains the same mutation. In some embodiments, the compositions described herein include 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 contains at least one amino acid of the second region, the first peptide is different from the second peptide, the first neoepitope contains a first mutation, and the second neoepitope contains 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 point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, and any combination thereof.

[0353] In some embodiments, the peptide may be derived from a protein having substitution mutations, such as KRAS G12C, G12D, G12V, Q61H or Q61L mutations, or NRAS Q61K or Q61R mutations. The substitution can be located at any position along the length of the peptide. For example, it can be located in the N-terminal third, the central third, or the C-terminal third of the peptide. In another embodiment, the substituted residue is located 2 to 5 residues away from the N-terminus or 2 to 5 residues away from the C-terminus. The peptide can similarly be derived from a tumor-specific insertion mutation, and the peptide includes one or more, or all, of the inserted residues. In some embodiments, the first neoepitope and / or the second neoepitope binds to the HLA protein with a higher affinity than the corresponding neoepitope without the substitution. In some embodiments, the first neoepitope and / or the second neoepitope binds to the HLA protein with a 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 neoepitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the first neoepitope. 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 neoepitope. In some embodiments, one or more of the at least one additional mutation is a mutation in the second neoepitope.

[0355] In some embodiments, the present disclosure provides a composition 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 composition provided by the present invention comprises one or more additional peptides, and the one or more additional peptides comprise a third novel epitope. 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 comprises a first 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, 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 comprises a first sequence of at least 8 or 9 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, 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 has a length of 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, 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 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 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% 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 contain at least one flanking sequence, and at least one flanking sequence is upstream or downstream of the neoepitope. 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 contains 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 is different from at least one flanking region of the second peptide. In some embodiments, at least one flanking residue contains a mutation.

[0358] In some embodiments, the peptide comprises a new epitope sequence that includes at least one mutated amino acid. In some embodiments, the peptide comprises a new epitope sequence that includes 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 new 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 new 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 at least one mutated amino acid. In some embodiments, the peptide comprises a new 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 at least one mutated amino acid.In some embodiments, the peptide comprises at least one mutant amino acid, 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-mutant amino acids upstream of the at least one mutant 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-mutant amino acids downstream of the at least one mutant amino acid, and comprises a novel epitope sequence derived from a protein.

[0359] In some embodiments, the peptide comprises at least one mutant amino acid and a sequence upstream of the at least one mutant amino acid 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%, 99%, or 100% sequence identity to the corresponding wild-type sequence, and comprises a neoepitope sequence derived from a protein. In some embodiments, the peptide comprises at least one mutant amino acid and a sequence downstream of the at least one mutant amino acid 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%, 99%, or 100% sequence identity to the corresponding wild-type sequence, and comprises a neoepitope sequence derived from a protein.In some embodiments, the peptide comprises at least one mutant amino acid, a sequence upstream of the at least one mutant amino acid having 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 the at least one mutant amino acid 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%, 99%, or 100% sequence identity to the corresponding wild-type sequence, and comprises a novel epitope sequence derived from a protein.

[0360] In some embodiments, the peptide comprises at least one mutant amino acid and a sequence upstream 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 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%, 99%, or 100% sequence identity to the corresponding wild-type sequence, and comprises a novel epitope sequence derived from a protein. In some embodiments, the peptide comprises at least one mutant amino acid 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 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%, 99%, or 100% sequence identity to the corresponding wild-type sequence, and comprises a novel epitope sequence derived from a protein.In some embodiments, the peptide comprises at least one mutant amino acid and at least one sequence upstream 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 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, and at least one 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 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, and comprises a novel epitope sequence derived from a protein.

[0361] Peptide modification In some embodiments, the present disclosure includes modified peptides. The modifications can include covalent chemical modifications that do not change the primary amino acid sequence of the antigenic peptide itself. The modifications can result in desired properties, such as an extended in vivo half-life, increased stability, reduced clearance, altered immunogenicity or allergenicity, enabling the rise of specific antibodies, cell targeting, antigen uptake, antigen processing, HLA affinity, HLA stability or antigen presentation. In some embodiments, the peptide can include one or more sequences that enhance the processing and presentation of epitopes by APCs, for example, for the generation of an immune response.

[0362] In some embodiments, the peptide 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 includes relatively small neutral molecules, such as amino acids or amino acid mimics, that are 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. Optionally, it will be understood that the spacer present need not be composed of the same residues and can thus be a hetero-oligomer or a homo-oligomer. The neoantigenic peptide can be linked directly or via a spacer to the T helper peptide at either the amino or carboxy terminus of the peptide. The amino terminus of either the neoantigenic 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 surrounding residues 382-398 and residues 378-389.

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

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

[0365] In some embodiments, peptides may be modified using a series of peptides having a single amino acid substitution to determine effects such as electrostatic charge, hydrophobicity, etc. 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 different 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-oligomers or hetero-oligomers. The number and type of residues substituted or added depend on the required spacing between essential contacts and the specific functional attributes required (e.g., hydrophobic vs. hydrophilic). An increase in binding affinity for an HLA molecule or T cell receptor 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 steric and charge interference that could, for example, disrupt binding. Amino acid substitutions are typically of a single residue. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final peptide.

[0366] In some embodiments, the peptides described herein may be modified by terminal - NH2 acylation, for example, alkanoyl (C1 - C 20 ) or thioglycolyl acetylation, terminal - carboxyl amidation, for example, by ammonia, methylamine, etc. In some embodiments, these modifications can provide sites for attachment to a support 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, for example, lipids, or may be chemically modified, for example, by acetylation. Further, the linkages in the peptide may be other than peptide linkages, for example, covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.

[0367] In some embodiments, the peptides described herein can include carriers such as those well - known in the art, for example, albumin such as thyroglobulin, human serum albumin, polyamino acid residues such as tetanus toxoid, poly - L - lysine and poly - L - glutamic acid, influenza virus proteins, hepatitis B virus core proteins, etc.

[0368] Peptides may be further modified to contain additional chemical moieties, which are usually not part of the protein. These derivatized moieties can improve solubility, biological half-life, protein absorption, or binding affinity. This moiety can also reduce or eliminate any desired side effects, such as those of the peptide. An overview 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, such as improved pharmacological properties, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide, binding to the desired HLA molecule, and activating appropriate T cells. For example, peptides can be subjected to various changes, such as either conservative or non-conservative substitutions, which can result in specific advantages in their use, such as improved HLA binding. Such conservative substitutions can involve replacing an amino acid residue with another amino acid residue that is biologically and / or chemically similar, for example, one hydrophobic residue for another amino acid, or one polar residue for another amino acid. The effect of single amino acid substitutions can also be investigated using D-amino acids. Such modifications can be carried out using well-known peptide synthesis procedures, as described, for example, in Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979), and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2d Ed. (1984).

[0369] In some embodiments, the peptides described herein can 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, leukotoxin molecules, inactivated bacteria, and dendritic cells.

[0370] Changes to the peptide can include, but are not limited to, conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation, HESylation, recombinant PEG mimetics, Fc fusions, albumin fusions, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusions, iron fusions, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, addition of a surfactant, addition of an amino acid mimetic, or addition of a non-natural amino acid.

[0371] Further suitable components and molecules for conjugation include, for example, molecules for targeting the lymphatic system, albumin such as thyroglobulin, human serum albumin (HSA), polyamino acids such as tetanus toxoid, diphtheria toxoid, poly(D-lysine:D-glutamic acid), the 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 to conjugate (e.g., link) 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, the fusion of albumin to a peptide or protein of the present disclosure can be achieved by genetic engineering such that, for example, DNA encoding HSA, or a fragment thereof, is joined to DNA encoding one or more polypeptide sequences. Subsequently, a suitable host can 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, for example, in vitro from prokaryotic or eukaryotic cells, or in vivo, for example, from transgenic organisms. In some embodiments of the present disclosure, the expression of the fusion protein is carried out in a mammalian cell line, such as a CHO cell line. Further, albumin itself can be modified to extend its circulation half-life. The fusion of modified albumin to one or more polypeptides can be achieved by the above genetic engineering techniques or by chemical conjugation, and the resulting fusion molecule has a half-life exceeding that of the fusion with unmodified albumin (see, for example, WO 2011 / 051489). As an alternative to direct fusion, including albumin binding via a conjugated fatty acid chain (acylation), several albumin binding strategies have been developed. Since serum albumin is a transport protein for fatty acids, these natural ligands having albumin binding activity have been used for the half-life extension of small protein therapeutics.

[0373] Additional candidate components and molecules for conjugation include those suitable for isolation or purification. Non-limiting examples include biotin (biotin-avidin specific binding pair), antibodies, receptors, ligands, binding molecules such as lectins, or molecules including solid supports such as, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test pieces, and membranes. To separate the conjugate by charge difference, purification methods such as cation exchange chromatography may be used, thereby effectively separating the conjugate by their various molecular weights. The content 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 by molecular weight.

[0374] In some embodiments, the amino or carboxyl terminus of the 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, and thus the biopharmaceutical may require less frequent dosing. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-released into circulation, maintaining the molecule in circulation for a longer time. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc fusion technologies link a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to conventional Fc fusion conjugates.

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

[0376] The stability of a peptide 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 prior to 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 turbid reaction sample is cooled for 15 minutes (4 °C) and then rotated to pellet the precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability-specific chromatographic conditions.

[0377] Problems associated with short plasma half-lives or susceptibility to proteolytic degradation can be overcome by various modifications, including conjugating or linking the peptide or protein sequence to any of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene (see, e.g., typically a linker covalently bound to both the protein and the non-proteinaceous polymer, such as via PEG). 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 circulation half-lives and decreased clearance, reduced immunogenicity and antigenicity, and reduced toxicity.

[0378] PEGs suitable for conjugation to a polypeptide or protein sequence are generally water soluble at room temperature and have the general formula R-(O-CH2-CH2) n-O-R, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has from 1 to 8 carbons. The PEG conjugated to the polypeptide sequence may 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 the PEGs have different n values and thus various different PEGs are present in a specific ratio. For example, some compositions include mixtures of conjugates where n = 1, 2, 3, and 4. In some compositions, the proportion of conjugate where n = 1 is 18 - 25%, the proportion of conjugate where n = 2 is 50 - 66%, the proportion of conjugate where n = 3 is 12 - 16%, and the proportion of conjugate where 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 fractions purified to contain, for example, conjugates with the desired number of PEGs attached, and to be free of unmodified protein sequences and conjugates with other numbers of PEGs attached can be identified.

[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 one or more free amino or carboxyl groups of the polypeptide sequence and the PEG. PEGs with spacers that can attach to free amino groups include N-hydroxysuccinimidyl PEG, which can be prepared by activating the succinate ester of PEG with N-hydroxysuccinimide. Another activated PEG that can attach 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 cyanogen chloride. Activated PEGs that attach to free carboxyl groups include polyoxyethylenediamine.

[0380] Conjugating one or more of the peptide or protein sequences of the present disclosure to PEG with a spacer can be done by a variety of conventional methods. For example, the conjugation reaction can be carried out in solution at a temperature of 4 °C to room temperature for 30 minutes to 20 hours, at a pH of 5 to 10, using a molar ratio of reagent to peptide / protein of 4:1 to 30:1. The reaction conditions can be selected mainly to induce the reaction to produce the desired degree of substitution. Generally, low temperature, low pH (e.g., pH = 5), and short reaction times tend to reduce the number of attached PEGs, while high temperature, neutral to high pH (e.g., pH > 7), and long reaction times tend to increase the number of attached PEGs. To terminate the reaction, various means known in the art can be used. 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 conferring several additional advantageous properties. As an example, simple polypeptide chains (e.g., containing Ala, Glu, Gly, Pro, Ser, and Thr) that can form an extended conformation similar to PEG can be recombinantly fused to the peptide or protein drug of interest and produced (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 epitope The new epitope comprises a neoantigenic determinant portion of a neoantigenic peptide or neoantigenic polypeptide that is recognized by the immune system. The new epitope refers to an epitope 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. This includes situations where the corresponding epitope is found in normal non-diseased cells or germline cells, but the sequence of the epitope is changed by one or more mutations in diseased cells, such as cancer cells, to result in a new epitope. The term "new epitope" is used interchangeably with "tumor-specific new epitope" herein and typically designates a series of residues, typically L-amino acids, that are connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The new epitopes can be of various lengths and can be in either their neutral (uncharged) form or in the form of salts, and contain no modifications such as glycosylation, side-chain oxidation, or phosphorylation, or contain these modifications subject to the condition that the biological activity of the polypeptides described herein is not destroyed.

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

[0384] In some embodiments, the compositions described herein include a first peptide comprising a first novel epitope of a protein and a second peptide comprising a second novel epitope of the same protein, wherein the first peptide is different from the second peptide, the first novel epitope comprises a mutation, and the second novel epitope comprises the same mutation. In some embodiments, the compositions described herein include a first peptide comprising a first novel epitope of a first region of a protein and a second peptide comprising a second novel 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 novel epitope comprises a first mutation, and the second novel 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 point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, and any combination thereof.

[0385] In some embodiments, the first novel epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the second novel epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second novel epitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first novel epitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the first novel epitope activates CD8 + T cells. In some embodiments, the first novel epitope activates CD4 + T cells. In some embodiments, the second novel epitope activates CD4 + T cells. In some embodiments, the second novel epitope activates CD8 + T cells. In some embodiments, CD4 +The TCR of the T cell binds to the class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to the class II HLA-peptide complex. In some embodiments, CD8 + The TCR of the T cell binds to the 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 neoepitope is longer than the first neoepitope. In some embodiments, the first neoepitope has a length of at least 8 amino acids. In some embodiments, the first neoepitope has a length of 8 to 12 amino acids. In some embodiments, the first neoepitope comprises a sequence of at least 8 contiguous amino acids, and at least 1 of the 8 contiguous amino acids differs at the corresponding position in the wild-type sequence. In some embodiments, the first neoepitope comprises a sequence of at least 8 contiguous amino acids, and at least 2 of the 8 contiguous amino acids differ at the corresponding position in the wild-type sequence. In some embodiments, the second neoepitope has a length of at least 16 amino acids. In some embodiments, the second neoepitope has a length of 16 to 25 amino acids. In some embodiments, the second neoepitope comprises a sequence of at least 16 contiguous amino acids, and at least 2 of the 16 contiguous amino acids differ at the corresponding position in the wild-type sequence. In some embodiments, the second neoepitope comprises a sequence of at least 16 contiguous amino acids, and at least 2 of the 16 contiguous amino acids differ at the corresponding position in the wild-type sequence.

[0387] In some embodiments, the new epitope binds to an HLA protein (e.g., HLA class I or HLA class II). In some embodiments, the new epitope binds to the HLA protein with a higher affinity than the corresponding wild-type peptide. In some embodiments, the new epitope has an IC 50 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.

[0388] In some embodiments, the new epitope 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 new epitope 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 new 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 new epitope binds to the HLA protein with a higher affinity than the corresponding wild-type new epitope. In some embodiments, the first and / or second new epitope has a K D or IC 50and binds to the HLA protein. In some embodiments, the first and / or second neoepitope 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 and binds to the HLA class I protein. In some embodiments, the first and / or second neoepitope has 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 and binds to the HLA class II protein.

[0390] In one aspect, the first and / or second neoepitope binds to a protein encoded by an HLA allele expressed by the subject. In another aspect, the mutation is not present in the non-cancerous cells of the subject. In yet another aspect, the first and / or second neoepitope is encoded by a gene or an expressed gene of the cancer cells of the subject.

[0391] Polynucleotide Alternatively, the nucleic acid (e.g., polynucleotide) encoding the peptide of the present disclosure can be used to produce the polypeptide using a host cell or in vitro, e.g., by in vitro translation. In some embodiments, in vitro translation is used to produce the peptide. Also contemplated are polynucleotides encoding polypeptides that can be used as a therapy, e.g., a polybody comprising a neoantigenic peptide. The polynucleotide can be, for example, DNA, cDNA, or RNA and can be either single-stranded and / or double-stranded.

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

[0393] The term “RNA” includes “mRNA” and, in some embodiments, relates to “mRNA.” The term “mRNA” means “messenger RNA” and relates to a “transcript” that is generated by using a DNA template and encodes a peptide or polypeptide. Typically, mRNA includes a 5′-UTR, a protein-coding region, and a 3′-UTR. mRNA has a limited half-life only intracellularly and in vitro. In some embodiments, the mRNA is self-amplifying mRNA. In the context of the present disclosure, mRNA can be generated by in vitro transcription from a DNA template. Methods for in vitro transcription are known to those of skill 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 an increased translation efficiency of the RNA. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of RNA used according to the present disclosure, it can be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, to increase the GC content to increase mRNA stability, perform codon optimization, thereby improving translation in cells, without changing the sequence of the expressed peptide or protein.

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

[0396] In some embodiments, the term "modified" relates to providing an RNA having a 5' cap or 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 connected 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 RNA 5' cap that occurs naturally relative to the 7-methylguanosine cap (m7G). In the context of the present disclosure, the term "5' cap" includes 5' cap analogs that are similar to the RNA cap structure and are modified to have the ability to stabilize the RNA and / or enhance the translation of the RNA when attached thereto in vivo and / or in cells.

[0397] In certain embodiments, the mRNA encoding the neoantigenic peptide of the present disclosure is administered to a subject in need thereof. In some embodiments, the present disclosure provides RNAs, oligoribonucleotides, and polynucleotide molecules comprising modified nucleosides, gene therapy vectors comprising them, gene therapy methods and gene transcription silencing methods comprising them. In some embodiments, the administered mRNA comprises at least one modified nucleoside.

[0398] The polynucleotides encoding the peptides described herein can be synthesized by chemical techniques, such as the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Polynucleotides encoding peptides comprising or consisting of analogs can be readily made by substituting the appropriate desired nucleobase(s) for those encoding the native epitope.

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

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

[0401] In some embodiments, the polynucleotide can include a coding sequence for a peptide or protein fused in-frame with a marker sequence, for example, that enables purification of the encoded peptide, and can then be incorporated into an individualized disease vaccine or immunogenic composition. For example, the marker sequence can be a hexa-histidine tag supplied by the pQE-9 vector to provide purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when using a mammalian host (e.g., COS-7 cells). Additional tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Sof tag 1, Sof tag 3, 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. The polynucleotide encoding the neoantigenic peptide described herein can also include a sequence encoding a ubiquitination signal sequence and / or a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.

[0402] In some embodiments, the polynucleotide can include the coding sequences of one or more of the presently described peptides or proteins fused in-frame to create a single concatenated neoantigenic peptide construct that can produce multiple neoantigenic peptides.

[0403] In some embodiments, the DNA sequence is constructed using recombinant techniques by isolating or synthesizing the DNA sequence encoding the wild-type protein of interest. Optionally, the sequence can be mutagenized by site-directed mutagenesis to obtain its functional analogs. See, for example, Zoeller et al., Proc. Nat’l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Patent No. 4,588,585. In some embodiments, the DNA sequence encoding the peptide or protein of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired peptide, and preferred codons can be selected in the host cell in which the recombinant polypeptide of interest is to be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding the isolated polypeptide of interest. For example, a reverse-translated gene can be constructed using the complete amino acid sequence. Additionally, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides 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 expression control sequences suitable for protein expression in the desired host. Appropriate 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 also encompasses vectors and expression vectors useful for the production and administration of the neoantigenic peptides and neoepitopes described herein, as well as host cells containing such vectors.

[0406] Vector In some embodiments, expression vectors capable of expressing the peptides or proteins 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, the DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. Optionally, the DNA can be ligated to appropriate transcriptional and translational regulatory nucleotide sequences recognized by the desired host (e.g., bacteria), but such controls are generally available in expression vectors. The vector is then introduced into the host bacteria for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0407] Numerous vectors and host systems suitable for the production and administration of the neoantigenic peptides described herein are known to those skilled in the art and are commercially available. The following vectors are provided as examples. Bacteria: 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). Eukaryotes: 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 the expression of the neoantigenic peptides described herein, the coding sequence is provided with an operably linked start codon and stop codon, a promoter region and a terminator region, and in some embodiments, a replication system for providing an expression vector for expression in the desired cell host. For example, a promoter sequence compatible with a bacterial host is provided in a plasmid containing convenient restriction sites for the insertion of the desired coding sequence. The resulting expression vector is transformed into a suitable bacterial host.

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

[0410] Using recombinant expression vectors, DNA encoding the peptides or proteins described herein can be amplified and expressed. A recombinant expression vector is a replicable DNA construct having a DNA fragment derived from synthetic or cDNA that encodes a peptide or biologically equivalent analog operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. The transcription unit generally includes (1) one or more gene elements having 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) the assembly of appropriate transcriptional and translational start and termination sequences as described in detail herein. Such regulatory elements may include operator sequences for controlling transcription. Usually, the ability to replicate in the host conferred by an origin of replication, and a selectable gene for facilitating the recognition of transformants, can be additionally incorporated. 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 the DNA of a polypeptide when expressed as a precursor involved in the secretion of the polypeptide, a promoter is operably linked to a coding sequence when controlling the transcription of the sequence, and a ribosome binding site is operably linked to a coding sequence when arranged to enable translation. Generally, operably linked means contiguous, and in the case of a secretory leader, it means contiguous and within the reading frame. Structural elements intended for use in a yeast expression system include leader sequences that allow for the extracellular secretion of the 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. This residue can optionally then be cleaved from the expressed recombinant protein to provide the final product.

[0411] Generally, a recombinant expression vector contains an origin of replication and a selectable marker that enable transformation of a host cell, such as the E. coli and S. cerevisiae TRP1 genes, and a promoter derived from a highly expressed gene to direct transcription of downstream structural sequences. Such promoters can be derived, inter alia, from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), acid phosphatase, or heat shock protein. Heterologous structural sequences are assembled at an appropriate stage with a translation initiation sequence and a termination sequence, and in some embodiments, a leader sequence that can direct secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that confers a desired characteristic, such as stabilization of the expressed recombinant product or simplified purification.

[0412] In some embodiments, the neoantigenic peptides described herein can also be expressed by a bacterial vector. 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 characterized far better than those of any other microorganism. Fusion proteins can be expressed in an E. coli expression vector and grown in various 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. Suitable bacteria, in many embodiments, include one or more mutations or other genetic modifications that increase the expression level.

[0413] Molecular cloning techniques for achieving these objectives are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well known to those skilled in the art. Examples of these techniques and descriptions sufficient to guide those skilled in the art through many cloning implementations 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), as well as Current Protocols in Molecular Biology, F.M. Ausubel 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 those skilled in the art through in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques, can be found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987) U.S. Patent No. 4,683,202.

[0414] Briefly stated, the method for protein expression in the E. coli system includes the following steps. Clone the nucleic acid encoding the target protein or polypeptide into the desired plasmid. Then, incorporate the plasmid into a bacterial host suitable for plasmid propagation (e.g., by transfection, or electroporation, or any other suitable method known to those skilled in the art). Culture and grow the bacteria in a culture medium while amplifying the protein product encoded by the nucleic acid within the bacteria (or, if the encoded protein is a secreted protein, secreting it into the medium). Then, collect the protein from the grown bacteria (for endogenous proteins) or the culture supernatant (for secreted proteins). High-copy number expression plasmids are mainly selected for this purpose. The basic elements of any plasmid for bacterial cloning include, among others, an ORI (origin of replication) site, a promoter at the 5' end, control elements such as enhancers, 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 the nucleic acid encoding the target protein is cloned within the designated cloning site of a commercially available vector with a suitable tag, the expressed protein is a fusion protein containing the tag. For example, plasmid pDEST-HisMBP contains MBP and a hexa-histidine tag. In this case, the MBP protein and the His tag are used for the isolation and purification of the protein from E. coli cells. Various such plasmids and expression systems are commercially available and known to those skilled in the art. For the construction of expression vectors, many promoters such as P lac 、P trp 、P tac 、λP L 、P T7 、and P BAD are commonly used. Among these, lacUV5, tac, and P T7The combination system with lacUV5 is widely used. This is because this expression can be easily regulated by changing the concentration of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG). Other promoters called λPL and λPR are generally induced by a temperature shift. To obtain high-level expression of the cloned gene, the expression cassette can include other sequences such as ribosome binding sites for translation initiation and transcription / translation termination sequences. High-level expression of the desired peptide or polypeptide can be achieved by using a bacterial expression vector containing a dual promoter. Cells can be grown in shake flasks or other containers, but for large-scale production, preparation of polypeptide growth in a fermenter is preferred. To obtain the maximum level of expression, galactose is added to the nutrient medium at an appropriate point in the growth cycle to induce an increase in the expression of the desired polypeptide. For example, the growth of host cells can be initiated in a medium containing fructose (0.25% final concentration) as a carbon source, and other sugars, glycerol, acetate) that cause an increase in the intracellular cAMP (adenosine 3’,5’-cyclic monophosphate) concentration 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 conveniently included in the expression vector. These genes encode proteins necessary for the survival or growth of transformed host cells grown in selective culture media. Host cells not transformed with a vector containing a selectable gene do not survive in the culture medium. Typical selectable 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 supply an essential nutrient not utilizable from complex media, the gene encoding D-alanine racemase for bacilli. Some 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, for example, over ampicillin selection. This is because ampicillin is rapidly degraded by β-lactamase in the medium, removing the selection pressure and allowing the culture to overgrow with cells that do not contain the vector.

[0417] The construction of suitable vectors containing one or more of the components listed above can utilize standard ligation techniques as described in the references above. The vector may contain other sequences to enable 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 cut, adjusted, and religated in the desired form to generate the required plasmid. To confirm the exact 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 the expressed protein. The basic steps of purification include protein lysis, binding of the protein to the matrix, washing, and elution. As an example, a polyhistidine (His6) tag is commonly placed at the N-terminus or C-terminus of the protein during cloning, and each target protein is expressed with the tag. Finally, His can be captured by the metal because the nitrogen of the imidazole moiety of polyhistidine interacts with the metal. Typically, the metal is immobilized to support the capture. Washing and elution are carried out with a suitable buffer. The tag can be designed to be cleaved from the expressed protein after purification. Similarly, various other tags such as GST tag, Halo tag, HA tag, etc. 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. These were originally developed to facilitate the detection and purification of recombinant proteins, but in recent years, it has become apparent that certain tags can also improve yield, improve solubility, and even promote proper folding of their fusion partners. The insolubility of recombinant proteins can be a particularly big problem in Escherichia coli in the production of bioactive substances for structural and functional studies, but the use of solubility-enhancing tags to avoid the formation of insoluble protein aggregates is gaining popularity and rapidly increasing. Many proteins that are highly soluble when overproduced in E. coli have been reported to have solubility-enhancing properties as fusion partners, but in most cases, the evidence to support these claims is scarce. Among the 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 tag, FLAG tag, HA tag, and others.

[0420] Examples of expression vectors capable of infecting mammalian cells include attenuated viral hosts such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector for expressing a nucleotide sequence encoding a novel antigenic peptide described herein. Methods useful for vaccinia vectors and 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 variety of other vectors useful for the therapeutic administration or immunization with the novel antigenic polypeptides described 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, will be apparent to those skilled in the art from the description herein. In some embodiments, the vector is a modified vaccinia Ankara (VA) (e.g., Bavarian Noridic (MVA-BN)).

[0422] Among the vectors that can be used in the practice of the present disclosure, integration into the host genome of a cell is possible by retroviral gene transfer methods, and often results in long-term expression of the inserted transgene. In some embodiments, the retrovirus is a lentivirus. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of the retrovirus can be altered by incorporating foreign envelope proteins and expanding the potential target population of the target cells. The retrovirus can also be engineered to allow conditional expression of the inserted transgene such that only specific cell types are infected by the lentivirus. A cell type-specific promoter can be used to target expression in a specific cell type. The lentiviral vector is a retroviral vector (thus, both lentiviral vectors and retroviral vectors can be used in the practice of the present disclosure). Furthermore, the lentiviral vector can transduce or infect non-dividing cells and typically can produce high viral titers. Thus, the choice of retroviral gene transfer system can depend on the target tissue. The retroviral vector is composed of cis-acting long terminal repeats that have the packaging capacity for foreign sequences of up to 6-10 kb. The minimal cis-acting LTR is sufficient for vector replication and packaging, and the vector then incorporates into the target cell using the desired nucleic acid to provide for permanent 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 (see, for example, Buchscher et al., (1992) J. Virol. 66:2731-2739, Johann et al., (1992) J. Virol. 66:1635-1640, Sommerfelt et al., (1990) Virol. 176:58-59, Wilson et al., (1998) J. Virol. 63:2374-2378, Miller et al., (1991) J. Virol. 65:2220-2224; see PCT / US94 / 05700).

[0423] Also useful in the practice of 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 that drives the expression of the target gene. Accordingly, the present disclosure contemplates viral vectors, including retroviral vectors and lentiviral vectors, among the vector(s) useful in the practice of the present disclosure.

[0424] Also useful in the practice of the present disclosure are adenoviral vectors. One advantage is the ability of recombinant adenoviruses to efficiently transcribe and express recombinant genes in a variety of mammalian cells and tissues in vitro and in vivo, resulting in high expression of the transcribed nucleic acid. Furthermore, the ability to infect quiescent cells productively expands the usefulness of recombinant adenoviral vectors. Additionally, the high expression levels ensure that the nucleic acid product is expressed to levels sufficient to generate an immune response (see, for example, U.S. Patent No. 7,029,848, which is incorporated herein by reference).

[0425] Regarding the adenovirus vectors useful for the practice of the present disclosure, reference is made to U.S. Patent No. 6,955,808. The adenovirus vectors used can 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; this content is incorporated herein by reference). The Ad35 vector is described in U.S. Patent Nos. 6,974,695, 6,913,922, and 6,869,794. The Ad11 vector is described in U.S. Patent No. 6,913,922. The C6 adenovirus vector is 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. The C7 vector is described in U.S. Patent No. 6,277,558. Adenovirus vectors that are E1 deficient or deleted, E3 deficient or deleted, and / or E4 deficient or deleted can also be used. Certain adenoviruses having mutations in the E1 region have an improved safety margin because the E1-deficient adenovirus mutants are replication-deficient or at least highly attenuated in non-permissive cells. Adenoviruses having mutations in the E3 region can enhance immunogenicity by disrupting the mechanism by which adenoviruses downregulate MHC class I molecules. Adenoviruses having E4 mutations can reduce the immunogenicity of adenovirus vectors for the suppression of late gene expression. Such vectors can be particularly useful when repeated revaccination with the same vector is desired. Adenovirus vectors deleted or mutated in E1, E3, E4; E1 and E3; and E1 and E4 can be used in accordance with the present disclosure.

[0426] Furthermore, "gutless" adenoviral vectors, in which all viral genes are deleted, can also be used according to the present disclosure. Such vectors require helper viruses for their replication and a special human 293 cell line that expresses both E1a and Cre, which is not present in the natural environment. Such "gutless" vectors are non-immunogenic and thus the vectors can be administered multiple times for re-vaccination. "Gutless" adenoviral vectors can be used for the insertion of heterologous inserts / genes such as the transgenes of the present disclosure, and can even be used for the simultaneous delivery of multiple heterologous inserts / genes.

[0427] In some embodiments, delivery is via an adenovirus, which may be a single booster dose. In some embodiments, the adenovirus is delivered via multiple doses. With respect to in vivo delivery, AAV is advantageous over other viral vectors due to its low toxicity and low potential to cause insertional mutagenesis as 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 ITR can serve as a promoter and is advantageous in eliminating the need for additional promoter elements.

[0428] For ubiquitous expression, the following promoters can be used. CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain, etc. For brain expression, the following promoters can be used. Synapsin I for all neurons, CaMK IIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis may include Pol III promoters such as U6 or H1. The use of Pol II promoters and intron cassettes can be used to express guide RNA (gRNA). Regarding AAV vectors useful in the practice of the present disclosure, reference is made to U.S. 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. Regarding AAV, AAV can be AAV1, AAV2, AAV5, or any combination thereof. AAV can be selected for the cells 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 the brain or nerve cells, and AAV4 can be selected to target heart tissue. AAV8 is useful for delivery to the liver. In some embodiments, delivery is via AAV. The 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, e.g., Verardi et al., Hum. Vaccin. Immunother. 2012 Jul;8(7):961-70, and Moss, Vaccine. 2013;31(39):4220-4222). Poxvirus expression vectors were described in 1982 and soon became widely used for vaccine development and research in numerous fields. 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 the practice of the present disclosure, such as the subfamily Chordopoxvirinae 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, particularly their synthetic or non-naturally occurring recombinants, their use, and methods for making and using such recombinants, can be found in scientific and patent literature.

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

[0431] In some embodiments, ALVAC is used as a vector in a disease vaccine or immunogenic composition. 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, an ALVAC virus expressing the tumor antigen CEA demonstrated an excellent safety profile and resulted in an increase in 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, a modified vaccinia Ankara (MVA) virus can be used as a viral vector for an antigen vaccine or immunogenic composition. MVA is a member of the orthopoxvirus family and has been generated by approximately 570 consecutive passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (see, for example, 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 compared to CVA and has highly restricted host cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extreme attenuation, i.e., a decrease in virulence or infectivity, but still has excellent immunogenicity. When tested in various animal models, MVA has been shown to be non-pathogenic even in immunosuppressed individuals. Furthermore, MVA-BN®-HER2 is a candidate immunotherapy designed for the treatment of HER-2 positive breast cancer and is currently in clinical trials. (Mandl et al., Cancer Immunol. Immunother. Jan 2012;61(1):19-29). Methods of making and using recombinant MVA are described (see, for example, U.S. Patent Nos. 8,309,098 and 5,185,146, which are incorporated herein by reference in their entirety).

[0433] Host cells suitable for the expression of polypeptides include prokaryotes, yeast, insects or higher eukaryotic cells under the control of appropriate promoters. 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. Appropriate 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, N.Y., 1985).

[0434] A variety of mammalian or insect cell culture systems are also advantageously utilized for the expression of recombinant proteins. Expression of recombinant proteins in mammalian cells can be carried out because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 strain of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing appropriate 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, as well as other 5' or 3' flanking non-transcribed sequences, and 5' or 3' untranslated sequences such as the necessary ribosome binding site, polyadenylation site, splice donor and acceptor sites, and transcription termination sequences. The baculovirus system for the production of heterologous proteins in insect cells is reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0435] A host cell is genetically engineered (transduced, transformed, or transfected) with a vector, which can be, for example, a cloning vector or an expression vector. The vector can be in the form of, for example, a plasmid, a viral particle, a phage, etc. The engineered host cell can be cultured in a conventional nutrient medium that has been modified as necessary for promoter activation, transformant selection, or polynucleotide amplification. Culture conditions such as temperature, pH, etc. are 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 within 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), and other cell lines capable of expressing compatible vectors such as the C127, 3T3, CHO, HeLa, and BHK cell lines or other cell lines capable of expressing Bowes melanoma; plant cells, etc. The selection of a suitable host is considered to be within the scope of those skilled in the art from the teachings of this specification.

[0437] Yeast, insect, or mammalian cell hosts can also be used, utilizing suitable 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), and other cell lines capable of expressing compatible vectors such as the 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 direct codon selection for each amino acid. Such polynucleotides can contain 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 + ) adjacent to the epitope and / or analog, Th (e.g., CD4 + ), and B cell epitopes).

[0439] Standard regulatory sequences well known to those of skill in the art can be included in the vector to ensure expression in human target cells. Several vector elements are desirable: a promoter having a cloning site downstream of the polynucleotide, e.g., a mini gene insertion; a polyadenylation signal for efficient transcription termination; an origin of replication for E. coli; and a selectable marker for E. coli (e.g., ampicillin or kanamycin resistance). A number of promoters, such as the human cytomegalovirus (hCMV) promoter, can be used for this purpose. For other suitable promoter sequences, see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466. In some embodiments, the promoter is the 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 plasmids derived from Escherichia coli, including pCR1, pBR322, pMB9, and their derivatives, and plasmids with a broader host range such as M13 and filamentous single-stranded DNA phages.

[0441] Vectors may 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 hosts by these two methods is illustrated in Scientific American (Weiner et al., (1999) Scientific American 281(1):34-41). Injection in saline is usually done intramuscularly (IM) or intradermally (ID) in skeletal muscle, and the DNA is delivered to the extracellular space. This can be assisted by electroporation by temporarily damaging muscle fibers with a myotoxin such as bupivacaine, or by using a hypertonic solution 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 the type of needle, needle alignment, injection rate, injection volume, muscle type, and the age, sex, and physiological state of the animal being injected (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 can include aerosol deposition of naked DNA on mucosal surfaces such as the nasal and lung mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129 - 88), as well as topical administration of pDNA to the eye 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 liposome 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, which can transiently permeabilize the cells. Such mild mechanical disruption of the membrane can be achieved by gently passing the cells through small openings (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 dispersions such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). When non-viral delivery systems are utilized, an exemplary delivery vehicle is a liposome. "Liposome" is a general term encompassing various single and multilamellar lipid vehicles formed by the generation 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 an aqueous medium. These are formed spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization prior to the formation of the closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5:505-10 (1991)). However, compositions having structures different from the normal vesicular structure in solution are also included. For example, the lipids can assume a micellar structure or 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 aspect, the nucleic acid can associate with the lipid. The nucleic acid that associates with the lipid can be encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that associates with both the liposome and the oligonucleotide, trapped within a liposome, complexed with a liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained as a suspension in the lipid, contain micelles, or be complexed with micelles, or otherwise associate with the lipid. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a “collapsed” structure. They may also simply be dispersed 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 lipids or synthetic lipids. For example, lipids include lipid droplets that naturally occur within the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0446] Suitable lipids can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo., dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.), cholesterol (“Choi”) can be obtained from Calbiochem-Behring, dimyristoyl 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 about -20° C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. In some embodiments, lipids are essential for delivering the nucleic acid encoding the protein of interest into cells. The nucleic acid constructs encoding the fusion polypeptides described herein can be delivered via a lipid composition, e.g., intracellular liposomes for the expression of the fusion polypeptides. In some embodiments, the liposomes contain one or more cationic lipids. In some embodiments, the liposomes contain 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. At least two different peptides can vary by length, amino acid sequence, or both. The peptides are derived from any protein known or found to contain tumor-specific mutations. 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, the first peptide being different from the second peptide, the first neoepitope comprising a mutation, and the second neoepitope comprising the same mutation. In some embodiments, the vector comprises 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, the first region comprising at least one amino acid of the second region, the first peptide being different from the second peptide, the first neoepitope comprising a first mutation, and the second neoepitope comprising a second mutation. In some embodiments, the first mutation and the second mutation are the same. In some embodiments, the mutations are selected from the group consisting of point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, 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, plasmid, phage, transposon, cosmid, virus, or virion. In some embodiments, the vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, poxvirus, alphavirus, vaccinia virus, hepatitis B virus, 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, cationic lipid, cationic polymer, metal nanopolymer, nanorod, liposome, micelle, microbubble, cell-penetrating peptide, or liposphere.

[0449] T cells and T cell receptors T cells are T lymphocytes, which are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells can be naive, i.e., not exposed to an antigen, and show an increase in the expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, as well as a decrease in the expression of CD45RO, compared to more mature T cells with antigen exposure and memory, i.e., memory T cells (T M )(antigen-experienced and long-lived), as well as effector cells (antigen-experienced, cytotoxic). T M can be further divided into subsets of central memory T cells (TC M , showing an increase in the expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, as well as a decrease in the expression of CD54RA compared to naive T cells), and effector memory T cells (T EM , showing a decrease in the expression of CD62L, CCR7, CD28, CD45RA, and an increase in the expression of CD127 compared to naive T cells or TC M ), and effector T cells (T E ) are TC MRefers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, and CD28 and are positive for granzyme and perforin. 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. By targeting antigen peptides to lymph nodes, exposure of resident naive T cells to antigens is promoted, and thus new priming T cells are generated. When the polypeptide is prepared with a neoantigen-concentrating peptide, the T cells then primed in the lymph nodes are primed against the neoantigen and become immunogenic against the tumor or cancer.

[0451] In another embodiment, the present disclosure provides a composition comprising a first peptide comprising a first neoepitope in a first region of a protein and a second peptide comprising a second neoepitope in 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 neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neoepitope binds to a class II HLA protein to form a class II HLA-peptide complex. In some embodiments, the second neoepitope binds to a class I HLA protein to form a class I HLA-peptide complex. In some embodiments, the first neoepitope is CD8 +Activates T cells. In some embodiments, the first new epitope is CD4 + Activates T cells. In some embodiments, the second new epitope is CD4 + Activates T cells. In some embodiments, the second new epitope is CD8 + Activates T cells. In some embodiments, CD4 + The TCR of T cells binds to class II HLA - peptide complexes. In some embodiments, CD8 + The TCR of T cells binds to class II HLA - peptide complexes. In some embodiments, CD8 + The TCR of T cells binds to class I HLA - peptide complexes. In some embodiments, CD4 + The TCR of T cells binds to class I HLA - peptide complexes.

[0452] In some embodiments, the first TCR is a first chimeric antigen receptor specific for the first new epitope, and the second TCR is a second chimeric antigen receptor specific for the second new epitope. 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 binds to the HLA - peptide complex with a K D or IC 50 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. In some embodiments, the first and / or second TCR binds to the HLA - peptide complex with a K D or IC 50and binds to the HLA class I - peptide complex. In some embodiments, the first and / or second TCR has 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 and binds to the HLA class II - peptide complex.

[0453] Antigen - presenting cell Neoantigenic peptides or proteins can be provided as antigen - presenting cells (e.g., dendritic cells) containing such peptides, proteins or polynucleotides as described herein. In other embodiments, such antigen - presenting cells are used to stimulate T cells for use in a patient. Thus, one embodiment of the present disclosure is a composition comprising at least one antigen - presenting cell (e.g., dendritic cell) pulsed or loaded with one or more neoantigenic peptides or polynucleotides as 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 ex vivo with neoantigenic peptides or polynucleotides. In related embodiments, such APCs or PBMCs are returned to and injected into the patient. In some embodiments, the antigen - presenting cell is a dendritic cell. In related embodiments, the dendritic cell is an autologous dendritic cell pulsed with a neoantigenic peptide or nucleic acid. The neoantigenic peptide can be any suitable peptide that elicits 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 cell is a CTL (e.g., CD8 + ). In some embodiments, the T cell is a helper T lymphocyte (Th (e.g., CD4 + ))

[0454] In some embodiments, the present disclosure provides a composition comprising a cell-based immunogenic pharmaceutical composition that can also be administered to a subject. For example, an antigen-presenting cell (APC)-based immunogenic pharmaceutical composition is suitable in the art and, as is understood, can be formulated using any of well-known techniques, carriers, and excipients. 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] The dendritic cell-based immunogenic pharmaceutical composition can be prepared by any method well-known in the art. In some cases, the dendritic cell-based immunogenic pharmaceutical composition can be prepared through ex vivo or in vivo methods. The ex vivo method can include using autologous DCs pulsed ex vivo with the polypeptides described herein to activate or load the DCs prior to administration to the patient. The in vivo method can include targeting specific DC receptors using antibodies coupled to the polypeptides described herein. The DC-based immunogenic pharmaceutical composition may further comprise DC activators such as TLR3, TLR-7-8, and CD40 agonists. The DC-based immunogenic pharmaceutical composition may further comprise 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, the APCs can be prepared from the blood of a human or non-human vertebrate. The APCs can also be isolated from a population of enriched white blood cells. The population of white blood cells can be prepared by methods known to those of skill in the art. Such methods typically include collection of heparinized blood, apheresis or leukapheresis, preparation of buffy coats, rosette formation, centrifugation, density gradient centrifugation (e.g., using Ficoll, colloidal silica particles, and sucrose), differential lysis of non-white blood cells, and filtration. The population of white blood cells can also be prepared by collecting blood from a subject, defibrinating to remove platelets, and lysing erythrocyte cells. The population of white blood cells can optionally be optionally enriched for monocytic dendritic cell precursors.

[0457] The population of blood cells can be obtained from a variety of subjects, depending on the desired use of the enriched population of white blood cells. The subject can be a healthy subject. Alternatively, the blood cells can be obtained from a subject in need of immune stimulation, such as, for example, a cancer patient, or other patient for whom immune stimulation is beneficial. Similarly, the blood cells can be obtained from a subject in need of immunosuppression, such as, for example, a patient having an autoimmune disorder (e.g., rheumatoid arthritis, diabetes, lupus, multiple sclerosis, etc.). The population of white blood cells can also be obtained from a healthy individual with a matching HLA.

[0458] When using blood as a source of APCs, blood leukocytes can be obtained using conventional methods that maintain their viability. According to one aspect of the present disclosure, the blood can be diluted in a medium that may or may not contain heparin or other suitable anticoagulants. The volume of blood to the medium can be about 1 to 1. The cells can be concentrated by centrifugation of the blood in the medium at about 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 for lysing red blood cells, such as ammonium chloride. For example, the mixture can be medium and ammonium chloride in a volume of about 1:1. The cells can be concentrated by centrifugation and washed in the desired solution until a population of leukocytes 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 can be phosphate buffered saline, Hank's balanced salt solution, and complete growth medium. APCs and / or APC precursor cells can also be purified by sedimentation.

[0459] In some embodiments, the APC can be a non-nominal APC under inflammatory conditions or other conditions that are activated. For example, non-nominal APCs 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 APCs can be prepared according to methods known in the art.

[0460] APCs can be cultured, grown, differentiated, and / or matured according to the type of APC, and thus, optionally. 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 medium 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 APCs. 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, and the like. Further, cytokines, growth factors, and / or hormones can be included in the growth medium. For example, for the maintenance and / or expansion of mature dendritic cells, cytokines such as granulocyte / macrophage colony-stimulating factor (GM-CSF) and / or interleukin 4 (IL-4) can be added. 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, and the like. Further, cytokines, growth factors, and / or hormones can be included in the growth medium.

[0462] Other immature APCs can similarly be cultured or propagated. Preparations of immature APCs can be matured to form mature APCs. Maturation of APCs can occur during or after exposure to new antigenic 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), and the like. In another aspect, isolated APC precursors can be used to prepare preparations of immature APCs. 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 monocytic dendritic cell precursors into immature dendritic cells. In some embodiments, APC precursors are isolated from PBMCs. PBMCs can be obtained from donors, e.g., human donors, 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, APCs include APCs loaded with first and second new antigenic peptides comprising first and second new epitopes, or polynucleotides encoding first and second new antigenic peptides comprising first and second new epitopes. In some embodiments, APCs are autologous APCs, allogeneic APCs, or artificial APCs.

[0463] In one embodiment, the present 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 present disclosure provides a composition comprising an APC comprising a first peptide comprising a first neoepitope in a first region of a protein and a second peptide comprising a second neoepitope in 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] Adjuvant An adjuvant can be used to enhance the immune response (humoral and / or cellular) induced in a patient receiving a composition provided herein. The active ingredient in the composition comprises an antigenic peptide. Incorporation of an adjuvant can further enhance the antigenic response of the composition. Optionally, the adjuvant can induce a Th1-type response. In other cases, the adjuvant can induce a Th2-type response. A Th1-type response can be characterized by the production of cytokines such as IFN-γ, as opposed to a Th2-type response, which can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0465] In some aspects, 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. Additionally, muramyl dipeptide (MDP) can also be used as a suitable adjuvant in combination 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 agonists, 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 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 derived from saponin (Aquila Biotech, Worcester, Mass., USA), bacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants, e.g., Ribi's Detox, Quil or Superfos, but are not limited thereto. Adjuvants also include incomplete Freund's or GM-CSF. Some immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparations have been previously described (Dupuis M, et al., Cell Immunol. 1998;186(1):18-27, Allison A C; 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.Some cytokines have been directly associated with affecting dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells 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. Patent No. 5,849,589, which is hereby incorporated by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich D I, 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 the following: CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ (lymphotoxin alpha (LTα)), GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), thymus-expressed chemokine (TECK), mucosa-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-1α, MIP-1β, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, 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, DR5, 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 gene, 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 the following: MCP-1, MIP-1α, MIP-1β, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant form of IL-18, CD40, CD40L, angiogenic factors, fibroblast growth factors, IL-7, IL-22, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, 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 functional fragments thereof.

[0469] In some embodiments, the adjuvant can be a modulator of toll-like receptors. Examples of modulators of toll-like receptors include, but are not limited to, TLR-9 agonists, such as imiquimod, small molecule modulators of toll-like receptors. Other examples of adjuvants used in combination with the immunogenic pharmaceutical compositions described herein can include, but are not limited to, saponins, CpG ODNs, etc. 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 can 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 have a diameter of less than 5 μm, and further can have a submicron diameter, and these small sizes are achieved using a microfluidizer to provide a stable emulsion. Droplets having a size of less than 220 nm can be subjected to filter sterilization.

[0470] Method for generating a polybody having an epitope peptide Polybodies can be generated by cloning. In one approach, a parental scaffold assembly containing an antigen peptide is first generated. The parental scaffold can be digested with a restriction enzyme, and then a peptide or polypeptide containing an epitope is cloned into the restriction site to obtain peptide scaffold unit(s), which are then inserted into a linearized expression vector. The peptide can contain an adjacent linker sequence. Alternatively, a peptide sequence with a linker can be synthesized or generated by PCR. Gibson assembly can be utilized to easily ligate the vector with the insert (the peptide or polypeptide inserted into the vector). The Gibson cloning method provides a fast and flexible cloning strategy regardless of the sequence, as any fragment can be ligated without having specific restriction sites. This involves the T5 exonuclease chewing back to generate single-stranded overhangs at the 5' and 3' ends of the DNA fragment (within the insert), while the vector usually has sticky ends. The insert overhangs thus created hybridize with the sticky ends of the vector upon contact, 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 ligated by hybridization of the overhangs and then ligated using a suitable ligation enzyme to form an intact vector. Variations from the above scheme are contemplated by those skilled in the art and are intended to be within the scope of the present disclosure.

[0471] Artificial mini-proteome vaccine Active tumor eradication immunotherapy requires therapeutics that are effective, rapidly generated, and widely adoptable. Personalized pharmaceuticals that use a subject's own neoantigen peptides to activate T cells are a newly promising area in immuno-oncology, but care must be taken in that the preparation of a neoantigen vaccine can take days to weeks, or even months. Further, this method is expensive. A more effective, time- and cost-effective method is needed here. The goal is to identify and develop a rapid and inexpensive vaccine that includes most neoantigens, as well as other potentially useful antigens from the subject, in a short period of time. Such a product should be easily integrated with any off-the-shelf combination of approved and developing immunotherapies, should be inexpensive to prepare from additional tumor samples after recurrence, or to reduce the effects of tumor heterogeneity between tumors, should incorporate a significantly broad representation of potentially useful epitopes, and should, in particular, activate both the subject's CD8+ and CD4+ cells. Provided herein are compositions and methods for vaccine manufacture that meet the above-described specific unmet needs.

[0472] In one aspect, provided herein is an efficient method for generating a subject-specific cancer vaccine that circumvents the step of neoantigen prediction. This method relates to the generation of an artificial m...

Claims

Claim 1 A composition comprising a fusion polypeptide, wherein the fusion polypeptide comprises (a) one or more antigen polypeptide sequences, and (b) one or more scaffold polypeptide sequences, wherein the scaffold polypeptide comprises Stefin A, the scaffold polypeptide sequence; comprising a polypeptide sequence, 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, the fusion polypeptide is configured to facilitate cleavage of the linker, cleavage of at least one of the one or more antigens, or presentation of at least one of the one or more antigens; the antigen polypeptide is a cancer antigen, and the one or more cancer antigen polypeptides comprise a plurality of neoantigen peptides or autoimmunity peptides; optionally, the neoepitopes of each peptide are unique, the composition. Claim 2 The scaffold polypeptide is (a) comprising a recombinant human polypeptide; (b) not configured to have target binding properties; (c) each of the scaffold polypeptides is Stefin A; (d) lacking (i) post-translational modification, (ii) intrapeptide disulfide bonds, or both; and / or (e) not configured to have enzyme activity, not having inhibitor activity, not having binding activity, or any combination thereof, The composition according to claim 1. Claim 3 The composition according to claim 1 or 2, wherein the fusion polypeptide comprises 6 scaffold polypeptides; and / or 1, 2, 3, 4, 5, or 6 antigen polypeptides. Claim 4 The composition according to any one of claims 1 to 3, wherein the molecular weight of the fusion polypeptide exceeds 75 kDa. Claim 5 The fusion polypeptide comprises a polypeptide sequence having the structure of formula (IIa) in the direction from the N-terminus to the C-terminus, (L-A-L) ｎ -S ｍ or S ｍ -(L-A-L) 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 of 2 or more, n is an integer of 1 or more; Optionally, n is an integer selected from 1 to 5, and m is 2, the composition according to any one of claims 1 to 4. Claim 6 The fusion polypeptide includes a polypeptide sequence having the structure of formula (IIc) in the N-terminal to C-terminal direction, S ｏ -(L-A-L) ｎ -S ｍ Formula (IIc), wherein each S independently represents a scaffold polypeptide sequence, each A independently represents an antigen polypeptide sequence, and each L independently represents a linker sequence or is absent, each of o, n, and m is independently an integer of 1 or more; optionally, o is 1 or 2 or 3, m is 1 or 2 or 3, and n is an integer selected from 1 to 5; further optionally, the fusion polypeptide has a polypeptide sequence having the structure of S-L-A-L-S, A-L-S-L-A-L-S, S-L-A-L-S-L-A, S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S, S-L-A-L-S-L-A-L-S-L-A-L-S-L-A-S, S-S-S-L-A-L-S-S-S, S-S-L-A-L-S-S-S, S-L-A-L-S-S-S, S-S-S-L-A-L-S-S, S-S-S-L-A-L-S, S-S-L-A-L-S-S, or S-L-A-L-S-L-A-L-S-L-A-L-S-L-A-S-L-A-L-S, the composition according to any one of claims 1 to 4.

7. The composition according to claim 6, wherein the scaffold polypeptide and the antigen polypeptide are located in the fusion polypeptide in an alternating pattern.

8. The one or more antigen polypeptides are HLA class I antigen polypeptides, and at least one of the linker sequences includes (a) a lysine residue, an arginine residue, or an alanine residue directly connected to at least one N-terminal of the antigen polypeptides; or (b) a serine residue, a lysine residue, an arginine residue, or an alanine residue directly connected to at least one C-terminal of the antigen polypeptides, the composition according to any one of claims 1 to 7.

9. The one or more antigen polypeptides are HLA class II antigen polypeptides, and at least one of the linker sequences: (a) contains an aspartic acid residue, a methionine residue, a leucine residue, a tyrosine residue, or a phenylalanine residue directly connected to at least one N-terminus of the antigen polypeptides; or (b) contains 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 at least one C-terminus of the antigen polypeptides. The composition according to any one of claims 1 to 8.

10. The fusion polypeptide comprises one or more binding moieties configured to bind to an antigen-presenting cell, an adjuvant, or a reagent; optionally, the antigen-presenting cell is a dendritic cell (DC), a macrophage, a Langerhans cell, or a B cell. The composition according to any one of claims 1 to 9.

11. The multivalent fusion polypeptide is functionalized by additional mixing with an anti-anchor antibody or a fragment thereof coupled to a functionalizing agent such as a DC-targeting domain, an adjuvant or other immunomodulatory agent. The composition according to any one of claims 1 to 9.

12. The one or more binding moieties are configured to bind to one or more receptors expressed on dendritic cells; optionally, the one or more receptors include a C-type lectin receptor, a scavenger receptor, a chemokine receptor, an F4 / 80 receptor, a DC-specific transmembrane protein (DC-STAMP), an Fc receptor, an internalization receptor, Clec9a, XCR1, or any combination thereof. The composition according to claim 10.

13. Each of the cancer neoantigen peptides or a part thereof binds to a protein encoded by an HLA allele expressed by the subject, is encoded by at least one expressed gene of the cancer cells of the subject, and at least one of the cancer neoantigen peptides or a part thereof contains one or more mutations that do not exist in the normal tissues of the subject; optionally, at least one of the one or more mutations a point mutation, wherein the cancer neoantigen peptide binds to the protein encoded by the HLA allele expressed by the subject with an IC50 of less than 500 nM, and with a higher affinity than the corresponding wild-type peptide, as required; a splice site mutation, a frameshift mutation, a read-through mutation, or a gene fusion mutation, the composition according to claim 1.

14. A pharmaceutical composition, comprising: a pharmaceutically acceptable excipient, carrier, or diluent, and the composition according to any one of claims 1 to 13, and further comprising an adjuvant as required. A pharmaceutical composition.

15. A pharmaceutical composition comprising the composition according to any one of claims 1 to 13 for treating or preventing cancer in a human subject in need of treating or preventing cancer.

16. Use of the composition according to any one of claims 1 to 13 in the manufacture of a medicament for treating or preventing cancer.

17. A composition comprising a fusion polypeptide, wherein the fusion polypeptide comprises: one or more antigen polypeptide sequences, and one or more scaffold polypeptide sequences, wherein the scaffold polypeptide comprises Stefin A, 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, the fusion polypeptide is configured to promote 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; the fusion polypeptide comprises one or more binding moieties configured to bind to one or more receptors expressed on dendritic cells; and optionally, the antigen polypeptide is a cancer antigen, an autoantigen, or a viral antigen.

18. The composition according to claim 17, wherein the one or more receptors comprise a C-type lectin receptor, a scavenger receptor, a chemokine receptor, an F4 / 80 receptor, a DC-specific transmembrane protein (DC-STAMP), an Fc receptor, an internalization receptor, Clec9a, XCR1, or any combination thereof.

19. A pharmaceutical composition, ​ (a)a pharmaceutically acceptable excipient, carrier, or diluent, and (b)the composition according to claim 17 or 18, and further comprising, if necessary, an adjuvant. A pharmaceutical composition. **Claim 20** A pharmaceutical composition comprising the composition according to any one of claims 17 to 19 for treating or preventing cancer in a human subject in need of treatment or prevention of cancer. **Claim 21** Use of the composition according to any one of claims 17 to 19 in the manufacture of a medicament for treating or preventing cancer.

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